Steps in PCR - (Apr/09/2008 )

Whats parametes are importnat for PCR three steps

Denaturation
why longer denaturation is avoided

Annealing
it should be longer or shorter
if longer then whats the disadvantage
if the shorter then whats the benefit

Extension
How to calculate how much times is requires to amplify the genes

How many cycles should be used

If higher cycles either yield is decreased or increased

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A longer denaturation steps means more template DNAs are depurinated at high temperature, DNA polymerase can't passing through abasic template.
Annealing for a too longer time, more non-specific annealing occurs, and more non-specific products. If too shorter, the products yield decreased.
Extension at 1 min/1 kb for Taq DNA polymerase, and 2 min/1 kb for Pfu DNA polymerase in most case.
Cycles are according to the amount of template, 20-35 are the most used. A nester PCR is prefered if insufficient products obtained at more than 40 cycles. PCR that over-cycled means it reached the plateau stage, the yield doesn't changed siginificantly, however, we should note that more mutations, more incompletely extended DNAs, more single-stranded DNA maybe produced.

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