Degenerate PCR

Warren Gallin wgallin at gpu.srv.ualberta.ca
Mon Mar 14 13:49:13 EST 1994


In Article <2m24e5$ps7 at news.u.washington.edu>, findley at u.washington.edu
(Seth Findley) wrote:
>Does anybody out there have experience with doing PCR with highly 
>degenerate oligo pairs?
>I was thinking degeneracy on the order of 128 or 256 complexity.
>
>(I have a number of "A or C or G or T" positions when I reverse 
>translate a segment of a protein I am looking for homologs of)
>
>Is this commonly problematic?
>Does one have to add much more of the primer to make up for the complexity,
>or do you just go a few more cycles?
>
>Any tips greatly appreciated from people who have had success with this.

   We've been doing this, and have been successful in pulling out the clones
we want, in particular ion channels from jellyfish.  So have a number of
other labs.  The short answer is, it works, but with problems.  The good
news is that you don't have to use any more primer than a normal PCR, nor do
you have to run more cycles; because you are using degenerate primers, once
you start priming off PCR product rather than template, there are always
several primers that only differe by one base from the PCR product template.
You do need to optimize the buffer and the annealing temperature.  Both of
these factors haved turned out to be critical in our hands, and totally
unpredictable.
   The bad news is that you will obtain lots of bands that are not what you
want; this isn't too surprising, since the degeneracy you are introducing is
decreasing the specificity.  You may have to sequence a lot of PCR products
to get the one that you are looking for, and that is probably the fastest
and most rigorous way to check your products.  There are a number of
chapters in handbooks about this approach.  We found that once we had basic
PCR down, it was just a matter of trying a lot of conditions and grinding
through the sequencing.
   Also, don't rely on just one pair of primers; you might get lucky, but
the incremental effort of running all of your optimizing reactions for a
number of pairs of primers is not very great, and it certainly is a lot
easier than going through the whole grind over and over again for each new
primer pair that you decide to use.
Warren Gallin,
Department of Zoology, University of Alberta
wgallin at gpu.srv.ualberta.ca



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