PCR mutagenesis

Zophonias O. Jonsson zjons at vetbio.unizh.ch
Fri Feb 3 05:05:18 EST 1995


In article <9502011533.AA12490 at umailsrv1.UMD.EDU>,
Lynne_A_WHITEHEAD at UMAIL.UMD.EDU (lw75) wrote:

> Hi there!
> I am interested in finding a protocol for PCR mutagenesis that will
> incorporate random mismatches while replicating.  Any references would be
> appreciated!
> Thanks!
> Lynne Whitehead
> Microbiology Dept. - U. of Maryland

Here are some references to get you started

Yuhong Zhou et al., NAR Vol. 19 No. 21 (1991), p. 6052,  Random mutagenesis
of gene-sized DNA molecules by use of PCR with Taq DNA polymerase

Johan H. Spee et al., NAR Vol. 21 No. 3 (1993), p. 777-778,  Efficient
random mutagenesis method with adjustable mutation frequency by use of PCR
and dITP

Marcel F. Brink et al., J. Mol. Biol. 237 (1994), p 368-377, Specialized
ribosomes allow for the study of mutations on funtionally important regions
in 16S rRNA, withour affecting cell growth


  There is not very much to it.  Just read any PCR manual, the chapter on
how to minimise error rates.  Then do everything wrong!  Getting mutations
with Taq is no problem, but adjusting the error frequency so that you get
enough single pont mutations takes a while.  If you don't have an easy and
reliable assay for your protein (assuming that you want single aa
substitutions in some enzyme or cofactor) this will be quite a demanding
task.

Good luck

Zophonias


_____________________________________________________________________________
Zophonias O. Jonsson
University of Zurich-Irchel



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