Perplexing result with site-directed mutagensis

Donald L. StGermain Donald.L.StGermain at dartmouth.edu
Fri Nov 18 23:02:56 EST 1994


Hello:  We would appreciate any insight you folks might have into a
problem we have recently run into with our site-directed mutagenesis
efforts.  We've been using Promega's Alterd-Sites kit for several
months to introduce single point mutations into several cDNAs with
excellent results.  Lately however, we have had three instances
involving three different mutational oligos and two different cDNA
templates where we have obtained incorrect base changes at the site of
mutation.  For example, in one case we wanted to mutate ...ctgAatt...
to ...ctgTatt..., but instead obtained  ...ctgGatt... in 4 of the 5
colonies that we picked and sequenced (the fifth colony had the
wild-type sequence).  I thought that possibly there was an error in the
sequence of the mutational oligo except that we got similar types of
errors with two other oligos as noted.  No other base changes in the
sequenced clones were observed.  Promega's tech support did not povide
us with much insight into this problem.  Has anyone had a similar
experience?  Do you know the explanation and is there a workaround so
that we can come up with the correct mutations??  Any help would be
much appreciated.

Don St. Germain



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