Standard Processes For PCR Reaction

The standard PCR process is divided into three steps:

DNA denaturation: (90 ℃ -96 ℃): Double stranded DNA template undergoes hydrogen bond cleavage under thermal action, forming single stranded DNA

Annealing: (60 ℃ -65 ℃): As the system temperature decreases, the primer binds to the DNA template, forming a local double strand.

Extension: (70 ℃ -75 ℃): Under the action of Taq enzyme (at around 72 ℃, with the best activity), dNTP is used as the starting material, extending from the 3 'end of the primer in the direction of 5' → 3 'end, to synthesize DNA strands complementary to the template.

After each cycle of denaturation, annealing, and extension, the DNA content doubles. Nowadays, some PCR can replicate in a short time even if Taq enzyme activity is not optimal due to the short amplification region. Therefore, a two-step method can be used, which involves annealing and stretching simultaneously between 60 ℃ and 65 ℃, to reduce one temperature rise and decrease process and improve reaction speed.