PCR

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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
    • 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

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