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

[peter]

On Mar 20, 3:46 pm, "WS" <novalidaddr... from nurfuerspam.de> wrote:
> 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

Taq will extend from a 3' mismatch primer, at least that is my
experience from early days of my career when someone mistakenly order
primers that I used for PCR. In the books it is written that Taq does
not have proof reading 3-5 activity - well that is relative to what
you compare it - it has less activity than most other PCR enzymes. So
to answer your question - yes it will extend, and you would see PCR
after 30-33 cycles.
Peter
P.S. when I do mutagenesis I always put a silent restriction site for
screening...