yeast one hybrid system

John Brunstein john.brunstein at helsinki.fi
Tue Nov 10 09:36:09 EST 1998


	Generally, as you point out, a one-hybrid screen is used to find a library
gene encoding an unknown protein binding to a known DNA sequence, or just
the reverse of what you are trying to do. 
	A more direct approach which has been successful in at least a couple of
papers as I recall was to use your known protein to gelshift a collection
of oligo 'randomers' which have flanking defined sequences. You mix
randomers and your protein, localize protein on the gel, isolate it.. and
then PCR amplify the shifted randomers by way of their flanking defined
sequences. If I recall correctly this process was repeated for three
enrichment cycles before the amplifed product(s) were analyzed by
sequencing.
	You might find this faster and easier than trying to locate or make a
library in which the library sequences lie proximal to the minimal promoter
as required for a one-hybrid screen. One possible problem with that
approach, even if you could find such a library, would be that your binding
site could easily be too far from the minimal promotor of the reporter to
allow for efficient transactivation.

-- 
Dr. John Brunstein
Dept. of Virology
Haartman Institute, University of Helsinki
Helsinki, FINLAND

gurumurthy d s <gmurthy at TIFRVAX.TIFR.RES.IN> wrote in article
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> Dear netters
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>     I need some help about the yeast one hybrid system. 



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