PCR fidelity (Taq error)
Matthew P. Hare
mhare at oeb.harvard.edu
Wed Mar 12 17:36:56 EST 1997
I have been trying various thermostable polymerases in order to find one
that gives me a low rate of nucleotide misincorporations. My assay for
fidelity is to amplify from a clone, TA clone that PCR product, and
sequence multiple clones.
Any variation among clones should be due to Taq error. I have tried
PE amplitaq (no "proofreading"), PE rTth XL (proofreads) and TaKaRa EX
(also proofreads). I invariably get an error rate (including all
variation) of 0.1 - 0.2 % (e.g. 1-2 substitutions in 300 bps sequenced
from three clones). This is at least an order of magnitude higher than
what has been reported as typical of Taq (without proofreading 3'-5'
exonuclease activity). I know that Taq fidelity is condition-dependent
and I have decreased the concentrations of dNTPs, MgCl and enzyme as much
as possible as well as keeping extension times as short as possible.
Similar error rates are being found in this same lab with a different
clone (different sequence motif, primers, etc.). So what gives?
Has anyone out there experienced these persistantly high Taq error rates
AND BEEN ABLE TO SOLVE THE PROBLEM?
Please respond to mhare at oeb.harvard.edu
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