Question

How is the amplification of a gene sample of interest carried out using PCR?

Answer 1

Denaturation, renaturation or primer annealing, and synthesis or primer extension, are the three steps involved in PCR. The double-stranded DNA of interest is denatured to separate into two individual strands by high temperature. This is called denaturation. Each strand is allowed to hybridize with a primer (renaturation or primer-annealing). The primer-template is used to synthesize DNA by using Taq DNA polymerase.

During denaturation, the reaction mixture is heated to 95 °C for a short time to denature the target DNA into single strands that will act as a template for DNA synthesis. Annealing is done by the rapid cooling of the mixture, allowing the primers to bind to the sequences on each of the two strands flanking the target DNA.

During primer extension or synthesis the temperature of the mixture is increased to 75°C for a sufficient period of time to allow Taq DNA polymerase to extend each primer by copying the single-stranded template. At the end of incubation, both single template strands will be made partially double-stranded. The new strand of each double-stranded DNA extends to a variable distance downstream. These steps are repeated again and again to generate multiple forms of the desired DNA. This process is also called DNA amplification.

                                      Steps involved in PCR