PCR errors:How many really?
troianovsk_s at MSDISK.WUSTL.EDU
Tue May 30 12:33:32 EST 1995
>Description: PCR errors:How many really?
>Okay, I know papers have been rejected because people have cloned using
>PCR and then either 1) not sequenced the clones, and/or 2)not tested
>independently isolated clones for whatever functional assay they are
>interested in. The usual line is that using PCR you take the risk of
>introducing PCR errors into the product. I seem to recall that Taq
>polymerase does have a relatively high error rate, about one in ten
>thousand bases. For most applications, I think this error rate is really
>quite low. Furthermore, anyone who has done any sequencing knows that
>standard sequencing errors exceed (easily) greater than one in ten
Here you are not absolutely right.
Lets assume that Taq has error rate 1:10`000, and you using Taq for PCR and
In PCR you start from very little amount of template and huge amount of
primers. Your final product is the result of amplification, so that chance
to introduce mistake is real, you close or even overcome the error rate.
During sequencing, however, you never even close to the error rate, since
you are using 1:1 molar ratio template:primers, and not increasing amount
of DNAs, but only elongating existing chains.
Therefore, assumption that for the same enzyme the probability to introduce
mistakes during these reaction would be the same is not correct.
>So, my questions: Anyone care to comment on the 1 in ten
>thousand rate? In my, all-be-it, limited PCR experience I have never
>seen a genuine, confirmed, PCR error. Am I lucky or what? Is template
>selection (e.g. GC content) or amount likely to affect error rates. My
>preference is hearing from voices of experience, rather than theoretical
>gobbeldy gook (sp?)
In our lab. it`s usual practice, when otherwise it is complicated to
generate chimeric constructs, we use convenient primers, and cloning of
amplified fragments. There was no mistake so far. But we use hi fidelity
DeepVent polymerase with proof reading activity (N.E.Biolabs), possibly
more template and possibly less cycles, and when the fragment to be cloned
was amplified from cDNA we used to sequence the resulted clone.
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