Fig 3-2.3.2.1: Basic Thermal Profile of PCR

3-2.3.2.1 Initial denaturation:

Initial denaturation involves heating of the reaction to a temperature of 94–96°C for 7-10 minutes (or 98°C if extremely thermostable polymerases are used). For specifically engineered DNA polymerases (Hot start Taq polymerases) activity requires higher range of temperature. The initial heating for such a long duration also helps in gradual and proper unfolding of the genomic DNA and subsequent denaturation, and thus exposing target DNA sequence to the corresponding primers.

3-2.3.2.2 Denaturation:

Denaturation requires heating the reaction mixture to 94–98°C for 20–30 seconds. It results in melting of the DNA template by disrupting the hydrogen bonds between complementary bases, yielding single-stranded DNA molecules.

Fig 3-2.3.2.2: Denaturation of double stranded DNA to single stranded DNA