Degenerate PCR...a complication..

douglas l feinstein dlfeins at cumc.cornell.edu
Tue Feb 8 18:59:19 EST 1994


Subject: Degenerate PCR...a complication..
From: Alejandro Abuin, aabuin at bcm.tmc.edu
Date: 8 Feb 1994 15:42:01 GMT
In article <2j8bs9$pvg at gazette.bcm.tmc.edu> Alejandro Abuin,
aabuin at bcm.tmc.edu writes:
>   Well, I want to do degenerate oligo PCR from a very conserved region
of
>a particular cDNA in the mouse. My problem is that the region between my
>hypothetical degenerate primers has been conserved IN SIZE from E.coli to
>humans...and there probably are several genes belonging to this family in
>the mouse...(one so far cloned).  
>   
>   If I do degenerate PCR across this region, it seems like all fragments
>will be of the same size, no matter what family member I'm amplifying. 
>The SIZE conservation is 100% from E.coli to humans, including yeast and
>the known mouse cDNA.  How would you go about separating different
species
>from a pool of PCR products of the same size?
>
>   Any ideas would be greatly appreciated...
>
>   Alex

An interesting problem. You definitely need to incorporate some type of
specificity into your screening and/or PCRing to avoid the other members
of the family. How large are your conserved regions? You would only have
to design a second, internal set of oligo(s) which extend 3 or 4 bases
(3' direction) to obtain some specificity.
	If they are large enough, you could make probes against an internal
region, label them, and screen all your cloned pcr fragments (plus/minus
screening perhaps).

dlf
>
Doug Feinstein																	 |  Voice:   212 570-2900
Dept Neurobiology															|	Fax:      212 988-3672
Cornell University Medical College    |  E-mail: dlfeins at cumc.cornell.edu
We're not the best at what we do, but we're the only ones who do it...



More information about the Methods mailing list