What Are the Steps in PCR?

By Staff WriterLast Updated Mar 26, 2020 6:05:47 AM ET

A polymerase chain reaction, or PCR, consists of three steps: DNA denaturation, primer annealing and extension. These steps are repeated between 20 and 35 times to synthesize the correct quantity of the DNA of interest. Each of these steps requires a different temperature range, which allows PCR machines to control the steps. PCR is typically done in small PCR reaction tubes containing all the necessary ingredients for DNA synthesis.

The first step in PCR, DNA denaturation, requires a high temperature, typically around 95 degrees Celsius. Denaturation causes the DNA to unzip and separate into single strands, exposing the DNA bases to the rest of the PCR mixture.

The second step, primer annealing, must occur at a lower temperature than the denaturation step. The PCR machine cools the solution to a temperature between 45 and 72 degrees Celsius. The specific temperature for annealing depends on the primers. Primers are short pieces of previously synthesized DNA that occur at the beginning and end of the DNA of interest. During primer annealing, the primers bind to the appropriate parts of the DNA strand.

The third step, extension, occurs at 72 degrees Celsius. This step entails the extension of new strands of DNA, starting with the primers.

After extension, the reaction is heated back to 95 degrees Celsius to start another cycle of PCR. The number of strands of DNA after each cycle of PCR steps doubles, so the amount of DNA produced is exponential. In this way, 20 to 35 cycles of PCR creates millions of strands of the DNA of interest.