Polymerase Chain Reaction

1. Polymerase Chain Reaction Mrs. Stewart Medical Interventions

2. 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.

4. KaryMullis • 1983 – Mullis and his colleagues invented the PCR technique • Nobel Prize in 1993

6. 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 

7. 3 steps in a PCR • Denature • Anneal • Extension

8. Denature • The DNA is heated to 95oC, causing the double stranded DNA to denature by breaking the hydrogen bonds between the strands.

9. Denaturation

10. Anneal • 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.

11. Anneal

12. Extension • 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)

13. Extension

14. 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.

15. How many cycles? • Most PCRs should include only 25 – 35 cycles. • Depends on the amount of starting material

16. 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).