re sequence polymorphisms

Callum Bell cbell at CBIL.HUMGEN.UPENN.EDU
Thu Nov 3 11:01:58 EST 1994



> One issue to bare in mind when sequencing PCR products is fidelity of the 
> the PCR reaction. Standard Taq is error prone, it has no editing function.
> Therefore an early error will create a poylmorphism in the finl products. 

I have heard several people make this argument against sequencing Taq
polymerase amplified DNA. True, it is well documented that Taq
misincorporates bases at an appreciable frequency. Consider, though,
the number of copies of the target sequence that are present before
amplification begins. 50 ng (a reasonable amount to use in a PCR
reaction) of human DNA contains tens of thousands of copies of the
genome, and therefore, as many copies of the target fragment.  Even if
a mutation occured during the first round of amplification, in one or a
few of these copies, the fraction of the total amount of DNA
represented by the mutated copies would be negligible. It seems to me
that only if the DNA template was derived from a single cell, or other
very small quantity of DNA, would misincorporations cause the apparent
polymorphisms. Otherwise, mutation detection based on PCR amplified DNA
would not be possible. 

Callum Bell

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