PCR: mutations in the primer region?

Ian A. York iayork at panix.com
Thu Aug 27 14:20:27 EST 1998


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.

Ian
-- 
    Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
    "-but as he was a York, I am rather inclined to suppose him a
     very respectable Man." -Jane Austen, The History of England



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