biotechnology
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Chapter 14: Genetic Engineering
-Modification of the DNA of an organism to produce new genes with new
characteristics
Biotechnology
• Use of organisms to benefit humanity
Recombinant DNA technology
• DNA from different organisms is spliced together
• Allows scientists to make many copies of any DNA segment (clone)
• Can introduce foreign DNA into cells of microorganisms
Recombinant DNA technology• Restriction enzymes – cut DNA– Bacteria produce for defense against viruses
• Vector – transports DNA into a cell– Ex: bacteriophage– Plasmid – separate, smaller circular DNA that
maybe be present and able to replicate inside bacteria
• Transformation – uptake of foreign DNA by cells– How plasmids can get into bacteria
Bacterial Conjugation
and Recombination
Recombinant DNA technology• Palindromic sequences – reads the same as
complement, in opposite direction– AAGCTT– TTCGAA– Many restriction enzymes cut these sequences
• Restriction enzymes cut on a stagger sticky ends (can pair with complementary single-stranded end of other DNA cut with same enzyme)
• DNA Ligase – links 2 fragments recombinant DNA
Fig. 20-3-3Restriction site
DNA
Sticky end
Restriction enzymecuts sugar-phosphatebackbones.
53
35
1
One possible combination
Recombinant DNA molecule
DNA ligaseseals strands.
3
DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.
2
Restriction Enzymes
Steps of Creating a Recombinant DNA Plasmid (Basic)
• 1. Plasmids and desired DNA cut by same restriction enzyme
• 2. Mix 2 types of DNA so sticky ends pair• 3. DNA ligase forms bonds between fragments
Fig. 20-2
DNA of chromosome
Cell containing geneof interest
Gene inserted intoplasmid
Plasmid put intobacterial cell
RecombinantDNA (plasmid)
Recombinantbacterium
Bacterialchromosome
Bacterium
Gene ofinterest
Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest
Plasmid
Gene ofInterest
Protein expressedby gene of interest
Basic research andvarious applications
Copies of gene Protein harvested
Basicresearchon gene
Basicresearchon protein
Gene for pest resistance inserted into plants
Gene used to alter bacteria for cleaning up toxic waste
Protein dissolvesblood clots in heartattack therapy
Human growth hor-mone treats stuntedgrowth
2
4
1
3
Cloning a Gene
Fig. 20-4-4
Bacterial cell
Bacterial plasmid
lacZ gene
Hummingbird cell
Gene of interest
Hummingbird DNA fragments
Restrictionsite
Stickyends
ampR gene
TECHNIQUE
Recombinant plasmids
Nonrecombinant plasmid
Bacteria carryingplasmids
RESULTS
Colony carrying non-recombinant plasmidwith intact lacZ gene
One of manybacterialclones
Colony carrying recombinant plasmid with disrupted lacZ gene
Cloning DNA
• Genome – total DNA per cell• Genomic library – collection of DNA fragments
more or less representative of all DNA in genome
• Genetic Probe – single stranded DNA or RNA that is radioactively labeled and can attach to target sequence by base pairing rules
• A probe can be synthesized that is complementary to the gene of interest
• For example, if the desired gene is
– Then we would synthesize this probe G5 3… …G GC C CT T TA A A
C3 5C CG G GA A AT T T
DNA Probe
Using a DNA probe
Polymerase Chain Reaction (PCR)
• Can amplify a small sample of DNA quickly• DNA replication in vitro• 2 strands separated by heating so special
heat-resistant DNA polymerase called Taq polymerase used (thermophile)
• MAJOR BONUS: Only specific sequences can be replicated
• Study: crime scenes, archaeological remains
PCR
Gel Electrophoresis• Separates fragments like DNA, RNA or
polypeptides (they carry charge and can migrate in an electrical field
• RNA and DNA (-) --- so they move to (+) pole• Smaller fragments go further• Compare sample to standard• Usually “blot” - transfer DNA from gel to
nitrocellulose filter for further analysis• DNA Fingerprinting
Gel Electrophoresis
Fig. 20-9a
Mixture ofDNA mol-ecules ofdifferentsizes
Powersource
Longermolecules
Shortermolecules
Gel
AnodeCathode
TECHNIQUE
1
2
Powersource
– +
+–
Fig. 20-9b
RESULTS
DNA Fingerprint
Transgenic Organisms
• Plants and animals in which foreign genes have been incorporated
• Animals– Inject DNA into nucleus of egg or stem cell– Eggs implanted in uterus; stem cells injected into
blastocysts + then implanted into foster mother
• Plants– Disease resistance– Pesticide resistance
Transgenics
Fig. 20-20
Culturedstem cells
Early human embryoat blastocyst stage
(mammalian equiva-lent of blastula)
Differentcultureconditions
Differenttypes ofdifferentiatedcells
Blood cellsNerve cellsLiver cells
Cells generatingall embryoniccell types
Adult stem cells
Cells generatingsome cell types
Embryonic stem cells
From bone marrowin this example
Fig. 20-18
TECHNIQUE
Mammarycell donor
RESULTS
Surrogatemother
Nucleus frommammary cell
Culturedmammary cells
Implantedin uterusof a thirdsheep
Early embryo
Nucleusremoved
Egg celldonor
Embryonicdevelopment Lamb (“Dolly”)
genetically identical tomammary cell donor
Egg cellfrom ovary
Cells fused
Grown inculture
1
33
4
5
6
2
Fig. 20-19
Cloning Video
GE Plants
Application of GE
• Human proteins– Insulin– Hormones - HGH
• Human treatments for disease– Multiple sclerosis, certain cancers, heart attacks,
forms of anemia
• Vaccines
Fig. 20-23
Fig. 20-24This photo shows EarlWashington just before his release in 2001,after 17 years in prison.
These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.
(a)
Semen on victim
Earl Washington
Source of sample
Kenneth Tinsley
STRmarker 1
STRmarker 2
STRmarker 3
(b)
17, 19
16, 18
17, 19
13, 16 12, 12
14, 15 11, 12
13, 16 12, 12
Forensics
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