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pfu and Taq PCR mutation percentage

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Tue Jan 11 06:43:31 EST 2000

In article <85eh2u$78b$1 at ssauraab-i-1.production.compuserve.com>, Gys de
Jongh <GysdeJongh at compuserve.com> writes
>Dr. Duncan Clark <Duncan at nospam.demon.co.uk> wrote in message
>news:9erByuAEgxd4EAXA at genesys.demon.co.uk...
>> Mutation frequency for Taq is 8 x 10E-6/bp/duplication
>> Mutation frequency for Pfu is 1.3 x 10E-6/bp/duplication



RT-PCR Accuracy Is Influenced by DNA Polymerase Fidelity 

The frequency of mutations in RT-PCR products is thought to be
influenced by the replication fidelity of both the RT and the DNA
polymerase, under the particular reaction conditions employed (e.g.,
number of duplications, pH, and [Mg2+]). The mutation frequency (M.F.)
of an RNA amplification procedure can be estimated from the RT error
rate, the DNA polymerase error rate, the number of cDNA template
duplications, and the amplicon size. For example, consider the situation
when an RNA template is reverse transcribed with MMLV-RT (error rate of
3.3 x10-5/base6), and then a 1-kb portion is PCR amplified for 20
duplications (106-fold amplification) using Stratagene’s PfuTurbo DNA
polymerase (error rate of 1.3 x 10-6 M.F./bp/duplication7). The
percentage of 1-kb cDNAs that contain errors is calculated to be 3.3%
(3.3 x 10-5/base x 1000 bases), while an additional 2.6% of the final
clones would contain mutations introduced by PfuTurbo DNA polymerase
[(1.3 x 10-6)(1000 bp)(20 duplications)]. In this example, the overall
M.F. would be 5.9%. In contrast, if the same RT-PCR reaction was carried
out with AMV-RT (error rate of 5.9 x 10-5/base6) and Taq DNA polymerase
(8 x 10-6 M.F./bp/duplication7), 5.9% of 1-kb cDNAs would contain RT-
generated errors, and an additional 16% of the final clones would
contain Taq-generated mutations [(8.0 x 10-6) x (1000 bp) x (20
duplications)]. The overall M.F. in this case would be 21.9%. Thus,
despite the relatively high error rates associated with RTs, the
accuracy of the PCR enzyme is expected to contribute to overall mutation
frequency due to the exponential amplification of the cDNA template
during the PCR portion of RT-PCR procedures. 

The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382

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