How is the amplification of a gene sample of interest carried out using polymerase
Chain Reaction (PCR)?
Polymerase Chain Reaction (PCR): Each cycle has three steps:
(i) Denaturation; (ii) Primer annealing; and (iii) Extension of primers
PCR stands for Polymerase Chain Reaction. In this reaction multiple copies
of the gene of interest are synthesised in vitro under three steps:
(i) Denaturation: In this double stranded DNA is converted to single stranded,
This is often achieved by heating or alkaline conditions. This is called melting of
(ii) Annealing: The two sets of primers (small chemical synthesized oligonucleotides
that are complementary to the regions of DNA) undergo biochemical process of
annealing at an optimum temperature of.
(iii) Extension: The enzyme DNA polymerase extends the primers using the nucleotide
provided in the reaction and the genomic DNA as template. If the process of replication
of DNA is repeated many times, the segment of DNA can be amplified to approximately
billion times. Such repeated amplification is achieved by the use of a thermostable DNA
polymerase and the amplified fragment if desired can be used to ligate with a vector
for further cloning.