degenerate oligos: I or everything?

Denise Melanson dmmelans at unity.ncsu.edu
Sun Dec 29 12:49:28 EST 1996


It probably depends on what the peptide sequence looks like in the area you
want to create primers specific to.  The more degeneracy you have the more
likely to have non-specific amplifications, however, if you create a set of
degenerate primers, you can calculate what percent of the pool will be 100%
homologous, and then set your annealing temperature to this. I would try to
find an amino acid sequence which contains the least amount of degeneracy
with the longest oligos possible and go for the degeneracy from there. I
would not use inosine, as you will amplify MANY non-specific products:

for example:

		AA sequence:   xxx.....FYHQNK......xxx

		oligo sequence:	         UUu/cUAu/cCAu/cCAa/gAAu/cAAa/g

This yields a set of degenerate 18mer oligos with 64 different sequences,
of which only 1 is exactly right. Therefore in any given oligo you could
have a maximum of 33% mismatch. So you could either set your annealing
temperature to allow for 33% mismatch or set it for no mismatch. 

In addition, I would stay away from amino acid sequences which include
arginine or serine, both of which have DNA sequences as follows

	u/aCa/g/u/c, which in itself is an 8% degeneracy

I hope this helped and didn't add more confusion to the problem

jstrassw at OPAL.TUFTS.EDU wrote in article
<Pine.PMDF.3.95.961228161453.1049B-100000 at OPAL.TUFTS.EDU>...
> Hello!
> I would like to know what the preferrred oligo strategy is for reverse
> genetics.  I have a peptide sequence and would like to PCR out the
> corresponding cDNA.  So, I have two choices: either put inosine in the
3rd
> position or include all 4 bases at the redundant position.  Any thoughts?
> Thanks,
> John
> 
> 
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