Polymerase Chain Reaction: Basic Protocols

The melding of a technique for repeated rounds of DNA synthesis with the discovery of a thermostable DNA polymerase has given scientists the very powerful technique known as polymerase chain reaction (PCR). PCR is based on three simple steps required for any DNA synthesis reaction: (1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers. These reactions may be carried out with any DNA polymerase and result in the synthesis of defined portions of the original DNA sequence. However, in order to achieve more than one round of synthesis, the templates must again be denatured, which requires temperatures well above those that inactivate most enzymes. Therefore, initial attempts at cyclic DNA synthesis were carried out by adding fresh polymerase after each denaturation step (1 ,2 ). The cost of such a protocol becomes rapidly prohibitive.