differential display kitsREGISTER

kitchingman at mbcf.stjude.org kitchingman at mbcf.stjude.org
Sat Jun 10 11:18:55 EST 1995


    Those of you who wish to get into differential display, or have been
frustrated by its tendency towards irreproducibility, might want to check out
Nucl. Acids Res., Dec. 25, 1994 issue.  There is an article by David Schatz
describing a modification of the representational difference analysis technique
for use with cDNA.  Basically, you make dscDNA, restrict with your four base
cutter of choice, ligate on adapters, PCR both a tester and driver, and then
subtract.  You have to go through 3-4 rounds of subtraction, but this takes
about 3-4 weeks and what you end up with is a few bands on an agarose gel that
can be directly cloned.  The method seems to be highly reproducible (I'm aware
of at least one other lab that has had no problems generating clones that are
differentially expressed in the tester and driver tissues), and, in theory,
allows one to examine a much higher percentage of the expressed repertoire at
one time than with differential display (with probably three different enzymes
you should be able to examine the majority of transcripts).  I know that many labs have succeeded
in identifying differentially expressed mRNAs with DD, but I also know that the
denominator is much larger.

Geoff Kitchingman
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