There are three major steps in a PCR, which are repeated for 20-35 cycles.
1. Denaturation at 94°C :
During the denaturation, the double strand melts open to single stranded DNA, all enzymatic reactions stop (e.g. the extension from a previous cycle). (usually 30 s) ●
2. Annealing at 54°C (45-60):
Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. The more stable bonds last longer (primers that fit exactly) – polymerase can attach and starts copying the template. Once there are a few bases built in, the hydrogen bond is so strong between the template and the primer, that it does not break anymore. (Usually 30 s) ●
3. Extension at 72°C :
This is the ideal working temperature for the polymerase. The bases (complementary to the template) are coupled to the primer on the 3′ side (the polymerase adds dNTP’s from 5′ to 3′, reading the template from 3′ to 5′ side, bases are added complementary to the template)
(usually 30 s to 1 min – depends on product length)
