PCR: mutations in the primer region?

Warren Gallin wgallin at gpu.srv.ualberta.ca
Thu Aug 27 14:13:33 EST 1998


In Article <6s4bhr$reg at panix3.panix.com>, iayork at panix.com (Ian A. York) wrote:
>In article <35E5AB66.41C6 at plum.niaid.nih.gov>,
>Tim Allison <allison at PLUM.NIAID.NIH.GOV> wrote:
>>
>>expression.  I've just received the sequences for a number of the
>>resulting clones and all of the sequences are wrong, but only within the
>>region that contained my PCR primers.
>
>I'm far from being an expert on this, but this seems to be a common
>observation; too common (in my humble opinion) to always be a problem with
>the primer manufacture.  It's happened to me several times, to colleagues
>in the lab, and the phenomenon has been posted here several times.
>
>I don't have a good explanation, though.  My only real idea, which isn't a
>very good one, is that mutations occur in the primer region and the primer
>manages to anneal to them anyway--but why they'd anneal, I don't know.
>This might predict that cranking up the annealing temp would reduce the
>problem, I suppose.
>
>Hopefully someone has a better answer.

    I think that bad primer synthesis is the only viable explanation.  The
sequence in the primer region in subcloned PCR products is from the
synthesized primers, not from any mismatched template (or rather the odds of
getting template-derived sequence differences in the primer region of a PCR
product are vanishingly small, certainly not in the 10-50% range).
    A possible explanation: with the advent of high throughput multiplexed
synthesis of primers from some comercial operations, I think that the
chances of getting this kind of mistake may be going up, or else it always
occured at a low rate but the increase in total number of primers being
produced is upping the total number of occurences.
Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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