PCR fidelity (Taq error)
gshuh at garnet.berkeley.edu
Thu Mar 20 05:19:48 EST 1997
In article <Pine.GSO.3.95.970312170837.19462J-100000 at oeb.harvard.edu>,
"Matthew P. Hare" <mhare at oeb.harvard.edu> wrote:
# 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
# Matt Hare
How many cycles are you using? If 40 cycles were used, then a given 300
base fragment will have been acted upon ~12000 times. 1-2 substitutions
in that context doesn't seem so bad.
Gene S. Huh, Ph.D.
Department of Molecular and Cell Biology
Life Sciences Addition, Room 221
University of California at Berkeley
Berkeley, California 94720-3200
Email: gshuh at socrates.berkeley.edu
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