How does temperature affect PCR?
Posted February 4, 2022
The PCR process involves repeatedly heating and cooling the reaction through a series of temperature changes to produce lots of copies of the DNA sequence of interest.
There are three main stages to this process. Temperature plays a key role in each of these stages:
Stage 1- Denaturing: In this first stage, the double-stranded DNA is heated to 94 - 95⁰C. That temperature is maintained for about 15 – 30 seconds. The high temperature breaks the hydrogen bonds between the bases in two strands of template DNA. This causes the two strands to separate, resulting in two single strands of DNA. These single DNA strands act as templates for the production of the new DNA strands.
Stage 2-Annealing: In this second stage, the temperature is lowered to 50 - 65⁰C, to allow the DNA primers to attach to a specific region on the single-stranded DNA template by way of hydrogen bonding. The optimum temperature used in this stage depends on the melting temperatures of the primers being used in the reaction.
Stage 3-Extending: During this final stage, the temperature is increased to 72⁰C to facilitate the synthesis of new DNA by the Taq DNA polymerase enzyme, which is very stable at high temperatures. Taq DNA polymerase facilitates the synthesis of new DNA by adding DNA bases to the template.
These three thermal cycling processes are repeated about 20 to 40 times to produce millions of copies of the DNA sequence of interest.
Temperature effect on polymerase fidelity