PCR

DNA from whole blood

  • white blood cells contain DNA

  • Approx 500 micrograms of DNA can be extracted from 10ml of whole blood

  • Samples are sometimes limited in size

  • One drop of blood is approx 50 microlitres

  • This could provide up to 2.5 micrograms of DNA assuming none is lost

PCR

  • polymerase chain reaction

  • Uses heat stable DNA polymerase to replicate DNA

    • Isolated from hot spring bacteria

      • Thermus aquaticus

    • Taq polymerase

  • primers

    • Short single stranded length of nucleotides

    • Usually about 20 nucleotides long

  • DNA nucleotides added

PCR stages

  • Denaturation - 95C

    • Heat DNA sample to disrupt hydrogen bonding

    • Strands denature

  • Annealing - 55C

    • Strands cool

    • Added primers form hydrogen bonds with complementary sequences on DNA strand

  • Extension - 72C

    • Taq polymerase adds free DNA nucleotides starting at the primer and extending along the denatured strand

  • Repeats around 30 times