semi-specific PCR

Dom Spinella dspinella at chugaibio.com
Wed Feb 25 15:25:50 EST 1998


Bryan Ford writes (in response to my earlier posting)
> > Undoubtedly, your smear contains your gene of interest in addition to a lot of other
> > products. You could always try cloning the smear and probing the clones
> 
> Ho Ho, and it is always possible to tow a Mercedes-Benz with a team of
> horses! 
> 
> If you have a probe then you have, or can easily have, the sequence
> necessary to design specific PCR primer(s) to keep the "Mercedes" on the
> road.
> 
> Sincerely,
> Bryan L. Ford


Well Bryan, if you think about this more carefully instead of instantly
being a smart ass, you might come up with reasons why you might want to
tow this particular Mercedes with a horse.  For example, suppose our
friend was looking for a full-length gene but had only partial sequence
information (lacking the 3' end for example).  He could then use this
strategy to enrich for a population of longer clones that could be
subsequently probed (in fact, this is pretty much what is done in the 3'
RACE protocols.)  The again, perhaps the PCR "smear" could be cloned in
an expression vector to look for function upon transfection with what is
now a much smaller, enriched population than the starting cDNA library
(notice I also alluded to the possibility of a functional assay rather
than a nucelic acid probe).  Since neither of us knows the particular
experimental details in question, let's not rule out possibilites a
priori. And Ho Ho yourself...
--Dom Spinella



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