PCR: Why is my TAQ unfaithful

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Fri Dec 2 11:45:26 EST 1994


> > In article <jwilber-221194142007 at jwilberding.chem.nd.edu>, 
> > jwilber at pop.nd.edu (Julie Wilberding) says:
> > >
> > >Hi,
> > >
> > >I have been tying to PCR three constructs.  I use Promega TAQ, 100uM
> > >dNTP's, buffer and the proper amount of primers and template (Double
> > >stranded circular plasmid).  My mutation rate is high ( 3 in one 180 bp
> > >fragment).  I have changed dNTP, TAQ, with no change.  This is a problem
> > >recently encountered in our lab.  The PCR conditions are 94 1 min., 55 1
> > >min., 72 2 min., 30 cycles with a final cycle b3ing a 10 min extension at
> > >72.  Is the circular template a possible cause???  Efficiency is good but
> > >fidelity sucks!  Any help or advice would be greatly appreciated.

In article <57400293wnr at genesys.demon.co.uk>, Duncan at genesys.demon.co.uk
(Duncan Clark) wrote:

> To further improve fidelity try adding 
> 1/64 unit Pfu (Taq extender!) per 2.5u Taq. Also does your template have 
> really bad secondary structure or 'cos that may be the problem.

Good points.  But if that doesn't do the trick, here are a few points that
might help.

1. dNTP concentration:  Altough 100uM should be quite O.K. you could easily
lower that and thereby increasing fidelity, if the fragment you are trying
to amplify is as short as 180 bp. I would try 20 mM.

2. Cutting down on the number of circles.  This will of course decrease
your yield drastically, but it may be better to have a little bit of the
right stuff than a lot of junk.

3. For short fragments TOO LONG EXTENSION TIME is a common source of
errors.  The incorporation rate of Taq pol has ben estimated 35-100 bp/sec.
 Two minutes for 180 pb is overkill.  A 3' end with a mismatched base is a
lot worse substrate for Taq than if the base at the end is paired. 
Misincorporations will therefore cause the reaction to pause.  If the
extension time is apropriate most products with errors will not be finished
and not serve as templates in the following rounds.

Still, the mutation rate you are getting sounds astonishing.  I have
actually used PCR for random mutagenesis.  And altough I used a lot higher
amounts of dNTP's, the same extension time and the same number of circles,
in addition to various amounts of MnCl2, I have never seen this kind of
error frequency.

Hope you can solve this.  Good luck.

Zophonias



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