PCR Notes

The Polymerase Chain Reaction (PCR)

Overview of PCR

  • What is PCR?
    • A technique to make many copies of a specific DNA region in vitro.
    • In vitro refers to processes performed outside of a living organism, typically in a lab setting.
    • The DNA region of interest can be a gene or a genetic marker.
  • Role of DNA Polymerase:
    • DNA polymerase synthesizes new DNA copies.
    • It reads the template DNA and adds the appropriate complementary base pairs from 5' to 3', similar to DNA replication.
    • This process is repeated in cycles to produce many copies of the original DNA.
  • Main Goal of PCR:
    • To make enough copies of target DNA for experimental uses, including:
      • Cloning a fragment of foreign DNA into a plasmid to create a recombinant DNA molecule.
      • Identifying different microorganisms in a patient sample by sequencing.
      • Disease diagnostics and detection of mutations in specific genes (RFLPs).
      • Generating forensic profiles and allele marker analysis (fingerprinting; STRs).

History of PCR

  • 1976:
    • Isolation of Taq DNA polymerase from Thermus aquaticus (T. aquaticus).
    • T. aquaticus is a thermophilic bacterium with high thermostability.
    • Found in hot springs; Taq polymerase's optimum activity temperature is 75-80 °C, and it remains stable up to 95 °C.
    • Human DNA polymerase optimum temperature is 37 °C.
    • Taq polymerase can replicate a 1000 bp strand of DNA in approximately 30 seconds.
  • 1983:
    • Kary Mullis created the PCR technique.
    • He used Taq polymerase to demonstrate that forward and reverse primers could produce many copies of a specific gene region.
  • 1985:
    • First publication using the PCR technique.
  • 1988:
    • Patent for Taq polymerase filed. First PCR thermocycler introduced.
  • 1989:
    • Taq polymerase labeled molecule of the year.
  • 1993:
    • Kary Mullis won the Nobel Prize.
  • Timeline Highlights:
    • 1953: Discovery of the DNA double helix structure.
    • 1967: Thomas Brock reports the isolation of the extremophilic bacterium Thermophilis aquaticus.
    • 1971: Kleppe and co-workers describe a method using an enzymatic assay to replicate a short DNA template with primers in vitro.
    • 1977: Frederik Sanger and colleagues introduce the