Gene of interest can be amplified by Polymerase chain reaction (PCR) using thermal cycle. It is in vitro replication of DNA to form multiple copies of desired DNA.
Principle.
1. In this, multiple copies of gene (or DNA) of interest are synthesised in vitro, using two sets of primers (small chemically synthesised oligonucleotides that are complimentary to the 3. region of template DNA) and the enzyme Taq DNA polymerase 2. The enzyme extends the primers using the nucleotides provided in reaction and genome DNA acts as template, to form two copies of DNA. 3. DNA polymerase enzyme used is thermostable enzyme isolated from a bacterium Thermus aquaticus, which remain active during the high temperature induced denaturation of double stranded DNA. 4. If the process of replication is repeated many times, the segment of DNA can be amplified to approximatelybillion times i.e., 1 billion copies are made. 5. The amplified fragments of desired gene can now be used to ligate with vector for further cloning 6. It involves different steps.
(a) Denaturation and` 94^@C` . (b) Annealing of primers at `54^@C` . (c) Extension by using Taq polymerase at `72^@C`.
