Question: When PCR is run, the m............... Edit
Answer: PCR involves two oligonucleotide primers usually between 17-30 nucleotides in lengths that flank
the DNA target sequence. The amplification of the selected region from a complex DNA mixture is
carried out in vitro by the enzyme Taq DNA Polymerase isolated from Thermus aquaticus. It is a
thermo stable enzyme that retains activity even after heating at 940C.
PCR consists of three basic steps:
Denaturation: Double stranded DNA melts to form single stranded DNA. This is
generally carried out at 920C-960C.
Annealing: Primers anneals to their complementary single stranded DNA. It is
usually carried out between 450C-600C.
Extension: The Polymerase adds dNTPs complementary to the template at the 3’end
of the primers.
i. Initial Denaturation Step: It is generally performed at 950 C for 1-3 min, depending
on GC content of template. Incomplete denaturation at the start of PCR results in inefficient
utilization of template and thus in a poor yield.
ii. Denaturation : It is performed for 30 sec -1 min at 940 C. Additives like
DMSO/Glycerol/Formamide are used to facilitate denaturation.
iii. Annealing: The optimal temperature is generally 50C lower than Tm of primer-template
DNA duplex, performed for 30 sec-1 min. Melting temp. can roughly be calculated by
iv. Extension: It is carried out at 720C and results in synthesis of new DNA strand. Usually 1
minute is required for amplification of 1kb target.
v. Final Extension:- As DNA synthesis proceeds, it becomes less efficient as most of the
components get used up. Hence, following the last cycle, enzyme is allowed to finish any
incomplete synthesis by carrying out a final extension at 720C for 5-15 minute.
Fig: PCR (1. Denaturation 2. Annealing 3. Extension)
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