Help: high mutation rate during RT-PCR

Karthik Aghoram kaghoram at unity.ncsu.edu
Tue Aug 15 07:49:30 EST 2000


I don't think Advantaq has proofreading activity (1 mutation per 10^5) 
bases.  So over 25-30 rds of PCR, that could be a big number.

Pfu has proofreading activity.  But it is not as efficient as Taq, and 
you may have to double your extension periods.

Roche Boehringer has an "Expand Hi-fidelity" system that has both Taq and 
a profreader (Pow?).  It has worked well for my colleagues here.

Good luck
Karthik (who is in no way affiliated to Roche or Boehringer or any other 
         manufacturer of anything)

Gillen, Dr. Clemens (Clemens.Gillen at grunenthal.de) wrote:
: Hi everybody,
: we are using the Advantage polymerase to amplify genes via RT-PCR and =
: we
: face the problems of various mutations. Do you know of certain =
: experimental
: conditions or better polymerases to overcome this problem?
: 
: 
: Clemens Gillen
: 
: Dr. Clemens Gillen			Tel.:	+49-(0)241-569-2494=09
: Gr=FCnenthal GmbH			Fax.:	+49-(0)241-569-2789
: Research & Development	email:Clemens.Gillen at grunenthal.de
: Molecular Pharmacology		http://www.grunenthal.com	=09
: Postfach 50 04 44=09
: D-52088 Aachen
: 
: 
: ---

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Karthik Aghoram				Postdoctoral Associate
					Dept of Crop Science
e-mail: kaghoram at unity.ncsu.edu		North Carolina St University
Phone (W): 919-515-2705			Raleigh, NC 27695-7620

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