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

Aawara Chowdhury via methods%40net.bio.net (by aawara from FEMA-trailer.org)
Tue Mar 20 21:34:19 EST 2007


In <1174419975.723250.263820 from l77g2000hsb.googlegroups.com>,
 WS <novalidaddress from nurfuerspam.de> wrote:

> Is there any residual intrinsic proofreading activity in Taq????? 

There isn't.  Full-length Taq does have a 5'->3' exo activity, but
that is irrelevant to proofreading.  The "KlenTaq" or "delta-Taq"
that most commercial manufacturers sell lacks this activity.

> 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.

In my experience, this is the reason you get an amplified product.  And
I have also see this happen.  I have mutated a "T" to a "C".  The wild-
type is amplified with a primer that ends in "G".

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

I did try raising the denaturation temperature, as well as adding
compounds like betaine and DMSO; neither worked.

AC
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