If a desired gene is identified in an organism for some experiments, explain the process of the following.
(i) Cutting this desired gene at specific location.
(ii) Synthesis of multiple copies of this desired gene.
.(i) The desired gene is cut by using the enzymes restriction endonucleases. Firstly, the restriction endonucleases that recognise the palindromic nucleotide sequence of the desired gene is identified. The endonuclease inspects the entire DNA sequences
to find and recognise the site. It cuts each of the double helix at a specific point, which is a little away from the centre of the palindromic site. The cutting site is between the same two bases on the opposite strands. This results in over-hanging of single stranded stretches, which act as sticky ends.
(ii) Multiple copies of the desired gene is synthesised by Polymerase Chain Reaction (PCR) method. In this method, the desired gene is synthesised in vitro. The double stranded DNA is denatured using high temperature of 95°C and the strands are separated. Each separated strand acts as a template.
Two sets of oligonucleotide primers are annealed to the denatured DNA strands. The thermostable taq polymerase extends the primers using nucleotides provided in the reaction mixture. Finally, the amplified fragments are ligated into recipient cells.