PCR

1. PCR By Staci Cutting and Mitch Gavazzi

2. What is PCR? • PCR is sometimes called Molecular photocopying the polymerase chain reaction is a fast and inexpensive technique used to make many copies of small segments of DNA.

3. The steps of PCR • Denature. The first step requires a high temperature to denature the dsDNA. This is typically done by briefly heating the sample to 92-94oC. • Anneal. The second step requires lowering the temperature to allow annealing of the primers to the ssDNA. The optimal annealing temperature depends upon the melting temperature of the primer-template hybrid. If the temperature is too high the primers will not anneal efficiently, and if the annealing temperature is too low the primers may anneal nonspecifically. Annealing is usually done, at 5oC below the Tm of the primers, typically about 45-55oC. (As a simple rule of thumb, the Tm of the primers can be estimated by adding 2oC for each A or T and 4oC for each G or C.) Because the primers are in vast excess to the template, the annealing reaction occurs very quickly once the proper temperature has been reached. To insure adequate specificity, the primers must be 20-30 nucleotides long. • Extend. The third step requires DNA synthesis by DNA polymerase. To withstand the repeated exposure to high temperatures, a thermostable DNA polymerase is used for PCR. A variety of thermostable DNA polymerases (isolated from different thermophilic Bacteria or Archae) are available, but the first thermostable DNA polymerase used for PCR, called Taq polymerase, was isolated from the bacterium Thermusaquaticus. The optimal temperature for Taq polymerase is about 75-80oC, but it is partially active even at typical annealing temperatures so primer extension begins during the annealing step. The rate of primer extension by Taq polymerase is about 50-100 nucleotides/sec. Thus, the time required for primer extension depends on the length of the sequence to be amplified.

4. Virtual PCR lab • http://learn.genetics.utah.edu/content/labs/pcr/