Ch 8 Recombinant DNA Technology

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Ch 8 Recombinant DNA Technology

• Principles of recombinant DNA technology
• Polymerase Chain Reaction (PCR)
• Gel electrophoresis
• Applications of recombinant DNA technology
• Gene cloning

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Terminology

• use of
  microbiological and biochemical techniques to
  solve practical problems and produce useful
  products
• 
  allow scientists to manipulate
  DNA ? alters organisms to give them useful traits
• 
  process of isolating genes from one organism ?
  manipulating DNA in vitro ?

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Yet more terminology

• extrachromosomal
  DNA that multiplies independently of the
  chromosome

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Polymerase Chain Reaction (PCR)

• PCR requires
• 
  contains region to be amplified (target region)
• Heat-stable
  (usually Taq)
• Short, ssDNA flanking
  target region (20 nucleotides)
• 3-step Amplification
• target DNA
• annealing of primers to
  target DNA
• synthesis of new DNA
  extends primers

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Polymerase Chain Reaction (PCR)
Gene of interest
Original DNA moleculewith gene of interest
3?
5?
5?
3?
One Cycle
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Each PCR cycle doubles the number of copies of
the DNA
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Agarose Gels

• DNA moves through electric field
• Shorter pieces move longer pieces move
• DNA ethidium bromide fluoresces under UV light

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Gel Electrophoresis

• Gel electrophoresis separates macromolecules on
  the basis of
• DNA-binding dye fluoresces orange in ultraviolet
  light, revealing

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Pharmaceutical and therapeutic applicatons

• Insulin used to treat diabetes
• Human growth hormone ? treat pituitary dwarfs
• Antibiotics
• Restriction enzymes
• Renin protein from calf stomach cheese
  production
• Identify genetic mutations associated with
  disease
• Identify viruses present in blood or other tissues

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Pharmaceutical and therapeutic applicatons

• Procure DNA sample
• PCR ?
• Cut with restriction enzymes
• Separate by
• Compare DNA fragment pattern
• Compare
• Paternity tests
• Identify human remains

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Pharmaceutical and therapeutic applicatons

• Recombinant protein
• Plants as vaccines
• DNA vaccines
• Replace missing/defective genes with normal
  copies
• Transplant animal organs/ cells/ tissues into
  humans
• Insert human genes into animals to prevent
  rejection by recipient

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Agricultural Applications

• Bt gene from Bacillus thuringiensis is a
  pesticide introduced into many plants

Pest-resistant transgenic vs. non-transgenic
plant
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How to Clone a Gene
Gene inserted into plasmid
Bacterium
Cell containing gene of interest
Bacterialchromosome
Plasmid
Gene of interest
Recombinant plasmid
DNA of chromosome
Plasmid put into bacterial cell
Recombinate bacterium
Host cell grown in culture, to form a clone of
cells containing the cloned gene of interest
Gene of interest
Protein expressed by gene of interest
Protein harvested
Copies of gene
Production of protein
Basic research, health applications
Production of DNA
Gene for pest resistance inserted into plants
Alter bacteria for toxic waste clean-up
Protein dissolvesblood clots in heart
Human growth hormone treats stunted growth
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DNA Fingerprinting (1)
Evidence is gathered Goal match evidence with
victim and suspect
DNA extracted from victims blood, unidentified
blood and suspects blood
REs cleave DNA into specific-sized fragments
Electrophoresis separates the DNA fragements
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DNA Fingerprinting (2)
DNA from the gel is transferred to a nylon
membrane
A radioactive probe binds to the target DNA
fragments
X-ray film is used to visualize the location of
the radioactive probe
Compare DNA banding pattern from victim,
unidentified and suspect blood