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. Kary Mullis 1983 – Mullis and his colleagues invented the PCR technique Nobel Prize in 1993 

5. PCR components DNA (Taq) polymerase PrimersNucleotides Target DNA sequence

6. Taq Polymerase The most widely used polymerase is that from Thermus aquaticus (Taq) – Thermophilic bacteria Thermophilic bacterium lives in hot springs and capable of growing at 70 -75 C 

7. 3 steps in a PCR 1. Denature 2. Anneal 3. Extension

8. Denature The DNA is heated to 95 o C, 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 32-72 o C, 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 72-75 o C, which is the optimal temperature for the Taq polymerase enzyme to function. Taq polymerase 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).