I've made many one-step gene replacements using sites from polylinkers and
I've never had a problem. I've had as many as 30 bases of nonhomology on the
ends. The explanation I've heard is that there is considerable exonuclease
activity nibbling at the ends of the linear fragment prior to the recombination
event, so that by the time rec. occurs the nonhomology regions are eliminated.
In a somewhat related mode, there is this new procedure for making gene
replacements that involves PCR-ing a selectable marker using oligos containing
homology flanking your locus on the 5' end and homology to the marker on the 3'
end (thus when PCR is complete you have a fragment with homology to your locus
on the ends and a selectable marker in the middle). Then you transform in this
fragment for a one-step replacement. This allows you to make any deletion you
choose (you're not a slave to your restriction sites).
We've tried this several times with zero success. We get transformants
but they are all conversions at the marker locus (we assume, they are not at
the desired locus). Has anyone had decent success with this? We've used
oligos with 50 bases of homology to our locus on either end. We have no
trouble with the PCR reaction, just with the gene replacement. We've tried
URA3 and LEU2.
Neigeborn at MBCL.rutgers.edu