Suggest and describe a technique to obtain multiple copies of a gene of interest in vitro. (3)
Polymerase Chain Reaction is the required technique
Polymerase Chain Reaction(PCR) : Each cycle has three steps:
(i) Denaturation: (ii) Primer annealing; and (iii) Extension of primers
PCR strands 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 DNA.
(ii) Annealing: The two sets of primers (small chemical synthesized oligonucleotides that are complementary to the regions of DNA) undergo boichemical process of annealing at an optimum temperature of C - C .
(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. (3)