5.1 The Reaction

Figure 41. The first step in PCR

The reaction mixture is heated to denature the DNA and separate the double strands. It is not necessary to have a pure DNA to start the procedure. It is necessary to know some of the base sequence at each end of the DNA that you wish to amplify and to prepare single-stranded primers containing the correct base sequences.

The temperature is lowered to allow the primers (added in excess) to bind to the 3' regions of the DNA strands.

DNA polymerase then synthesizes the complementary strand on each DNA. The DNA polymerase was isolated from an organism that lives at high temperatures so that the enzyme is stable to heating to over 90° and is enzymatically active at 70°

The heating and cooling cycle is repeated many times. Each time the concentration of the newly-amplified DNA should double. Notice that the first round of synthesis produces new strands of indeterminate length, but the products become more uniform as the reaction proceeds. This happens because the products are soon defined by the primers at each end of the desired sequence. Commercial thermal cyclers are available that will precisely automate the many heating and cooling cycles.

A particular gene often may be amplified to microgram quantities in as little as four hours.

Figure 42. Multicycle PCR