How to Avoid PCR Errors

Brian Foley brianf at med.uvm.edu
Thu Feb 17 15:31:47 EST 1994


Helen McBride (Helen_McBride at hlthsci.med.utah.edu) wrote:
: I have used Pfu polymerase for amplifications of around 1.8 kb without
: any errors. I too had a problem in getting coding errors when I used Taq
: for amplification. Pfu can actually use the same conditions as Taq and
: even the same standard Taq buffer with the addition of 10% DMSO. If you
: want more information. Please E-mail me at the address below. Good Luck!

: Helen_McBride at hlthsci.med.utah.edu

	I have now sequenced 23 kb of PCR-amplified cDNA (RT-PCR) and
I have found on average one PCR-induced mutation per 1.9 kb of sequence
(12 mutations found among 23 kb sequenced).
	Due to the Poisson distribution of these errors, I have found some 
products as small as 600 bases long with TWO artifact mutations in the
same clone!
	I was using TAQ polymerase, 200 micromolar dNTP concentrations, and
20 cycles of PCR following RT with MoMULV reverse transcriptase.  The errors
could have come either from the RT or the TAQ-PCR.
	I do not have similar data for other polymerases or other dNTP
concentrations.

--
********************************************************************
*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *
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