PCR: mutations in the primer region?
z-suldan at ski.mskcc.nospam.org
Fri Sep 4 13:14:27 EST 1998
> I don't think it can. The DNA made complementary to either primer in
> the PCR is NEVER copied, no matter what the template is or how Taq
> reacts to mismatches. It only ever acts as an annealing site for a
> primer in successive cycles. So any mutant strands created in the way
> you suggest (with errors in the primer region) are dead in the water
> (since the primer region mutation is cancelled out in any daughter
> strand by the sequence of the next primer that anneals) and can only
> arise stochastically.
Well... you're right, but... if the polymerase introduces an error in the
region of the primer that does not affect the annealing of the wt primer
then it is possible for the errors to be introduced by the polymerase
while the primers are fine. This is not so far fetched as you might think.
A recent reaction I did had a primer that added both a tag and a
restriction site. The tm of the region of the primer complimentary to the
starting template was approx 50 C (this would be for rounds 1&2). The Tm
of the primer with all the added sequence was somewhere up in the
80's-90's. If you don't adjust your annealing temp in the PCR rxn for
rounds 3 to the end, it would be very easy for the primers to anneal to a
mismatch (or 2 or 3 or 5 or more!) and errors be introduced.
Have a nice holiday weekend,
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