AT to GC mutations

delisior at rnisd0.DNET.roche.com delisior at rnisd0.DNET.roche.com
Tue Apr 13 19:11:17 EST 1993


margolin at darkwing.uoregon.edu (Brian Margolin) writes:

>I would like to create a large number of random AT to GC mutations in  a
specific regin in vitro.  I have been able to find a couple of chemical
methods that would allow me to create GC to AT mutations such as
bisulfite or hydroxylamine modification, but have not
been able to come up with any chemical modifications that would
specifically create AT to GC mutations.  Does anybody out there have any
suggestions?

The other idea that I had for doing this was to try polymerizing across
the region of interest with one nucleotide absent in the mix and one in
great excess.  The idea is that the polymerase would misincorporate
the nucleotide in excess if the correct nucleotide was not there.  In
my case either A or T could be absent and either G or C could be in
excess.  I don't know whether or not I can expect to find a polymerase
that would do this or if the polymerase would just stall when the
correct base was missing.  Does anybody know what I can expect?
Furthermore can anybody recommend a particular polymerase that might be
usefull to me?

Thanks in advance,
Brian Margolin

<
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Brian:

I've been using the method described in Yeast vol.8: 79-82(1992) by Denise
Muhlrad, Rosalyn Hunter, and Roy Parker with great success. If you are not
working in yeast then see the article in TECHNIQUE-A JOURNAL OF METHODS IN CELL
AND MOLECULAR BIOLOGY, Vol.1, No.1(August), 1989: pp 11-15.

Both articles describe TARGETED RANDOM MUTAGENESIS USING PCR. The reaction
is essentially PCR with 10% DMSO AND MnCl2. For you purposes you can adjust
the concentrations of the individual dNTPs to increase the probability of
the polymerase misincorporating the prefered base.

If your library cannot retrieve these articles for you then e-mail me your
FAX number and I'll send you a copy.

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Robert DeLisio                  |  Internet:
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