Pfu polymerase in PCR

Tom Duncan duncant at vax.cs.hscsyr.edu
Mon Feb 17 11:36:23 EST 1997


Colin Dolphin wrote:
> 
> (This does happen as PFU is very good at correcting your
> mutation-containing primer if your trying to use it to do PCR-mutagenesis
> resulting in all wild-type products.)
> 

I have used Pfu polymerase for PCR-based site-specific mutagenesis a
number of times with good success. In my last round I did four separate
mutagenic reactions, and I had NO wild-type clones in the first twenty
clones screened (5 screened per mutation). I think the "repair" of
mutant oligos by Pfu exo activity can usually be avoided if enough
complementary bases are included at the 3' end of the oligo.

Also, an earlier post in this thread (by Duncan Clark, I believe)
indicated that un-annealed primers would be degraded by Pfu's exo
activity; my impression was that most proofreading polymerases only
"chewed back" on 3' ends of dsDNA. Are there references for Pfu
degrading ssDNA?

	Cheers,
	Tom
-- 
	Thomas M. Duncan, PhD
	Dept. Biochemistry & Molecular Biology
	SUNY Health Science Center
	750 E Adams St,	Syracuse, NY 13210
	Email:	duncant at vax.cs.hscsyr.edu



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