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

chovek69 via methods%40net.bio.net (by ivanoov from gmail.com)
Fri Mar 23 01:36:17 EST 2007


On Mar 21, 3:51 pm, Michael Sullivan <mlsul... from wisc.edu> wrote:
> 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
>
> > _______________________________________________
> > Methods mailing list
> > Meth... from net.bio.net
> >http://www.bio.net/biomail/listinfo/methods
>
> ---
> 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)- Hide quoted text -
>
> - Show quoted text -

Did you try dNTP concentration gradient and lowering Mg2+ ? Maybe it's
worth trying (e.g. 0.05mM, 0.1mM, 0.125mM, 0.15mM etc.....).

Ivan



More information about the Methods mailing list