Problem with Site-Directed Mutagenesis

Tue May 2 20:30:59 EST 1995

Todd DeZwaan wrote:

> While creating a series of site-directed mutations in a specific region of
> my gene of interest I have generated a mutant which gives two
> different phenotypes; lethal and temperature sensitive.  .... 

> My question is this, is it possible that I have a sub-population of
> mutagenic oligonucleotides which would account for the presence of
> two different mutant constructs in a single prep (i.e., what is the
> possibility that a base was misincorporated in some of my mutagenic
> oligonucleotides during synthesis) ?  

Although anything is possible, as long as the synthesizer was set-up
and programed correctly, misincorporated oligos during synthesis are
pretty unlikely.  However, depending on the oligo structure, it is possible
to produce an occasional "anomalous" mutation (i.e., 2-3 base inserts or
deletions), especially if there is a short repeat in the oligo.  Also,
sometimes, if more than one base is introduced by the oligo, not all the
mutants will have incorporated all the mutations.

However, it sounds as though you do not know where the mutation is
located.  Have you sequenced through the region to which the mutagenic
oligo bound?  It is possible there is a second "spontaneous"
mutation outside this region.  Although T4-polymerase has a low
mismatch error rate (i.e., <1x 10-6, Kunkel et al. 1984. J. Biol Chem
259:1539-1545), a mutation may have been introduced during the
synthesis and ligation step.  More likely, however, a second mutation
may occured in the E.coli BMH71-18 mutS which are mismatch-repair
negative and Rec A positive.  Although uncommon, spontaneous
mutations can occur during any mutagenesis.

Try retransforming your plasmid(s) into DH5alpha (or equivalent strain),
and grow it up in a well isolated colony.  Sequence to confirm the
mutation you introduced, and redo your assay.

Paul Diehl, Ph.D.
CLONTECH Technical Service
800-662-2566 x141
pauld at  

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