PCR
- Created by: kpritchard16
- Created on: 21-12-18 11:56
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- PCR
- What PCR requires
- Correct buffer
- Add Mg to buffer - right conc can make big different in yield
- Two oligonucleotide primers
- should be close to 50% G/C content and have 'GC clamp' at 3' end
- made of synthetic ssDNA, typically 18 bases long
- they anneal to denatured template DNA by complementary base pairing
- Thermal cycling machine
- Target DNA
- Thermostable DNA polymerase
- Taq DNA polymerase used mostly
- catalyses DNA synthesis at 72oC
- can withstand 92oC for short periods of time
- has no 3'-5' exonuclease - no proof reading
- Taq DNA polymerase used mostly
- Correct buffer
- Typical protocol
- 3. 72oC for 1min to synthesise new DNA by extending the primers with dNTPs
- 2. 55oC for 30sec to anneal the primers to DNA
- Repeat for 30 cycles, take around 1hr 30mins
- 2^30 = 1.07x10^9 copies assuming 100% efficiency
- 1. 95oC for 30sec to denature DBA
- A procedure that generates a large amount of target DNA
- Each cycle doubles the number of copies of DNA
- Applications
- Introducing novel sequences - mutagenesis which introduces mutation into centre of primer to be incorporated in all copied molecules
- Straightforward amplification for gene cloning, medical detection, criminal detection and ancient DNA
- Other lab techniques include RT PCR, QPCR, inverse PCR, gene synthesis and molecular evolution
- What PCR requires
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