Vent DNA polymerase and asymmetric PCR

Tim Buss tbuss at fred.fhcrc.org
Tue Mar 28 20:40:22 EST 1995


Q: Will the 3' -> 5' exonuclease activity of Vent destroy the single
stranded product of an asymmetric PCR?

I have been using Taq polymerase in asymmetric PCR reactions to generate
long mutagenic 'oligos' (approx. 800 bases long). Reducing the
concentration of one of the primers 50X to 0.5 picomoles was about right.
I would get the main double stranded product, and see the single stranded
running a little faster on a 1.5 to 2% agarose gel.

The error rate with Taq has caused some problems so I tried using Vent.
Once the PCR conditions had been optimized the symmetric worked very
well, but the asymmetric does not work. The double stranded product is
there, but no single stranded. I have tried varying amounts of primer,
but only the amount of double stranded product varies.

So, am I flogging a dead horse here, the exonuclease activity simply
chewing it up, or do you think it is a matter of further optimizing the
conditions??

Many thanks,

Tim.



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