Perplexing result with site-directed mutagensis

Duncan Clark Duncan at genesys.demon.co.uk
Thu Nov 24 06:46:20 EST 1994


In article: <3ajtdg$5jf at dartvax.dartmouth.edu>  Donald.L.StGermain at dartmouth.edu (Donald L. StGermain) writes:
> 
> 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.


This may be daft but it has been known for phosphoramidites to be labelled 
incorrectly by the manufacturer or for the wrong one to be put on a 
DNA synthesizer hence incorrect oligo. Alternatively could your 
mutation in some way be lethal and the E.coli is making it less 
lethal?

Duncan
 
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