Module 6: Sec 4 - Manipulating Genomes

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  • Created by: Simbaaax
  • Created on: 15-05-17 21:41

PCR (Polymerase Chain Reaction)

=amplify DNA; enabling it to be analysed. (amount of DNA increases exponetially)

Relies on fact that;

  • DNA has 2 anti-parallel strands
  • DNA strand has 5' and 3' ends
  • DNA only grows from 3' end
  • Base pairs = complementary

1) DNA sample mixed with; DNA nucleotides, primers, Mg2+ ions, Taq Polymerase (enzyme)

2) Heated to 95oC ---> break H bonds + form 2 single strands

3) Cooled to 68oC ---> primers anneal at end of single stranded DNA

4) Taq Polymerase binds near primers at 72oC

5) Taq Polymerase catalyses addition of DNA nucleotides to single stranded DNA from primer

6) Taq Polymerase reaches end - new double strand of DNA made - CYCLE REPEATED

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DNA Probes

Probe = short single stranded DNA (50-80 nucleotides)

Can be labelled via;

  • radioactive marker (e.g. P-32; once annealed, shows on photographic film)
  • flourescent marker (emits colour on exposure to U.V. light)

Uses;

  • locate specific gene for genetic engineering
  • identify same gene in variety of genomes
  • identify presence of specific allele for genetic disease
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Replica Plating

Identifying transformed bacteria.

1) Reverse transcriptase makes single stranded cDNA. 

DNA Polymerase makes it double stranded.

2) Free nucleotides add on and make sticky ends.

3) Ligase enzymes help put Insulin gene into plasmids. --> now called Recombinant plasmids.

4) E Coli mixed with recombinant and heat shocked (in presence of CaCl2)

Identify recombinant plasmids by looking at Agar of tray with both antibiotics compared to tray with one. The spots where a bacteria colony is missing is where the recombinant plasmids are.

Remember; recombinant plasmid only resistant to one of the two antibiotics, adding the insulin gene made the other resistance gene break up.

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Sangers Chain Termination Method

In 4 seperate tubes; 

  • single stranded DNA template
  • DNA primer
  • DNA polymerase
  • Free nucleotides
  • add one of each modified nucleotide to each test tube; modified because it has radioactive label attached.

DNA fragments of varying lengths produced.

Passed through electrophoresis.

Nucleotide at the end of each fragment read off.

Good because; efficient + safe

Bad because; time consuming + costly

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Pyrosequencing

1) Nebuliser cuts DNA into 300-800 fragments.

2) Lengths degraded to single stranded DNA. (TEMPLATES)

3) Incubated with; primer, DNA polymerase, ATP sulfurylase, luciferase, apyrase, APS, luciferin and one of 4 activated nucleotides. 

4) When activated nucleotide incorporated;

  • 2 phosphoryls released
  • in APS presence, phosphoryls converted to ATP
  • in presence of ATP, luciferase (enzyme) converts luciferin to oxyluciferin. 
  • conversion generates light; detected via camera.

Amount of light generated = proportional to amount of ATP released (depends on diff nucleotides)

Unincorporated nucleotides broken down via Apyrase.

Software will package the strands into longer sequences.

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