Ch 8 Recombinant DNA Technology

1
Ch 8 Recombinant DNA Technology

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

2
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 ? transfer genes into
  another organism

3
Yet more terminology

• extrachromosomal
  DNA that multiplies independently of the
  chromosome

4
Polymerase Chain Reaction (PCR)

• PCR requires
• 
  contains region to be amplified (target region)
• 
  (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

5
Polymerase Chain Reaction (PCR)
Gene of interest
Original DNA moleculewith gene of interest
3?
5?
5?
3?
Heat to 94C
5?
3?
One Cycle
1
5?
3?
Deoxyribonucleotidetriphosphates
2
DNA primer
DNA polymerase
3?
5?
5?
3?
DNA polymerase
Cool at 65C
3?
5?
DNA primer
3
3?
5?
6
Each PCR cycle doubles the number of copies of
the DNA
7
Agarose Gels

• DNA moves through electric field
• DNA ethidium bromide

8
Gel Electrophoresis

• Gel electrophoresis separates macromolecules
• DNA-binding dye fluoresces orange in ultraviolet
  light, revealing the separated bands to which it
  binds.

9
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

10
Pharmaceutical and therapeutic applicatons

• Procure DNA sample
• PCR ? many copies
• Cut with restriction enzymes
• Separate by gel electrophoresis
• Compare DNA fragment pattern

11
Pharmaceutical and therapeutic applicatons

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

12
Agricultural Applications

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

Pest-resistant transgenic vs. non-transgenic
plant