Help: high mutation rate during RT-PCR
Dr. Duncan Clark
Duncan at nospam.demon.co.uk
Tue Aug 15 09:29:47 EST 2000
In article <B7868D2D2540D311BED900105AD9DBA957E41B at n4s03451.grunenthal.c
om>, Gillen, Dr. Clemens <Clemens.Gillen at grunenthal.de> writes
>we are using the Advantage polymerase to amplify genes via RT-PCR and we
>face the problems of various mutations. Do you know of certain experimental
>conditions or better polymerases to overcome this problem?
Using Pfu would reduce the mutations from PCR but you will still be left
with mutations from the RT and the RT's are poor in that regard. You are
also going to be limited to shorter fragments with Pfu but PfuTurbo may
overcome that limitation.
All you can really do is use the best fidelity enzyme and most
importantly reduce the amount of amplification you need in the PCR side
i.e. less cycles with more template.
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....
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