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

Michael Sullivan via methods%40net.bio.net (by mlsulliv from wisc.edu)
Wed Mar 21 08:51:52 EST 2007


Is it possible that your PCR reactions have gone to completion long  
before the 30 cycles you are running? I'm thinking that one  
possibility is that if the mismatched primers are able to work,  
albeit very inefficiently, running the PCR to completion would mask  
the inefficiency. For example, if the positive control reactions have  
gone to completion after only 20 cycles, running 30 cycles would take  
care of a 1000X difference in efficiency with your experimental sample.

If this seems like a possibility, you might try real time qPCR or the  
poor man's version (collecting samples after various numbers of  
cycles, say 10, 15, 20, 25, 30). Either of these might reveal  
substantial differences in priming efficiency.

Mike

On Mar 20, 2007, at 2:46 PM, WS 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
>
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---
Michael L. Sullivan
Plant Research Molecular Geneticist
US Dairy Forage Research Center
ARS-USDA
1925 Linden Drive West
Madison, WI 53706
(608) 890-0046 (Phone)
(608) 890-0076 (FAX)



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