The following message was sent to me as a reply to my PCR-KO question. Steve
asked that I forward it for posting:
I have used this technique several times and have had some success.
In my hands, I get about 2% of my transformants integrating at the correct
locus, as opposed to gene conversions of the marker locus, using oligos
with 40-bp extensions that match the target gene. My success has been with
scoring the transformants. For one case, I created a deletion of my gene
of interest that was marked with HIS3, that also eliminated a URA3
disruption. Therefore, I replica printed my HIS(+) transformants onto an
FOA plate after several days to determine which colonies were integrations
at the desired locus. I pick both FOA(s) and FOA(r) colonies for a
followup PCR analysis to show that the pattern of FOA resistance is
mirrored by the PCR fragment length pattern.
This technique has been used extensively by the Boeke lab, who
discussed the tecnique at the Mid-Atlantic Yeast Genetics Meeting this
Spring. In that lab, they have used a strain that carries a complete
deletion of the HIS3 locus, and uses primers that are internal to the
deletion endpoints, thereby totally eliminating the HIS3 target site.
The origin of this technique was an abstract in Yeast, the exact
citation of which unfortunately escapes me right now, but Jef Boeke has
said that he has been in contact with the author, and has encouraged him to
write up the technique as a short paper.
Steven A. Haney
Dept. of Molecular Biology
shaney at watson.princeton.edu