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Polymerase Chain Reaction. Mrs. Stewart Medical Interventions. Polymerase Chain Reaction. a lab technique that produces numerous copies of a specific segment of our DNA in a relatively short period of time. three -step process repeated over and over

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polymerase chain reaction

Polymerase Chain Reaction

Mrs. Stewart

Medical Interventions

polymerase chain reaction1
Polymerase Chain Reaction
  • a lab technique that produces numerous copies of a specific segment of our DNA in a relatively short period of time.
    • three-step process
    • repeated over and over
    • produces identical copies of the target sequence.
kary mullis
  • 1983 – Mullis and his colleagues invented the PCR technique
  • Nobel Prize in 1993
taq polymerase
Taq Polymerase
  • The most widely used polymerase is that from Thermusaquaticus (Taq) – Thermophilic bacteria
  • Thermophilicbacterium lives in hot springs and capable of growing at 70 -75 C 
3 steps in a pcr
3 steps in a PCR
  • Denature
  • Anneal
  • Extension
  • The DNA is heated to 95oC, causing the double stranded DNA to denature by breaking the hydrogen bonds between the strands.
  • The temperature of the sample is lowered to between 32-72oC, causing the primers to hybridize or "anneal" to their complementary sequences on either side of the target sequence.
  • The temperature of the sample is heated to between 72-75oC, which is the optimal temperature for the Taq polymerase enzyme to function.
  • Taqpolymerase binds and extends a complementary DNA strand from each primer (adding approximately 60 bases per second, using the free-floating nucleotides)

As amplification proceeds, the DNA sequence between primers doubles after each cycles

  • (The amplification of the target sequence proceeding in an exponential fashion ( 1 2 4 8 16................) up to million of times the starting amount until enough is present to be seen by gel electrophoresis.
how many cycles
How many cycles?
  • Most PCRs should include only 25 – 35 cycles.
    • Depends on the amount of starting material

Advantages of PCR

    • Useful, non-invasive procedure
    • Simplicity of the procedure
    • Sensitivity of the PCR
  • Disadvantages of PCR
    • False positive results (cross contamination).
    • False negative results (e.g. rare of circulating fetal cells).