SuperScript error rate?
ehovig at radium.uio.no
Mon Mar 5 18:41:37 EST 2001
In article <xIsiH$CNI3o6EAXi at genesys.demon.co.uk>, "Dr. Duncan Clark"
<d_clark at genesys.demon.co.uk> wrote:
> In article <ehovig-59017E.11355905032001 at nntp.uio.no>, the eminent
> Eivind Hovig at DNR wrote
> >Does anyone have relevant pointers to estimates of the error rate of
> >reverse transcriptases, and SuperScript in particular, with respect to
> >in vitro reverse transcription?
> Poor! Stratagene published something in a Strategies maybe a year or
> more ago. Have a look on their web site as they are all there via pdf
Thanks for the tip. I found some lines of relevant text:
"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)]."
But this does not really shed that much scientific light (they don't
state that the numbers are precise). I have not been able to locate any
references where anybody have performed systematic assays of this. The
reason of my interest are the various amplification techniques for
microarray analysis and their reliability, and also the lower limit of
biologically relevant mutation analysis based on small amounts of
naturally occuring mRNA (in vivo). Given the flow of "enhanced" reverse
transcriptases recently, this is an even more pressing question for me.
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