Vent DNA polymerase and assymmetric PCR

molpath at chmeds.ac.nz molpath at chmeds.ac.nz
Wed Mar 29 16:37:41 EST 1995


In article <3ladq6$var at mule.fhcrc.org>, Tim Buss <tbuss at fred.fhcrc.org> writes:
> 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.

	Hi Tim. Could be you are right - the exo activity of Vent is probably
	wiping out your single-stranded product. If this is the case, what
	about getting some of the new 9 degrees N DNA polymerase from NEB.
	It is supposed to be genetically engineered to have only 1-5% of the 
	wild type exonuclease activity. Enough to provide proofreading, but not
	enough to excessivley degrade single-stranded DNA or primers.
	Catalog #260 (S or L).

-- 
Andrew Fellowes                                             
Hospital Scientist                                          
Canterbury Health Laboratories, PO Box 151, Christchurch, New Zealand
 Ph: 64 3 364 0550 | Fax: 64 3 364 0525 | Email: molpath at chmeds.ac.nz



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