Lecture Presentation by Nicole Tunbridge andKathleen Fitzpatrick
19DNA Technology
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The DNA Toolbox
a) Recently the genome sequences of two extinct speciesmdashNeanderthals and wooly mammothsmdashhave been completed
b) Advances in sequencing techniques make genome sequencing increasingly faster andless expensive
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Figure 191
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Figure 191a
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a) Biotechnology is the manipulation of organisms or their components to make useful products
b) The applications of DNA technology affect everything from agriculture to criminal lawto medical research
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Concept 191 DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry
a) The complementarity of the two DNA strands is the basis for nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand
b)Genetic engineering is the direct manipulation of genes for practical purposes
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DNA Sequencing
a) Researchers can exploit the principle of complementary base pairing to determine a genersquos complete nucleotide sequence called DNA sequencing
b) The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger
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Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
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Figure 192a
(a) Standard sequencing machine
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Figure 192b
(b) Next-generation sequencing machines
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Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
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Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
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Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
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Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
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a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
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Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
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Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
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Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
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Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
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a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
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Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
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Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
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a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
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Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
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Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
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TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
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Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
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Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
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Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
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Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
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Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
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Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
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Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
The DNA Toolbox
a) Recently the genome sequences of two extinct speciesmdashNeanderthals and wooly mammothsmdashhave been completed
b) Advances in sequencing techniques make genome sequencing increasingly faster andless expensive
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Figure 191
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Figure 191a
copy 2015 Pearson Education Ltd
a) Biotechnology is the manipulation of organisms or their components to make useful products
b) The applications of DNA technology affect everything from agriculture to criminal lawto medical research
copy 2015 Pearson Education Ltd
Concept 191 DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry
a) The complementarity of the two DNA strands is the basis for nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand
b)Genetic engineering is the direct manipulation of genes for practical purposes
copy 2015 Pearson Education Ltd
DNA Sequencing
a) Researchers can exploit the principle of complementary base pairing to determine a genersquos complete nucleotide sequence called DNA sequencing
b) The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger
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Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
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Figure 192a
(a) Standard sequencing machine
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Figure 192b
(b) Next-generation sequencing machines
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Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
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Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
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Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
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Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
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Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 191
copy 2015 Pearson Education Ltd
Figure 191a
copy 2015 Pearson Education Ltd
a) Biotechnology is the manipulation of organisms or their components to make useful products
b) The applications of DNA technology affect everything from agriculture to criminal lawto medical research
copy 2015 Pearson Education Ltd
Concept 191 DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry
a) The complementarity of the two DNA strands is the basis for nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand
b)Genetic engineering is the direct manipulation of genes for practical purposes
copy 2015 Pearson Education Ltd
DNA Sequencing
a) Researchers can exploit the principle of complementary base pairing to determine a genersquos complete nucleotide sequence called DNA sequencing
b) The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger
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Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
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Figure 192a
(a) Standard sequencing machine
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Figure 192b
(b) Next-generation sequencing machines
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Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
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Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 191a
copy 2015 Pearson Education Ltd
a) Biotechnology is the manipulation of organisms or their components to make useful products
b) The applications of DNA technology affect everything from agriculture to criminal lawto medical research
copy 2015 Pearson Education Ltd
Concept 191 DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry
a) The complementarity of the two DNA strands is the basis for nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand
b)Genetic engineering is the direct manipulation of genes for practical purposes
copy 2015 Pearson Education Ltd
DNA Sequencing
a) Researchers can exploit the principle of complementary base pairing to determine a genersquos complete nucleotide sequence called DNA sequencing
b) The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger
copy 2015 Pearson Education Ltd
Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
copy 2015 Pearson Education Ltd
Figure 192a
(a) Standard sequencing machine
copy 2015 Pearson Education Ltd
Figure 192b
(b) Next-generation sequencing machines
copy 2015 Pearson Education Ltd
Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Biotechnology is the manipulation of organisms or their components to make useful products
b) The applications of DNA technology affect everything from agriculture to criminal lawto medical research
copy 2015 Pearson Education Ltd
Concept 191 DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry
a) The complementarity of the two DNA strands is the basis for nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand
b)Genetic engineering is the direct manipulation of genes for practical purposes
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DNA Sequencing
a) Researchers can exploit the principle of complementary base pairing to determine a genersquos complete nucleotide sequence called DNA sequencing
b) The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger
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Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
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Figure 192a
(a) Standard sequencing machine
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Figure 192b
(b) Next-generation sequencing machines
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Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
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Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
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Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
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Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
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Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
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Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Concept 191 DNA sequencing and DNA cloning are valuable tools for genetic engineering and biological inquiry
a) The complementarity of the two DNA strands is the basis for nucleic acid hybridization the base pairing of one strand of nucleic acid to the complementary sequence on another strand
b)Genetic engineering is the direct manipulation of genes for practical purposes
copy 2015 Pearson Education Ltd
DNA Sequencing
a) Researchers can exploit the principle of complementary base pairing to determine a genersquos complete nucleotide sequence called DNA sequencing
b) The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger
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Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
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Figure 192a
(a) Standard sequencing machine
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Figure 192b
(b) Next-generation sequencing machines
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Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
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a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
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Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
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Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
DNA Sequencing
a) Researchers can exploit the principle of complementary base pairing to determine a genersquos complete nucleotide sequence called DNA sequencing
b) The first automated procedure was based on a technique called dideoxy or chain termination sequencing developed by Sanger
copy 2015 Pearson Education Ltd
Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
copy 2015 Pearson Education Ltd
Figure 192a
(a) Standard sequencing machine
copy 2015 Pearson Education Ltd
Figure 192b
(b) Next-generation sequencing machines
copy 2015 Pearson Education Ltd
Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
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Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 192
(a) Standard sequencing machine
(b) Next-generation sequencing machines
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Figure 192a
(a) Standard sequencing machine
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Figure 192b
(b) Next-generation sequencing machines
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Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
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Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
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a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
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a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
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Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
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Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
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Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
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TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
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Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
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Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
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Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 192a
(a) Standard sequencing machine
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Figure 192b
(b) Next-generation sequencing machines
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Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
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a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
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Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
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Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 192b
(b) Next-generation sequencing machines
copy 2015 Pearson Education Ltd
Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
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Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
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Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 193 DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
DNApolymerase
DNA (template strand)
Labeled strands
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strand
Shortest Longest
Technique
Results
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
CTGACTTCGACAA
CTGACTTCGACAA
TGTT
CTGTT
dATP
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
dddd
dddd
dddd
dddd
dd
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 193a
DNA(template strand)
Primer Deoxyribo-nucleotides
Dideoxyribonucleotides(fluorescently tagged)
Technique
DNApolymerase
5prime
5prime
3prime
3prime
CTGACTTCGACAA
TGTT
dATP ddATPdCTP ddCTPdTTP ddTTPdGTP ddGTP
P P P P P PG G
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
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Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
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Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
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Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
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Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 193b
DNA (template strand)
Labeled strands
Shortest Longest
5prime
5prime 5prime3prime
3prime
3primeCTGACTTCGACAA
CTGTT
GCTGTT
CTGTT
CTGTT
CTGTT
CTGTT
CTGTT
GACTGAAGCTGTT
ACTGAAG
CTGAAG
TGAAG
CTGTT
GAAG
AAG
AG
dddd
dddd
dddd
dddd
dd
Technique
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
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Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
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Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
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Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 193c
Directionof movementof strands
Longest labeled strand
Detector
Laser Shortest labeled strandResults
Last nucleotideof longestlabeled strand
Last nucleotideof shortestlabeled strand
GACTGAAGC
Technique
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) ldquoNext-generation sequencingrdquo techniques use a single template strand that is immobilized and amplified to produce an enormous number of identical fragments
b) Thousands or hundreds of thousands of fragments (400ndash1000 nucleotides long) are sequenced in parallel
c) This is a type of ldquohigh-throughputrdquo technology
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 194 Technique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
Results
6 7 8
5
4
3
2
1
4-mer
3-mer
2-mer
1-mer
ATGC
Template strandof DNA
Primer
3prime
A3prime5prime 5primeT G C
A T G C A T G C A T G C A T G C
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP dGTP dCTP
PPi
PPi
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
CC
CC
AA
AA
T
T
T
G
GG
G
A A A AC
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
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Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
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Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 194aTechnique
Genomic DNA is fragmented
Each fragment is isolated witha bead
Using PCR 106 copies of eachfragment are made each attachedto the bead by 5prime end
The bead is placed into a well withDNA polymerases and primers
4
Template strandof DNA
Primer3prime
A
3prime5prime 5prime
T G C
3
2
1
5 A solution of each of the four nucleotidesis added to all wells and then washed offThe entire process is then repeated
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
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a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
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Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
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Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
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Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
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Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
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Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 194b
6 7If a nucleotide is joined to a growing strand PPi is released causing a flashof light that is recorded
If a nucleotide is notcomplementary to thenext template baseno PPi is released andno flash of light is recorded
Templatestrandof DNA
Primer
DNApolymerase
dATPdTTP
PPi
CC
CC
AA
AA
T
T
T
GG
G
G
TechniqueA T G C A T G C
A
CC
CC
AA
AA
T
T
T
GG
G
GA
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
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Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
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Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
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a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
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Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
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Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
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Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
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Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
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Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
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Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 194c
8
TechniqueA T G C A T G C
CC
CC
AA
AA
T
T
T
GG
G
G
CC
CC
AA
AA
T
T
T
GG
G
GA A
C
dGTP dCTP
PPi
The process is repeated until everyfragment has a complete complementarystrand The pattern of flashes reveals thesequence
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
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Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
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Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 194d
Results
4-mer
3-mer
2-mer
1-mer
ATGC
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
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Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
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a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
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Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) In ldquothird-generation sequencingrdquo the techniques used are even faster and less expensive thanthe previous
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
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Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Making Multiple Copies of a Gene or Other DNA Segment
a) To work directly with specific genes scientists prepare well-defined DNA segments in multiple identical copies by a process called DNA cloning
b)Plasmids are small circular DNA molecules that replicate separately from the bacterial chromosome
c) Researchers can insert DNA into plasmids to produce recombinant DNA a molecule withDNA from two different sources
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Reproduction of a recombinant plasmid in a bacterial cell results in cloning of the plasmid including the foreign DNA
b) This results in the production of multiple copies of a single gene
c) The production of multiple copies of a single gene is a type of DNA cloning called gene cloning
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 195 Bacterium
4
3
2
1
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone ofcells containing the ldquoclonedrdquo gene of interest
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteriafor cleaning up toxic waste
Protein dissolves blood clotsin heart attack therapy
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 195a Bacterium
Bacterialchromosome
Plasmid
Gene insertedinto plasmid
Cell containing geneof interest
RecombinantDNA (plasmid)
Gene ofinterest
DNA ofchromosome(ldquoforeignrdquo DNA)
Plasmid put intobacterial cell
Recombinantbacterium
Host cell grown in culture to form a clone of cells containing the ldquoclonedrdquo gene of interest
Gene ofinterest
Gene ofinterest Protein expressed
from gene of interest
1
2
3
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 195b
4
Gene ofinterest Protein expressed
from gene of interest
Copies of gene
Gene for pest resistanceinserted into plants
Protein harvested
Basic researchand variousapplications
Human growth hormonetreats stunted growth
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolves blood clots in heart attack therapy
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
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Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) A plasmid used to clone a foreign gene is called a cloning vector
b) Bacterial plasmids are widely used as cloning vectors because they are readily obtained easily manipulated easily introduced into bacterial cells and once in the bacteria they multiply rapidly
c) Gene cloning is useful for amplifying genes to produce a protein product for research medical or other purposes
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
a) Bacterial restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites
b) A restriction enzyme usually makes many cuts yielding restriction fragments
c) The most useful restriction enzymes cut DNAin a staggered way producing fragments with ldquosticky endsrdquo
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
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Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
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Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
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Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
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a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
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Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
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Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
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a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
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Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
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Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
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Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Sticky ends can bond with complementary sticky ends of other fragments
b)DNA ligase is an enzyme that seals the bonds between restriction fragments
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
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Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
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Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
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Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 196Bacterialplasmid
Restriction site
DNA
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
1
Base pairing of stickyends produces variouscombinations
2
DNA ligaseseals the strands
3
Sticky end
Fragment from different DNA moleculecut by the same restriction enzyme
One possible combination
Recombinant DNA molecule
GG
5prime
GG
5prime
5prime5prime
5prime5prime
5prime
5prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime 3prime
3prime
3prime
3prime
3prime
3prime
5prime 5prime 5prime
5prime5prime 5prime
5prime
5prime
G AAT T CGAATTC
G AATT CGAATTC
Recombinant plasmid
GAATTCCTTAAG
CTTAAAATTC
AATTC
CTTAA
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
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Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
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a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
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Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 196a
Bacterialplasmid
Restriction site
Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow
DNA
G
5prime
5prime
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3prime
3prime
3primeSticky end
G
1
CTTAAAATTC
C T TA A GG A AT T C
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
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Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
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Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 196b
2
G5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeSticky end
G
3prime
3prime
3prime
3prime 3prime
3prime 3prime
3prime
5prime
5prime
5prime 5prime 5prime
5prime 5prime 5prime
Base pairing of stickyends produces variouscombinations Fragment from different
DNA molecule cut by the same restriction enzyme
One possible combination
G G
G CGC
G CGC
AATT AATTTTAATTAA
CTTAA
CTTAA
AATTC
AATTC
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
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Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
TTAA
Figure 196c
3prime
3prime 3prime
3prime 3prime
3prime
5prime 5prime 5prime
5prime 5prime 5primeOne possible combination
G AATT CGC
G CGC
Recombinant DNA molecule
Recombinantplasmid
3prime
5prime3prime
5prime
3 DNA ligaseseals the strands
AATTTTAA
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
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Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
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Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
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a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
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Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
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a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Animation Restriction Enzymes
>
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a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) To check the recombinant plasmid researchers might cut the products again using the same restriction enzyme
b) To separate and visualize the fragments produced gel electrophoresis would be carried out
c) This technique uses a gel made of a polymer to separate a mixture of nucleic acids or proteins based on size charge or other physical properties
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
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Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
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Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 197
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
(a) Negatively charged DNA molecules movetoward the positive electrode
Powersource
(b) Shorter molecules are slowed down less thanlonger ones so they move faster through the gel
Restriction fragments(size standards)
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 197a
AnodeCathode
Wells
Gel
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
(a) Negatively charged DNA molecules movetoward the positive electrode
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
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Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 197b
Restriction fragments(size standards)
(b) Shorter molecules are slowed down less than longer ones so they move faster through the gel
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
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Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
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Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
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Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Video Biotechnology Lab
>
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
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a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
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Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
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a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
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a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
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Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
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Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
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Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
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Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
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a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
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Figure 1918
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Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
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Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
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Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use in DNA Cloning
a) The polymerase chain reaction PCR can produce many copies of a specific target segment of DNA
b) A three-step cyclemdashheating cooling and replicationmdashbrings about a chain reaction that produces an exponentially growing population of identical DNA molecules
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
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Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
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a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
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Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
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Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
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Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
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Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
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Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
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Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
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Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
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Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
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Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
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Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
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Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
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Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
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Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
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Figure 1923a
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Figure 1923b
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Video Pronuclear Injection
>
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Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
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Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
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a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
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a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
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a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
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Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) The key to PCR is an unusual heat-stable DNA polymerase called Taq polymerase
b) PCR uses a pair of primers specific for the sequence to be amplified
c) PCR amplification occasionally incorporates errors into the amplified strands and so cannot substitute for gene cloning in cells
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 198
Targetsequence
Genomic DNA
Technique
Denaturation
5prime
5prime
5prime
5prime
3prime
3prime
3prime
3primeAnnealing
Extension
Cycle 1yields
2molecules
Primers
Newnucleotides
1
2
3
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 198a
Targetsequence
Genomic DNA
Technique5prime
5prime
3prime
3prime
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
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Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
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Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 198b-1
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
1
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 198b-2
1
2
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Primers
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 198b-3
1
2
3
Denaturation
Technique
5prime
5prime
3prime
3prime
Cycle 1yields
2molecules
Annealing
Extension
Primers
Newnucleotides
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 198c
Technique
Cycle 2yields
4molecules
Cycle 3 yields 8molecules2 molecules
(in white boxes)match target
sequence
Results After 30 more cycles over 1 billion (109) molecules matchthe target sequence
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors
b) The resulting clones are sequenced anderror-free inserts selected
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
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Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 199DNA fragments obtainedby PCR with restrictionsites matching those inthe cloning vector
Cut with same restrictionenzyme used on cloningvectorA gene that makes bacterial
cells resistant to an antibioticis present on the plasmid
Cloning vector(bacterial plasmid)
Only cells that take upa plasmid will survive
Mix and ligate
RecombinantDNA plasmid
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Animation Cloning a Gene
>
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
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Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Expressing Cloned Eukaryotic Genes
a) After a gene has been cloned its protein product can be produced in larger amounts for research
b) Cloned genes can be expressed as protein in either bacterial or eukaryotic cells
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Bacterial Expression Systems
a) Several technical difficulties hinder expression of cloned eukaryotic genes in bacterial host cells
b) To overcome differences in promoters and other DNA control sequences scientists usually employ an expression vector a cloning vector that contains a highly active bacterial promoter
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes
b) Researchers can avoid this problem by using cDNA complementary to the mRNA which contains only exons
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Eukaryotic DNA Cloning and Expression Systems
a) Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells such as yeasts as hosts for cloning and expressing genes
b) Even yeasts may not possess the proteins required to modify expressed mammalianproteins properly
c) In such cases cultured mammalian or insect cells may be used to express and study proteins
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
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a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
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a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
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Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
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a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
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Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
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Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
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Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
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Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
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Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) One method of introducing recombinant DNA into eukaryotic cells is electroporation applying a brief electrical pulse to create temporary holes in plasma membranes
b) Alternatively scientists can inject DNA into cells using microscopically thin needles
c) Once inside the cell the DNA is incorporated into the cellrsquos DNA by natural genetic recombination
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Cross-Species Gene Expression and Evolutionary Ancestry
a) The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species
b) For example Pax-6 is a gene that directs formation of a vertebrate eye the same gene in flies directs the formation of an insect eye (which is quite different from the vertebrate eye)
c) The Pax-6 genes in flies and vertebrates can substitute for each other
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Concept 192 Biologists use DNA technology to study gene expression and function
a) Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Analyzing Gene Expression
a) The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Studying the Expression of Single Genes
a) mRNA can be detected by nucleic acid hybridization with complementary molecules
b) These complementary molecules of either DNA or RNA are nucleic acid probes
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1910
Head
50 microm
Thorax Abdomen
Segmentboundary
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1910a
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
5prime 3prime
3prime3prime
3prime
5prime 5prime
5primeTAACGGTTCCAGCATTGCCAAGGTCG
CTCAAGTTGCTCTGAGTTCAACGAGA
Cellsexpressingthe wg gene
Cellsexpressingthe en gene
wg mRNA en mRNA
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1910b
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
Head Thorax Abdomen
Segmentboundary
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1910c
50 microm
Head Thorax Abdomen
T1 T2 T3 A1 A2 A3 A4 A5
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Reverse transcriptase-polymerase chain reaction (RT-PCR) is useful for comparing amounts of specific mRNAs in several samples at the same time
b) Reverse transcriptase is added to mRNA to make complementary DNA (cDNA) which serves as a template for PCR amplification of the gene of interest
c) The products are run on a gel and the mRNA of interest is identified
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1911-1
DNA innucleus
mRNAs incytoplasm
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1911-2
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime5prime3prime
3primeA A A A A AT T T T T
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1911-3
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1911-4
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1911-5
DNA innucleus
mRNAs incytoplasm
Reverse transcriptasePoly-A tailmRNA
DNA strand
Primer(poly-dT)
5prime
5prime
5prime
5prime
3prime
3prime3prime
3primeA A A A A A
A A A A A A
T T T T T
T T T T T
5prime3prime 5prime
3prime
DNA polymerase
5prime3prime 5prime
3prime
cDNA
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1912
cDNA synthesis mRNAs
cDNAs
PCR amplificationPrimers
Specificgene
Gel electrophoresis
Results Embryonic stages1 2 3 4 5 6
Technique1
2
3
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Studying the Expression of Interacting Groups of Genes
a) Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays
b)DNA microarray assays compare patterns of gene expression in different tissues at different times or under different conditions
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1913
Genes expressedin first tissue
Genes expressedin second tissue
Genes expressedin both tissues
Genes expressedin neither tissue
DNA microarray(actual size)
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1913a
Each dot is a well containing identical copiesof DNA fragments that carry a specific gene
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) With rapid and inexpensive sequencing methods available researchers can also just sequence cDNA samples from different tissues or embryonic stages to determine the gene expression differences between them
b) By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Determining Gene Function
a) One way to determine function is to disable the gene and observe the consequences
b) Using in vitro mutagenesis mutations are introduced into a cloned gene altering or destroying its function
c) When the mutated gene is returned to the cellthe normal genersquos function might be determined by examining the mutantrsquos phenotype
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Gene expression can also be silenced using RNA interference (RNAi)
b) Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the genersquos mRNA
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) In humans researchers analyze the genomes of many people with a certain genetic condition to try to find nucleotide changes specific to the condition
b) These genome-wide association studies test for genetic markers sequences that vary among individuals
c) SNPs (single nucleotide polymorphisms) single nucleotide variants are among the most useful genetic markers
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene
b) SNPs are rarely directly involved in the disease they are most often in noncoding regions of the genome
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1914
DNA
SNPNormal allele
Disease-causingallele
A
T
C
G
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Concept 193 Cloned organisms and stem cells are useful for basic research and other applications
a) Organismal cloning produces one or more organisms genetically identical to the ldquoparentrdquo that donated the single cell
b) A stem cell is a relatively unspecialized cell that can reproduce itself indefinitely or under certain conditions can differentiate into one or more types of specialized cells
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Cloning Plants Single-Cell Cultures
a) In plants cells can dedifferentiate and thengive rise to all the specialized cell types ofthe organism
b) A totipotent cell such as this is one that can generate a complete new organism
c) Plant cloning is used extensively in agriculture
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1915
Cross sectionof carrot root
Smallfragments
Fragmentswere culturedin nutrientmediumstirringcaused singlecells to shearoff into theliquid
Singlecells free insuspensionbegan todivide
Embryonicplantdevelopedfrom aculturedsingle cell
Plantlet wascultured onagar mediumLater it wasplanted in soil
Adult plant
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Cloning Animals Nuclear Transplantation
a) In nuclear transplantation the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell
b) Experiments with frog embryos have shown thata transplanted nucleus can often support normal development of the egg
c) However the older the donor nucleus the lower the percentage of normally developing tadpoles
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1916
Experiment Frog embryo
UV
Frog egg cell Frog tadpole
Less differ-entiated cell
Fully differ-entiated(intestinal) cell
Donornucleustrans-planted
Enucleatedegg cell
Donornucleustrans-plantedEgg with donor nucleus
activated to begindevelopmentResults
Most developinto tadpoles
Most stop developingbefore tadpole stage
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Reproductive Cloning of Mammals
a) In 1997 Scottish researchers announced the birth of Dolly a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell
b) Dollyrsquos premature death in 2003 as well as her arthritis led to speculation that her cells were not as healthy as those of a normal sheep possibly reflecting incomplete reprogramming of the original transplanted nucleus
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1917 Technique
1 2
3
4
5
6
Mammarycell donor
Results
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1917a
Technique
1
Mammarycell donor
Egg celldonor
Egg cellfrom ovary
Culturedmammarycells
Nucleus removed
Cells fused
Nucleus frommammary cell
2
3
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1917b
4
5
6
Technique
Results
Nucleus frommammary cell
Grown in culture
Early embryo
Implanted in uterusof a third sheep
Surrogatemother
Embryonicdevelopment Lamb (ldquoDollyrdquo)
genetically identical tomammary cell donor
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Since 1997 cloning has been demonstrated in many mammals including mice cats cows horses mules pigs and dogs
b) CC (for Carbon Copy) was the first cat cloned however CC differed somewhat from herfemale ldquoparentrdquo
c) Cloned animals do not always look or behave exactly the same
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1918
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Faulty Gene Regulation in Cloned Animals
a) In most nuclear transplantation studies only a small percentage of cloned embryos have developed normally to birth and many cloned animals exhibit defects
b) Many epigenetic changes such as acetylation of histones or methylation of DNA must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Stem Cells of Animals
a) Stem cells are relatively unspecialized cells that can both reproduce indefinitely and under certain conditions differentiate into one or more specialized cell types
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1919
Stem cell
Cell division
Stem cell and Precursor cell
Fat cells or Bonecells
or Whiteblood cells
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Embryonic and Adult Stem Cells
a) Many early embryos contain stem cells capableof giving rise to differentiated embryonic cells ofany type
b) In culture these embryonic stem cells reproduce indefinitely
c) Depending on culture conditions they can be made to differentiate into a variety of specialized cells
d) Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace nonreproducing cells as needed
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1920
Cells that can generateall embryonic cell types
Cultured stem cells
Cells that generate a limitednumber of cell types
Different cultureconditions
Liver cells Nerve cells Blood cells
Different types ofdifferentiated cells
Embryonicstem cells
Adult stemcells
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Embryonic stem (ES) cells are pluripotent capable of differentiating into many differentcell types
b) The ultimate aim of research with stem cellsis to supply cells for the repair of damaged or diseased organs
c) ES cells present ethical and political issues
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Induced Pluripotent Stem (iPS) Cells
a) Researchers can treat differentiated cells and reprogram them to act like ES cells
b) Researchers used retroviruses to induce extra copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells
c) iPS cells can perform most of the functions ofES cells
d) iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1921 Stem cell Precursor cellExperiment
Skinfibroblastcell
Four ldquostem cellrdquo master regulatorgenes were introduced usingthe retroviral cloning vector
Induced pluripotentstem (iPS) cell
Oct34 Sox2
c-MycKlf4
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Concept 194 The practical applications of DNA-based biotechnology affect our lives in many ways
a) Many fields benefit from DNA technology and genetic engineering
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Medical Applications
a) One benefit of DNA technology is identification of human genes in which mutation plays a role in genetic diseases
b) Researchers use microarray assays or other tools to identify genes turned on or off in particular diseases
c) The genes and their products are then potential targets for prevention or therapy
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Diagnosis and Treatment of Diseases
a) Scientists can diagnose many human genetic disorders using PCR and sequence-specific primers then sequencing the amplified product to look for the disease-causing mutation
b) SNPs may be associated with a disease-causing mutation
c) SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Human Gene Therapy
a) Gene therapy is the alteration of an afflicted individualrsquos genes
b) Gene therapy holds great potential for treating disorders traceable to a single defective gene
c) Vectors are used for delivery of genes into specific types of cells for example bone marrow
d) Gene therapy provokes both technical and ethical questions
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1922Clonedgene 1
2
3
4
Insert RNA version of normal alleleinto retrovirus or other viral vector
Viral RNA
Viralcapsid
Let virus infect bone marrow cellsthat have been removed from thepatient and cultured
Viral DNA carrying the normalallele inserts into chromosome
Bonemarrowcell frompatient
Inject engineeredcells into patient
Bonemarrow
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Pharmaceutical Products
a) Advances in DNA technology and genetic research are important to the developmentof new drugs to treat diseases
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Synthesis of Small Molecules for Use as Drugs
a) The drug imatinib is a small molecule that inhibits overexpression of a specific leukemia-causing receptor
b) This approach is feasible for treatment of cancers in which the molecular basis is well-understood
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Protein Production in Cell Cultures
a) Host cells in culture can be engineered to secrete a protein as it is made simplifying the task of purifying it
b) This is useful for the production of insulin human growth hormones and vaccines
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Protein Production by ldquoPharmrdquo Animals
a) Transgenic animals are made by introducing genes from one species into the genome of another animal
b) Transgenic animals are pharmaceutical ldquofactoriesrdquo producers of large amounts of otherwise rare substances for medical use
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1923
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1923a
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1923b
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Video Pronuclear Injection
>
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Forensic Evidence and Genetic Profiles
a) An individualrsquos unique DNA sequence or genetic profile can be obtained by analysis of tissue or body fluids
b) DNA testing can identify individuals with a high degree of certainty
c) Genetic profiles are currently analyzed using genetic markers called short tandem repeats (STRs)
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) STRs are variations in the number of repeats of specific DNA sequences
b) PCR and gel electrophoresis are used to amplify and then identify STRs of different lengths
c) The probability that two people who are not identical twins have the same STR markers is exceptionally small
d) As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1924 (a) Earl Washington justbefore his release in 2001after 17 years in prison
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1924a
(a) Earl Washington justbefore his release in 2001after 17 years in prison
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 1924b
(b) These and other STR data (not shown) exoneratedWashington and led Tinsley to plead guilty to the murder
Source ofsample
STRmarker 1
STRmarker 2
STRmarker 3
Semen on victim 1719 1316 1212
Earl Washington 1618 1415 1112
Kenneth Tinsley 1719 1316 1212
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Environmental Cleanup
a) Genetic engineering can be used to modify the metabolism of microorganisms
b) Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Agricultural Applications
a) DNA technology is being used to improve agricultural productivity and food quality
b) Genetic engineering of transgenic animals speeds up the selective breeding process
c) Beneficial genes can be transferred between varieties or species
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Agricultural scientists have endowed a number of crop plants with genes for desirable traits
b) The Ti plasmid is the most commonly used vector for introducing new genes into plant cells
c) Genetic engineering in plants has been used to transfer many useful genes including those for herbicide resistance increased resistance to pests increased resistance to salinity and improved nutritional value of crops
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Safety and Ethical Questions Raised by DNA Technology
a) Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures
b) Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) Most public concern about possible hazards centers on genetically modified (GM) organisms used as food
b) Some are concerned about the creation of ldquosuper weedsrdquo from the transfer of genes from GM crops to their wild relatives
c) Other worries include the possibility that transgenic protein products might cause allergic reactions
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
a) As biotechnology continues to change so does its use in agriculture industry and medicine
b) National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 19UN01
5prime 3primeCUCAUCACCGGC
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 19UN02
5prime3prime GAGTAGTGGCCG
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 19UN03
Regulatory region Promoter
A
A
A
B
B
B
C
C
C
A B C
A B C
Gene
Treatments
Segments being tested
Hoxd13 mRNAHoxd13
Blue = Hoxd13mRNA whitetriangles = futurethumb locations
Relative amount ofHoxd13 mRNA ()
0 20 40 60 80 100
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 19UN04
Sticky end
5prime
5prime
5prime
5prime
3prime
3prime 3prime
3primeG
GC T TA AA AT T C
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 19UN05
Mix and ligate
Recombinant DNA plasmids
Cloningvector(often abacterialplasmid)
DNA fragments obtainedby PCR or from another source(cut by same restrictionenzyme used on cloning vector)
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 19UN06
Plasmid
Aardvark DNA
5prime 3prime3prime 5prime
GAATTCTAAAGCGCTTATGAATTCCTTAAGATTTCGCGAATACTTAAG
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
(25)
(26)
Figure 19UN01
Figure 19UN02
Figure 19UN03
Figure 19UN04
Figure 19UN05
Figure 19UN06
Figure 19UN07
copy 2015 Pearson Education Ltd
Figure 19UN07
Slide 1
The DNA Toolbox
Figure 191
Figure 191a
Concept 191 DNA sequencing and DNA cloning are valuable tools
DNA Sequencing
Figure 192
Figure 192a
Figure 192b
Figure 193
Figure 193a
Figure 193b
Figure 193c
(2)
Figure 194
Figure 194a
Figure 194b
Figure 194c
Figure 194d
(3)
Making Multiple Copies of a Gene or Other DNA Segment
(4)
Figure 195
Figure 195a
Figure 195b
(5)
Using Restriction Enzymes to Make a Recombinant DNA Plasmid
(6)
Figure 196
Figure 196a
Figure 196b
Figure 196c
Animation Restriction Enzymes
(7)
Figure 197
Figure 197a
Figure 197b
Video Biotechnology Lab
Amplifying DNA The Polymerase Chain Reaction (PCR) and Its Use
(8)
Figure 198
Figure 198a
Figure 198b-1
Figure 198b-2
Figure 198b-3
Figure 198c
(9)
Figure 199
Animation Cloning a Gene
Expressing Cloned Eukaryotic Genes
Bacterial Expression Systems
(10)
Eukaryotic DNA Cloning and Expression Systems
(11)
Cross-Species Gene Expression and Evolutionary Ancestry
Concept 192 Biologists use DNA technology to study gene expre
Analyzing Gene Expression
Studying the Expression of Single Genes
(12)
Figure 1910
Figure 1910a
Figure 1910b
Figure 1910c
(13)
Figure 1911-1
Figure 1911-2
Figure 1911-3
Figure 1911-4
Figure 1911-5
Figure 1912
Studying the Expression of Interacting Groups of Genes
Figure 1913
Figure 1913a
(14)
Determining Gene Function
(15)
(16)
(17)
Figure 1914
Concept 193 Cloned organisms and stem cells are useful for ba
Cloning Plants Single-Cell Cultures
Figure 1915
Cloning Animals Nuclear Transplantation
Figure 1916
Reproductive Cloning of Mammals
Figure 1917
Figure 1917a
Figure 1917b
(18)
Figure 1918
Faulty Gene Regulation in Cloned Animals
Stem Cells of Animals
Figure 1919
Embryonic and Adult Stem Cells
Figure 1920
(19)
Induced Pluripotent Stem (iPS) Cells
Figure 1921
Concept 194 The practical applications of DNA-based biotechno
Medical Applications
Diagnosis and Treatment of Diseases
Human Gene Therapy
Figure 1922
Pharmaceutical Products
(20)
(21)
(22)
Figure 1923
Figure 1923a
Figure 1923b
Video Pronuclear Injection
Forensic Evidence and Genetic Profiles
(23)
Figure 1924
Figure 1924a
Figure 1924b
Environmental Cleanup
Agricultural Applications
(24)
Safety and Ethical Questions Raised by DNA Technology
