CAMPBELL

BIOLOGY Reece • Urry • Cain • Wasserman • Minorsky • Jackson

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TENTH EDITION

CAMPBELL

BIOLOGY Reece • Urry • Cain • Wasserman • Minorsky • Jackson

TENTH EDITION

20 DNA Tools and Biotechnology

Lecture Presentation by Nicole Tunbridge and Kathleen Fitzpatrick

The DNA Toolbox • Recently the genome sequences of two extinct

species—Neanderthals and wooly mammoths—have been completed

• Advances in sequencing techniques make genome sequencing increasingly faster and less expensive

Figure 20.1 How can the technique shown in this model speed up DNA sequencing?

Mammoths DNA sequencing

Overview: DNA Terminology • Sequencing of the human genome was completed by

2007. • Sequencing of the genomes of more than 7,000 species

was under way in 2010 • DNA sequencing has depended on advances in

technology, starting with making recombinant DNA • Recombinant DNA: nucleotide sequences from two

different sources, often two species, are combined in vitro into the same DNA molecule

• Methods for making recombinant DNA are central to genetic engineering, the direct manipulation of genes for practical purposes

• Biotechnology: the manipulation of organisms or their genetic components to make useful products

• An example of DNA technology is the microarray, a measurement of gene expression of thousands of different genes

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DNA sequencing and DNA cloning are valuable tools for genetic engineering

and biological inquiry • 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

• Researchers can exploit the principle of complementary base pairing to determine a gene’s complete nucleotide sequence, called DNA sequencing

• The first automated procedure was based on a technique called dideoxy or chain termination sequencing, developed by Sanger

Figure 20.2

(a) Standard sequencing machine

(b) Next-generation sequencing machines

DNA Sequencing identifies the exact nucleotide sequence of a DNA fragment

• Relatively short DNA fragments can be sequenced by the dideoxy chain termination method

• Modified nucleotides called dideoxyribonucleotides (ddNTP) attach to synthesized DNA strands of different lengths

• Each type of ddNTP is tagged with a distinct fluorescent label that identifies the nucleotide at the end of each DNA fragment

• The DNA sequence can be read from the resulting spectrogram

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Methodology of DNA Sequencing

1. The DNA is denatured to a single strand and is incubated with a Primer, DNA polymerase, and fluorescently labeled dd-nucleotides.

2. Synthesis of a complimentary strand begins at the 3’ end of the primer. The fluorescently labeled ddNTP’s will be added randomly to the 3’ end of the growing strand. This prevents and further elongation of that strand, so each strand has the fluorescent tag of the last ddNTP.

3. The labeled strands are separated by gel electrophoresis using a capillary tube. The shortest strands more the furthest. A fluorescent detector measures the tags.

Figure 20.3 DNA (template strand)

Primer Deoxyribo- nucleotides

Dideoxyribonucleotides (fluorescently tagged)

DNA polymerase

DNA (template strand)

Labeled strands

Direction of movement of strands

Longest labeled strand

Detector

Laser Shortest labeled strand

Shortest Longest

Technique

Results

5′

5′

5′

5′

5′

3′

3′

3′

3′

3′

C T G A C T T C G A C A A

C T G A C T T C G A C A A

T G T T

C T G T T

dATP

G C T G T T

C T G T T

C T G T T

C T G T T

C T G T T

C T G T T

G A C T G A A G C T G T T

A C T G A A G

C T G A A G

T G A A G

C T G T T

G A A G

A A G

A G

ddATP dCTP ddCTP dTTP ddTTP dGTP ddGTP

P P P P P P G G

Last nucleotide of longest labeled strand

Last nucleotide of shortest labeled strand

G A C T G A A G C

dd dd

dd dd

dd dd

dd dd

dd

DNA sequencing • “Next-generation sequencing” techniques use

a single template strand that is immobilized and amplified to produce an enormous number of identical fragments

• Thousands or hundreds of thousands of fragments (400–1,000 nucleotides long) are sequenced in parallel

• This is a type of “high-throughput” technology • In “third-generation sequencing,” the

techniques used are even faster and less expensive than the previous

Figure 20.4 Technique

Genomic DNA is fragmented.

Each fragment is isolated with a bead.

Using PCR, 106 copies of each fragment are made, each attached to the bead by 5′ end.

The bead is placed into a well with DNA polymerases and primers.

A solution of each of the four nucleotides is added to all wells and then washed off. The entire process is then repeated.

If a nucleotide is joined to a growing strand, PPi is released, causing a flash of light that is recorded.

If a nucleotide is not complementary to the next template base, no PPi is released, and no flash of light is recorded.

The process is repeated until every fragment has a complete complementary strand. The pattern of flashes reveals the sequence.

Results

6 7 8

5

4

3

2

1

4-mer

3-mer

2-mer

1-mer

A T G C

Template strand of DNA

Primer

3′

A 3′ 5′ 5′ T G C

A T G C A T G C A T G C A T G C

Template strand of DNA

Primer

DNA polymerase

dATP dTTP dGTP dCTP

PPi

PPi

C C

C C

A A

A A

T

T

T

G

G G

G

C C

C C

A A

A A

T

T

T

G

G G

G

C C

C C

A A

A A

T

T

T

G

G G

G

C C

C C

A A

A A

T

T

T

G

G G

G

A A A A

C

DNA cloning yields multiple copies of a gene or other DNA segment

• DNA molecules are very long and contain several genes.

• To study a specific gene, scientists make several gene-sized pieces of DNA in identical copies, a process called DNA cloning. – Cloned genes are useful for making several copies of

a particular gene and producing a protein product. • DNA (and gene) cloning utilizes bacteria and their

plasmids. – Plasmids are small circular DNA molecules

that replicate separately from the bacterial chromosome

Gene cloning involves using bacteria to make multiple copies of a gene.

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Gene cloning involves using bacteria to make multiple copies of a gene

• DNA is inserted into a plasmid, and the recombinant plasmid is inserted into a bacterial cell

• Reproduction in the bacterial cell results in cloning of the plasmid including the foreign DNA

• This results in the production of multiple copies of a single gene

• A plasmid used to clone a foreign gene is called a cloning vector

• 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

• Gene cloning is useful for amplifying genes to produce a protein product for research, medical, or other purposes

Figure 20.5

Using Restriction Enzymes to Make Recombinant DNA

• $ ---Restriction enzymes cut DNA molecules at specific DNA sequences called restriction sites. eg. EcoRI, BamHI, HindIII

• A restriction enzyme usually makes many cuts in DNA, yielding restriction fragments

• The most useful restriction enzymes cut DNA in a staggered way, producing fragments with “sticky ends” that bond with complementary sticky ends of other DNA fragments.

• Most of restriction enzymes are bacterial origin.

• DNA ligase is an enzyme that seals the bonds between restriction fragments

Sticky ends Blunt ends

EcoRI PstI SmaI

EcoRI= Escherichia coli PstI= Providencia stuartii SmaI= Serratia mercescens

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Figure 20.6 Bacterial plasmid

Restriction site

DNA

Restriction enzyme cuts the sugar-phosphate backbones at each arrow.

1

Base pairing of sticky ends produces various combinations.

2

DNA ligase seals the strands.

3

Sticky end

Fragment from different DNA molecule cut by the same restriction enzyme

One possible combination

Recombinant DNA molecule

5′

5′

5′ 5′

5′ 5′

5′

5′

3′

3′

3′

3′ 3′

3′

3′

3′

3′

3′ 3′

3′

3′

3′

3′

3′

5′ 5′ 5′

5′ 5′ 5′

5′

5′

G A A T T C G A A T T C

G A A T T C G A A T T C

Recombinant plasmid

GAATTC CTTAAG

Gel Electrophoresis and Southern Blotting

• One indirect method of rapidly analyzing and comparing genomes is gel electrophoresis

• $ --- This technique uses a gel to separate nucleic acids or proteins by size

• A current is applied that causes charged molecules to move through the gel

• Molecules are sorted into “bands” by their size • Smaller DNA (or proteins) migrate through the gel much easier (and

faster) than larger DNA/protein. • To check the recombinant plasmid, researchers might cut the products

again using the same restriction enzyme • To separate and visualize the fragments produced, gel

electrophoresis would be carried out • 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 © 2014 Pearson Education, Inc.

Figure 20.7

Anode Cathode

Wells

Gel

Mixture of DNA mol- ecules of different sizes

(a) Negatively charged DNA molecules move toward the positive electrode.

Power source

(b) Shorter molecules are slowed down less than longer ones, so they move faster through the gel.

Restriction fragments (size standards)

Methods for Analysis of DNA – Negative charge of molecule causes DNA to move

toward positive pole – Rate of movement is dependent on size of fragment –

larger fragments move more slowly

• Southern blotting combines gel electrophoresis separation of DNA fragments with nucleic acid hybridization

• Specific DNA fragments can be identified by Southern blotting, using labeled probes that hybridize to the separated DNA fragments after they have been transferred to a paper-like membrane i.e. DNA immobilized on a “blot” of gel.

Southern Blotting

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DNA + restriction enzyme

3 2 1

4

TECHNIQUE

I Normal β-globin allele

II Sickle-cell allele

III Heterozygote

Restriction fragments Nitrocellulose

membrane (blot)

Heavy weight

Gel

Sponge

Alkaline solution Paper

towels

II I III

II I III II I III

Preparation of restriction fragments

Gel electrophoresis DNA transfer (blotting)

Radioactively labeled probe for β-globin gene

Nitrocellulose blot

Probe base-pairs with fragments

Fragment from sickle-cell β-globin allele

Fragment from normal β- globin allele

Film over blot

Hybridization with labeled probe Probe detection 5

Amplifying DNA in Vitro: The Polymerase Chain Reaction (PCR)

• The polymerase chain reaction (PCR) can produce millions of copies of a specific target segment of DNA

• A three-step cycle—heating, cooling, and replication—brings about a chain reaction that produces an exponentially growing population of identical DNA molecules.

• The key to PCR is an unusual, heat-stable DNA polymerase called Taq polymerase obtained from hot-spring bacteria.

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Figure 20.8

Target sequence

Genomic DNA

Technique

Denaturation

5′

5′

5′

5′

3′

3′

3′

3′ Annealing

Extension

Cycle 1 yields

2 molecules

Primers

New nucleotides

1

2

3

Cycle 2 yields

4 molecules

Cycle 3 yields 8 molecules; 2 molecules

(in white boxes) match target

sequence

PCR

• PCR primers can be designed to include restriction sites that allow the product to be cloned into plasmid vectors

• The resulting clones are sequenced and error-free inserts selected

Figure 20.9 DNA fragments obtained by PCR with restriction sites matching those in the cloning vector

Cut with same restriction enzyme used on cloning vector A gene that makes bacterial

cells resistant to an antibiotic is present on the plasmid.

Cloning vector (bacterial plasmid)

Only cells that take up a plasmid will survive

Mix and ligate

Recombinant DNA plasmid

Bacterial Expression Systems • Several technical difficulties hinder expression of cloned

eukaryotic genes in bacterial host cells • 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

• Another difficulty with eukaryotic gene expression in bacteria is the presence of introns in most eukaryotic genes

• Researchers can avoid this problem by using cDNA, complementary to the mRNA, which contains only exons

Eukaryotic DNA Cloning and Expression Systems

• Molecular biologists can avoid eukaryote-bacterial incompatibility issues by using eukaryotic cells, such as yeasts, as hosts for cloning and expressing genes

• Even yeasts may not possess the proteins required to modify expressed mammalian proteins properly.

• The use of cultured eukaryotic cells as host cells and yeast artificial chromosomes (YACs) as vectors helps avoid gene expression problems – YACs behave normally in mitosis and can carry more

DNA than a plasmid – Eukaryotic hosts can provide the post-translational

modifications that many proteins require • In such cases, cultured mammalian or insect cells may

be used to express and study proteins

• One method of introducing recombinant DNA into eukaryotic cells is electroporation-applying a brief electrical pulse to create temporary holes in plasma membranes

• Alternatively, scientists can inject DNA into cells using microscopically thin needles

• Once inside the cell, the DNA is incorporated into the cell’s DNA by natural genetic recombination

• Eukaryotic expression is a good way to test the function of a specific protein

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Storing Cloned Genes in DNA Libraries Genomic library is made by cloning DNA fragments from an

entire genome into four different kinds of vector which depend on size of clone.

1. A genomic library is made by cloning DNA fragments from an entire genome into plasmids then growing the plasmids in bacteria. Each plasmid-containing bacterial cell carries copies of a particular segment of the genome.

2. A genomic library can also be made using bacteriophages ( Phage).

3. A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert used to make genomic libraries. BACs are another type of vector used in DNA library construction.

4. A yeast artificial chromosome (YAC) is a large plasmid used to make genomic libraries.

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Bacterial clones

Recombinant plasmids

Recombinant phage DNA

or

Foreign genome cut up with restriction enzyme

(a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

Phage clones

Large plasmid Large insert with many genes

BAC clone

Cross-Species Gene Expression and Evolutionary Ancestry

• The remarkable ability of bacteria to express some eukaryotic proteins underscores the shared evolutionary ancestry of living species

• 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)

• The Pax-6 genes in flies and vertebrates can substitute for each other

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Biologists use DNA technology to study gene expression and function

• Analysis of when and where a gene or group of genes is expressed can provide important clues about gene function

• Analyzing Gene Expression- The most straightforward way to discover which genes are expressed in certain cells is to identify the mRNAs being made Labeled nucleic acid probes that hybridize with mRNAs can provide information about the time or place in the organism at which a gene is transcribed.

Studying the Expression of Single Genes • Changes in the expression of a gene during embryonic

development can be tested using the following method to compare mRNA from different developmental stages. 1. Northern blotting-Scientists carry out gel

electrophoresis on samples of mRNA from different stages of development, or following treatment using drugs or growth factors, etc. Then the separated mRNA is transferred to a nitrocellulose membrane, and then allow the mRNAs on the membrane to hybridize with a labeled probe that recognizes a specific mRNA.

2. In situ hybridization uses fluorescent dyes attached to probes to identify the location of specific mRNAs in place in the intact organism

3. Reverse transcriptase-polymerase chain reaction (RTPCR)

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Figure 20.10

Head

50 µm

Thorax Abdomen

Segment boundary

Head Thorax Abdomen

T1 T2 T3 A1 A2 A3 A4 A5

5′ 3′

3′ 3′

3′

5′ 5′

5′ TAACGGTTCCAGC ATTGCCAAGGTCG

CTCAAGTTGCTCT GAGTTCAACGAGA

Cells expressing the wg gene

Cells expressing the en gene

wg mRNA en mRNA

• Reverse transcriptase-polymerase chain reaction (RT-PCR) is quicker and more sensitive than Northern blotting and is useful for comparing amounts of specific mRNAs in several samples at the same time

• Reverse transcriptase is added to mRNA to make cDNA, which serves as a template for PCR amplification of the gene of interest

• The products are run on a gel and the mRNA of interest identified

RT-PCR

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Figure 20.11

DNA in nucleus

mRNAs in cytoplasm

Reverse transcriptase Poly-A tail mRNA

DNA strand

Primer (poly-dT)

5′

5′

5′

5′

3′

3′ 3′

3′ A A A A A A

A A A A A A

T T T T T

T T T T T

5′ 3′ 5′

3′

DNA polymerase

1. mRNA is isolated and reverse transcriptase is added to the tube. 2. RT makes the first DNA strand. Then the mRNA is degraded. 3. DNA polymerase syn- thesizes the second DNA strand using an added primer. 4. The resulting cDNA carries the complete coding sequence of the gene.

A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell

A cDNA library

represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

cDNA

Figure 20.12

cDNA synthesis mRNAs

cDNAs

PCR amplification Primers

Specific gene

Gel electrophoresis

Results Embryonic stages 1 2 3 4 5 6

Technique 1

2

3

Studying the Expression of Interacting Groups of Genes

• Automation has allowed scientists to measure the expression of thousands of genes at one time using DNA microarray assays

• DNA microarray assays compare patterns of gene expression in different tissues, at different times, or under different conditions

Genome-wide expression studies are made possible by the use of DNA microarray assays

• A DNA microarray consists of tiny amounts of a large number of single-stranded DNA fragments representing different genes fixed to a glass slide in a tightly spaced array, or grid, also called a DNA chip.

• Ideally, these fragments represent all the genes of an organism.

• The DNA fragments on a microarray are tested for hybridization with cDNA molecules that have been prepared from the mRNAs in particular cells of interest and labeled with fluorescent dyes.

• In addition to uncovering gene interactions and providing clues to gene function, DNA microarray assays may contribute to a better understanding of certain diseases and suggest new diagnostic techniques or therapies. – Comparing patterns of gene expression in cancer tumors and

noncancerous tissue has resulted in more effective treatment protocols.

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Figure 20.13

Genes expressed in first tissue.

Genes expressed in second tissue.

Genes expressed in both tissues.

Genes expressed in neither tissue.

DNA microarray (actual size)

Each dot is a well containing identical copies of DNA fragments that carry a specific gene.

►

• 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

• By uncovering gene interactions and clues to gene function DNA microarray assays may contribute to understanding of disease and suggest new diagnostic targets

Determining Gene Function • One way to determine function is to disable the

gene and observe the consequences • Using in vitro mutagenesis, mutations are

introduced into a cloned gene, altering or destroying its function

• When the mutated gene is returned to the cell, the normal gene’s function might be determined by examining the mutant’s phenotype

• Gene expression can also be silenced using RNA interference (RNAi)

• Synthetic double-stranded RNA molecules matching the sequence of a particular gene are used to break down or block the gene’s mRNA

• In humans, researchers analyze the genomes of many

people with a certain genetic condition to try to find nucleotide changes specific to the condition

• These genome-wide association studies test for genetic markers, sequences that vary among individuals

• SNPs (single nucleotide polymorphisms), single nucleotide variants, are among the most useful genetic markers

• SNP variants that are found frequently associated with a particular inherited disorder alert researchers to the most likely location for the disease-causing gene

• SNPs are rarely directly involved in the disease; they are most often in noncoding regions of the genome

Figure 20.14

DNA

SNP Normal allele

Disease-causing allele

A

T

C

G

Cloned organisms and stem cells are useful for basic research and

other applications • Organismal cloning produces one or more

organisms genetically identical to the “parent” that donated the single cell

• 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

Cloning Plants: Single-Cell Cultures

• In plants, cells can dedifferentiate and then give rise to all the specialized cell types of the organism

• A totipotent cell, such as this, is one that can generate a complete new organism

• Plant cloning is used extensively in agriculture

Cloning a Eukaryotic Gene in a Bacterial Plasmid

• In gene cloning, a plasmid is used as a cloning vector

• A cloning vector is a circular DNA molecule that can carry foreign DNA into a host cell and replicate there

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Figure 20.15

Cross section of carrot root

Small fragments

Fragments were cultured in nutrient medium; stirring caused single cells to shear off into the liquid.

Single cells free in suspension began to divide.

Embryonic plant developed from a cultured single cell.

Plantlet was cultured on agar medium. Later it was planted in soil.

Adult plant

Cloning Animals: Nuclear Transplantation

• In nuclear transplantation, the nucleus of an unfertilized egg cell or zygote is replaced with the nucleus of a differentiated cell

• Experiments with frog embryos have shown that a transplanted nucleus can often support normal development of the egg

• However, the older the donor nucleus, the lower the percentage of normally developing tadpoles

Figure 20.16 Experiment Frog embryo

UV

Frog egg cell Frog tadpole

Less differ- entiated cell

Fully differ- entiated (intestinal) cell

Donor nucleus trans- planted

Enucleated egg cell

Donor nucleus trans- planted Egg with donor nucleus

activated to begin development Results

Most develop into tadpoles.

Most stop developing before tadpole stage.

DNA technology applications for transgenic animals

• DNA technology permits the genetic engineering of transgenic animals, which speeds up the selective breeding process.

• The goals of creating a transgenic animal are often the same as the goals of traditional breeding—for instance, to make a sheep with better quality wool, a pig with leaner meat, or a cow that will mature in a shorter time.

• Scientists might, for example, identify and clone a gene that leads to the development of larger muscles (muscles make up most of the meat we eat) in one variety of cattle and transfer it to other cattle or even to sheep.

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Cloning of Mammals using Nuclear Transplantation- Reproductive Cloning of

Mammals • In 1997, Scottish researchers announced the birth of Dolly,

a lamb cloned from an adult sheep by nuclear transplantation from a differentiated mammary cell

• These researchers cultured donor mammary cells in a nutrient-poor medium and then fused these cells with enucleated sheep eggs.

• The resulting diploid cells divided to form early embryos, which were implanted into surrogate mothers.

• Out of several hundred implanted embryos, one successfully completed normal development, and Dolly was born.

• Later analyses showed that Dolly’s chromosomal DNA was identical to that of the nucleus donor.

• Dolly’s mitochondrial DNA came from the egg donor. • Dolly’s 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 20.17 Technique

1 2

3

4

5

6

Mammary cell donor

Results

Egg cell donor

Egg cell from ovary

Cultured mammary cells

Nucleus removed

Cells fused

Nucleus from mammary cell

Grown in culture

Early embryo

Implanted in uterus of a third sheep

Surrogate mother

Embryonic development Lamb (“Dolly”)

genetically identical to mammary cell donor

• Since 1997, cloning has been demonstrated in many mammals, including mice, cats, cows, horses, mules, pigs, and dogs

• CC (for Carbon Copy) was the first cat cloned; however, CC differed somewhat from her female “parent”

• Cloned animals do not always look or behave exactly the same

Figure 20.18 CC, the first cloned cat (right), and her single parent

Faulty Gene Regulation in Cloned Animals

• In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth, and many cloned animals exhibit defects

• 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

Figure 20.19

Stem cell

Cell division

Stem cell and Precursor cell

Fat cells or Bone cells

or White blood cells

Stem Cells of Animals Stem cells are relatively unspecialized cells that can both reproduce indefinitely and, under certain conditions, differentiate into one or more specialized cell types

Embryonic and Adult Stem Cells • Many early embryos contain stem cells capable

of giving rise to differentiated embryonic cells of any type

• In culture, these embryonic stem cells reproduce indefinitely

• Depending on culture conditions, they can be made to differentiate into a variety of specialized cells

• Adult stem cells can generate multiple (but not all) cell types and are used in the body to replace non reproducing cells as needed

Stem Cells of Animals • A stem cell is a relatively unspecialized cell that can

reproduce itself indefinitely and differentiate into specialized cells of one or more types

• Stem cells isolated from early embryos at the blastocyst stage are called embryonic stem (ES) cells; these are able to differentiate into all cell types (pluripotent)

• The adult body also has stem cells, which replace non reproducing specialized cells

• With the addition of specific growth factors, cultured stem cells from adult animals have been made to differentiate into multiple types of specialized cells.

• This technology may provide cells for the repair of damaged or diseased organs, such as insulin-producing pancreatic cells for people with diabetes or certain kinds of brain cells for people with Parkinson’s disease or Huntington’s disease.

Figure 20.20

Cells that can generate all embryonic cell types

Cultured stem cells

Cells that generate a limited number of cell types

Different culture conditions

Liver cells Nerve cells Blood cells

Different types of differentiated cells

Embryonic stem cells

Adult stem cells

• Embryonic stem (ES) cells are

pluripotent, capable of differentiating into many different cell types

• The ultimate aim of research with stem cells is to supply cells for the repair of damaged or diseased organs

• ES cells present ethical and political issues

Induced Pluripotent Stem (iPS) Cells • Researchers can treat differentiated cells, and

reprogram them to act like ES cells • Researchers used retroviruses to induce extra

copies of four stem cell master regulatory genes to produce induced pluripotent stem (iPS) cells

• iPS cells can perform most of the functions of ES cells

• iPS cells can be used as models for study of certain diseases and potentially as replacement cells for patients

Figure 20.21 Stem cell Precursor cell Experiment

Skin fibroblast cell

Four “stem cell” master regulator genes were introduced, using the retroviral cloning vector.

Induced pluripotent stem (iPS) cell

Oct3/4 Sox2

c-Myc Klf4

The practical applications of DNA-based biotechnology affect our lives

in many ways • Many fields benefit from DNA technology

and genetic engineering. • Medical Applications:- • One benefit of DNA technology is identification of human

genes in which mutation plays a role in genetic diseases • Researchers use microarray assays or other tools to

identify genes turned on or off in particular diseases • The genes and their products are then potential targets for

prevention or therapy

Diagnosis and Treatment of Diseases

• 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

• SNPs may be associated with a disease-causing mutation

• SNPs may also be correlated with increased risks for conditions such as heart disease or certain types of cancer

• Cloned disease genes include those for sickle-cell disease, hemophilia, cystic fibrosis, Huntington’s disease, and Duchenne muscular dystrophy (DMD).

Human gene therapy • Gene therapy—introducing genes into an afflicted

individual for therapeutic purposes—holds great potential for treating genetic disorders caused by a single defective gene.

• In theory, a normal allele of the defective gene could be inserted into dividing somatic cells of the tissue affected by the disorder, such as bone marrow cells.

• Bone marrow cells, which include the stem cells that give rise to all the cells of the blood and immune system, are prime candidates for gene therapy.

• 10 young patients with severe combined immunodeficiency (SCID), which is caused by a single defective gene, have been treated in gene therapy trials. – After two years, nine of these patients showed

significant improvement. However, three of the patients subsequently developed leukemia and one died.

• Gene therapy provokes both technical and ethical questions.

Figure 20.22 Cloned gene 1

2

3

4

Insert RNA version of normal allele into retrovirus or other viral vector.

Viral RNA

Viral capsid

Let virus infect bone marrow cells that have been removed from the patient and cultured.

Viral DNA carrying the normal allele inserts into chromosome.

Bone marrow cell from patient

Inject engineered cells into patient.

Bone marrow

• Transgenic animals are made by introducing genes from one species into the genome of another animal

• Transgenic animals are pharmaceutical “factories,” producers of large amounts of otherwise rare substances for medical use

• “Pharm” plants are also being developed to make human proteins for medical use

Protein Production by “Pharm” Animals and Plants

Figure 20.23 Goat milk can produce silk to make bullet proof vest.

Pharmaceutical Products • Advances in DNA technology and genetic

research are important to the development of new drugs to treat diseases

Synthesis of Small Molecules for Use as Drugs • The drug imatinib is a small molecule that

inhibits overexpression of a specific leukemia-causing receptor

• This approach is feasible for treatment of cancers in which the molecular basis is well-understood

Protein Production in Cell Cultures • Host cells in culture can be engineered to

secrete a protein as it is made, simplifying the task of purifying it.

• Cells in culture can also be used to produce vaccines, which stimulate the immune system to defend against specific pathogens.

• This is useful for the production of insulin, human growth hormones, and vaccines

Genetic profiles may be important forensic evidence

• Body fluids or small pieces of tissue may be left at the scene of a violent crime or on the clothes of the victim or assailant.

• DNA testing, in contrast, can identify the guilty individual with a high degree of certainty because the DNA sequence of every person is unique (except for identical twins).

• Genetic markers that vary in the population can be analyzed for a given person to determine that individual’s unique set of genetic markers, or genetic profile. Genetic profiles can be analyzed using RFLP(Restriction Fragment Length Polymorphism)analysis or STR (short tandem repeats).

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STR’s • STRs (short tandem repeats) are variations in the

lengths number of repeats of specific DNA sequences in specific regions of the genome.

• PCR is used to amplify particular STRs using sets of primers that are labeled with different colored fluorescent tags; the length of the region, and thus the number of repeats, can then be determined by electrophoresis.

• Forensic scientists test only a few selected portions of the DNA—about 13 STR markers.

• The probability that two people (who are not identical twins) would have exactly the same set of STR markers is somewhere between one chance in 10 billion and one in several trillion.

• As of 2013 more than 300 innocent people have been released from prison as a result of STR analysis of old DNA evidence

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Figure 20.24 (a) Earl Washington just before his release in 2001, after 17 years in prison.

(b) These and other STR data (not shown) exonerated Washington and led Tinsley to plead guilty to the murder.

Source of sample

STR marker 1

STR marker 2

STR marker 3

Semen on victim 17,19 13,16 12,12

Earl Washington 16,18 14,15 11,12

Kenneth Tinsley 17,19 13,16 12,12

Microorganisms can be used for environmental cleanup

• Many bacteria can extract heavy metals, such as copper, lead, and nickel, from their environments and incorporate the metals into compounds such as copper sulfate or lead sulfate, which are readily recoverable.

• Genetically engineered microbes may become important in both mining minerals (especially as ore reserves are depleted) and cleaning up highly toxic mining wastes.

• Biotechnologists are also trying to engineer microbes that can be used in wastewater treatment plants to degrade chlorinated hydrocarbon.

• Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

•

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Biofuels can supplement or replace fossil fuels

• Biofuels from crops such as corn, soybeans, and cassava have been proposed as a supplement or even replacement for fossil fuels.

• To produce “bioethanol,” starch is converted to sugar and then fermented by microorganisms; a similar process can produce “biodiesel” from plant oils. Either product can be mixed with gasoline or used alone to power vehicles.

• Critics charge that the environmental effects of growing these crops makes their total impact greater than that of fossil fuels.

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Agricultural Applications • DNA technology is being used to improve agricultural

productivity and food quality • Genetic engineering of transgenic animals speeds up the

selective breeding process • Beneficial genes can be transferred between varieties or

species • Agricultural scientists have endowed a number of crop

plants with genes for desirable traits • The Ti plasmid is the most commonly used vector for

introducing new genes into plant cells • 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

Figure 20-25

Site where restriction enzyme cuts

T DNA

Plant with new trait

Ti plasmid

Agrobacterium tumefaciens

DNA with the gene of interest

Recombinant Ti plasmid

TECHNIQUE

RESULTS

Safety and Ethical Questions Raised by DNA Technology

• Potential benefits of genetic engineering must be weighed against potential hazards of creating harmful products or procedures

• Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology

• As biotechnology continues to change, so does its use in agriculture, industry, and medicine

• National agencies and international organizations strive to set guidelines for safe and ethical practices in the use of biotechnology

Ethical Questions Raised by DNA Technology

• The increasing speed and decreasing cost with which genome sequences of individual people can be determined are raising significant ethical questions.

• Who should have the right to examine someone else’s genetic information?

• How should a person’s genetic information be used?

• Should a person’s genome be a factor in suitability for a job or eligibility for insurance?

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Safety and Ethical Questions Raised by DNA Technology

• Most public concern about possible hazards

centers on genetically modified (GM) organisms used as food like GM Corn, Golden Rice.

• Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives.

• Other worries include the possibility that transgenic protein products might cause allergic reactions.

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Producing Clones of Cells Carrying Recombinant Plasmids

• Several steps are required to clone the hummingbird β-globin gene in a bacterial plasmid:

The hummingbird genomic DNA and a bacterial plasmid are isolated – Both are digested with the same restriction enzyme – The fragments are mixed, and DNA ligase is added

to bond the fragment sticky ends – Some recombinant plasmids now contain

hummingbird DNA – The DNA mixture is added to bacteria that have

been genetically engineered to accept it – The bacteria are plated on a type of agar that selects

for the bacteria with recombinant plasmids – This results in the cloning of many hummingbird

DNA fragments, including the β-globin gene

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restriction site

Sticky ends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carrying plasmids

RESULTS

Colony carrying non- recombinant plasmid with intact lacZ gene

One of many bacterial clones

Colony carrying recombinant plasmid with disrupted lacZ gene

Screening a Library for Clones Carrying a Gene of Interest

• A clone carrying the gene of interest can be identified with a nucleic acid probe having a sequence complementary to the gene

• This process is called nucleic acid hybridization • The DNA probe can be used to screen a large number of clones

simultaneously for the gene of interest • Once identified, the clone carrying the gene of interest can be

cultured

Gene of interest Nucleic acid probe

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Fig. 20.7

Probe DNA

Radioactively labeled probe

molecules

Film

Nylon membrane

Multiwell plates holding library clones

Location of DNA with the complementary sequence

Gene of interest

Single-stranded DNA from cell

Nylon membrane

TECHNIQUE

•

Screening a Library for Clones

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DNA technology allows us to study the

sequence, expression, and function of a gene

• DNA cloning allows researchers to – Compare genes and alleles between

individuals – Locate gene expression in a body – Determine the role of a gene in an organism

• Several techniques are used to analyze the DNA of genes

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• Restriction fragment analysis- DNA fragments produced by restriction enzyme digestion of a DNA molecule are separated/ or sorted by gel electrophoresis.

• Restriction fragment analysis is useful for comparing two different DNA molecules, such as two alleles for a gene if the nucleotide difference alters a restriction site.

• Variations in DNA sequence are called polymorphisms

• Sequence changes that alter restriction sites are called RFLPs (restriction fragment length polymorphisms)

Restriction Fragment Analysis

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Figure 20.10

Normal β-globin allele

Sickle-cell mutant β-globin allele

Large fragment

Normal allele

Sickle-cell allele

201 bp 175 bp

376 bp

(a) DdeI restriction sites in normal and sickle-cell alleles of the β-globin gene

(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

201 bp 175 bp

376 bp

Large fragment

Large fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI

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