oligo mutations

[JMILLER%VXBIO.SPAN at STAR.STANFORD.EDU]

 In our lab, we have made >250 mutagenic oligos in the last year. One
can get dowstream mutants, usually deletions, if (1)the oligo sequence
has homology to something downstream, and (2)it is a fairly long oligo
>30 bases. Short oligos with one or two mismatches should never cause
any trouble. Long oligos, especially if they have a repetitive sequence
such as GLY-ALA-GLY-ALA frequently cause secondary insertions/deletions
both at the mutagenic site, and downstream. For some very large replacements
we are doing (ie., 10 amino acids) it was necessary to screen the mutants
for function prior to sequencing. This gets rid of all the deletion/insertion
mutants. Point mutations downstream don't seem to happen.

-Peter Markiewicz