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FW: PCR gene disruptions

Neigeborn, Lenore neigeborn at OCELOT.RUTGERS.EDU
Mon Nov 7 14:15:43 EST 1994


The following message was sent to me as a reply to my PCR-KO question.  Steve
asked that I forward it for posting:
______________________________________________________________________________
Hello all,

	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.  
	

Cheers,

Steven A. Haney
Broach Lab
Dept. of Molecular Biology
Princeton Univ.

shaney at watson.princeton.edu









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