Principles of PCR
These reaction components go through multiple cycles of the three main steps of PCR. Denaturation is the first step in the PCR reaction. The sample is heated at a high temperature between 94 and 98 degrees Celcius to separate the double stranded DNA into single strands. The next step is annealing at a temperature between 55 and 72 degrees Celcius, where the primers bind specifically to complementary sequences of both strands of the DNA template. The primers are designed to bracket the region that is targeted for amplification. The optimal annealing temperature for a particular primer pair can be determined experimentally by testing a range of temperatures around 3 to 5 degrees lower than the lowest melting temperature of the two primers. The final step is extension at 68 degrees Celcius or 72 degrees Celcius, depending on the enzyme’s optimal temperature. In this step, the enzyme extends the primer molecules by incorporation of the building blocks, the dNTPs. The polymerase can only start to replicate on the template where a primer has annealed to it. That means that the primer sequence is crucial for amplification of the correct part of the DNA. In some cases, the annealing and extension steps can be combined. This is known as a 2-step cycling protocol.