3' single base primer mismatch with Taq still yields products?

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Tue Mar 20 14:46:15 EST 2007


Hi folks,

I have some PCRs that drive me mad:

Did some mutagenesis on a plasmid and now want to screen for the
mutants by PCR. I made primers with a single base 3' mismatch
expecting only a PCR product when the mutation was present (the second
primer is a standard primer). Strangely, I get bands even with those 3
primers that are designed to detect mutations in other constructs,
they can bind but there still is this 3' mismatch, so there should not
be any extension.

I ran the reactions (30 cycles) in a gradient cycler at 60+-10 degC
annealing temperature and can see the bands in all 4 reactions at
temperatures up to 60. The primers have an overlap of 17 bases. My Taq
I is some ordinary enzyme, without any special features. Plasmids were
prepped by some Maniatis' style kit-less homebrew miniprep that never
made any problems before.

Is there any residual intrinsic proofreading activity in Taq????? As
oligos are synthesized from the 3' end, there should be no problem be
caused by eventually existing N-{1,2,3,...} products as they are
missing the 5' end. I realize, of course, that a few extended primers
in the first rounds will yield a product that is happily extended in
the further cycling and thus will be able to generate this band.

I think, I missed something important. Maybe non-textbook behaviour of
PCR? Should I try a touch down PCR?

Any clues? No Trolls, please!

Wo



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