re sequence polymorphisms

Roger Wiegand rcwieg at ccmail.monsanto.com
Thu Nov 3 14:40:47 EST 1994


In article <9411031557.AA26524 at cbil.humgen.upenn.edu>,
cbell at CBIL.HUMGEN.UPENN.EDU (Callum Bell) wrote:

 
> 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. 
> 


Indeed! Even if the amplification begins from one molecule and a mistake
occurs in the first round, only one of the four templates for the second
round will contain the mutation, resulting in 25% of the product being
mutant. I've been trying to sequence mixed populations and 25% of a second
sequence is close to the lower limit of what you can even see with any
reliability. (Note that this a gross simplification of what probably goes
on in the PCR reaction, but I think the premise is sound.) If you clone
and sequence individuals you will routinely find mutations, but I doubt
that you could ever see a PCR-induced mutation by sequencing the
unseparated PCR product.

-- 
Thanks,
Roger

mailto::rcwieg at ccmail.monsanto.com



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