Mutagenesis using straight PCR?
Donald J Roufa
droufa at matt.ksu.ksu.edu
Mon Apr 20 08:54:56 EST 1992
txt15 at po.CWRU.Edu (Tao Tao) writes:
>To PCR specialists on the net,
> I would like to know whether it is feasible to use standard PCR to
>random mutagenize a DNA fragment. Since Taq polymerase has not editing
>function, this should be possible.
> My concern is the low rate (around 10^-4?), which means mutants would
>be hard to get, especially in the case of short target size. So the question
> What parameters should I change in order to increase the error rate
>of Taq polymerase? Also, is there any reagents useful in inreasing this error
>rate (base derivatives, etc)?
> Thanks in advance for your help!
We have indeed used PCR to randomly mutagenize cloned DNAs
efficiently if we first pretreat the cloned DNAs with a standard
chemical mutagen, e.g. nitrous acid. Taq polymerase efficiently
replicates DNAs containing deaminated bases and results in random
transition mutations. For a more complete description of the
procedure we use, see: J.J. Diaz, D.D. Rhoads and D.J. Roufa.
PCR-Mediated Chemical Mutagenesis of Cloned Duplex DNAs.
BioTechniques 11:204-211 (1991).
Don Roufa DROUFA at MATT.KSU.KSU.EDU
Division of Biology Kansas State Univ. Manhattan, KS 66506
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