Pfu polymerase in PCR

Tom Duncan duncant at vax.cs.hscsyr.edu
Wed Feb 19 19:05:42 EST 1997


Dr. Duncan Clark wrote:
> 
> In article <33088907.41C6 at vax.cs.hscsyr.edu>, Tom Duncan
> <duncant at vax.cs.hscsyr.edu> writes
> >Are there references for Pfu degrading ssDNA?
> 
> Wayne Barnes refers to this problem with Vent in his KlenTaq Gene paper
> and there was a detailed paper in NAR about a year or more ago showing
> that primer degradation by the proof-reading pol. could be ovecome by
> putting a thiol linkage in an oligo close to the 3' end. The paper was
> solely on overcoming primer degradation during PCR using proof-reading
> pols. As normal, somewhere in a filing cabinet I have the paper but have
> I filed it under PCR, methods, oligos etc etc?

My main point was whether any references show that Pfu or other
proof-reading polymerases degrade primers in the single-stranded form
(that is, primers not annealed to their template). The thiol linkage near
the 3' end would block degradation of annealed primers, so that does not
show that primers are degraded directly. My understanding from earlier
refs was that proof-reading polymerases must be bound to dsDNA with a
3'-OH (annealed template/primer) in order to chew back on the exposed
3'-OH. In the absence of nucleotides, if you left a template/primer sample
with polymerase long enough, more and more primer might be degraded as the
initially-bound primers are destroyed and more primers bind to the low
number of templates.
Again, I've had no problem with degradation or "repair" of mutagenic
primers when using Pfu for PCR-based site-directed mutagenesis, as long as
additions are made in the proper order.

	Sincerely,

	Thomas M. Duncan
	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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