Not a component of PCR ?
Correct Answer: Restriction enzyme
Description: Steps in PCRPCR uses DNA polymerase to repetitively amlify targeted poion of DNA. Each cycle doubles the amout of DNA in the sample, leading to exponential increase with repeated cycles of amplification. Thus amplification after 'n' number of cycle in (2)". Twenty cycles provide an amplification of 106 (million) and 30 cycles of 109 (billion).PCR occurs in following steps -i) Isolation of target DNA sequence :- A DNA containing the sequences to be amplified is isolated.ii) Primers construction:- The sequence of flanking region (nucleotide sequence of sho segments on each side of target DNA sequence) are used to construct 2 single stranded oligonucleotides which are complementary to the respective flanking region. These oligonucleotides functions as primers in PCR reaction.iii) Denaturation of DNA :- Target DNA is heated to separate the double strand of DNA into single strands.iv) Annealing of primers to single stranded DNA :- The separated strands are cooled and allowed to anneal to the two primers, one for each strand.v) Chain extension :- DNA polymerase and deoxyribonucleotides (dNTP) are added to the mixture. DNA polymer?ase (Taq polymerase) then uses the primer for DNA synthesis using target strands as templates. On both strands, Primer is amplified in 5'-43' direction by addition of nucleotides (dNTP) at 3' end. The mixture is once again heated to melt (separate) all double stranded structure. There are now four strands of DNA.A second cycle of cooling, allows fuher binding of the primers and a second round of polymerization is repeated. The process of heating (denaturation), cooling (annealing) and polymerization is repeated many times.As Taq polymerase is not denatured on heating and therefore does not have to be added at each successive cycle.Thus following are required in PCR :- Target double stranded DNA, two specific primers, a thermostable DNA polymerase (Taq polymerase), deoxyribonucleotides (dNTP).
Category:
Biochemistry
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