Mutagenesis using straight PCR?

[Donald J Roufa]

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
>is this:
>	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!

>Tao Tao

	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
-- 
    Don Roufa        DROUFA at MATT.KSU.KSU.EDU
Division of Biology  Kansas State Univ.   Manhattan, KS  66506