PCR fidelity (Taq error)

[Gene Huh]

In article <Pine.GSO.3.95.970312170837.19462J-100000 at oeb.harvard.edu>,
"Matthew P. Hare" <mhare at oeb.harvard.edu> wrote:

# Aloha,
# 
# 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
# 
# Cheers,
# 
# Matt Hare

Matt,

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