degenerate oligos: I or everything?

parakeet s535290 at
Sun Dec 29 16:35:09 EST 1996

> 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

I personally only use Inosines if the stretch of AA that I am reverse
translating is going to be very degenerate (>256 fold degenerate, although
I have been able to use primers that are *a lot* more degenerate than
this, conventional wisdom is that you try and keep the primer as
non-degenerate as possible). 

In order to get a nice 20mer, you need a heptapeptide with no more than 4
or 5 fourfold degenerate residues. If you have a couple of two fold
degenerate sites, you can still be around 512-fold degenerate *without*
using inosines and I have been able to go down to as much as 1024-fold
degenerate with good results. You can also split a primer into two
different pools during synthesis and during PCR in order cut the
degeneracy in half. The thing about Inosines is that while they mostly
don't hinder pairing between the primer and the target, they also don't
contribute to base pairing. So a 20mer with two inosines behaves like an
18mer in terms of stability. If you stick an inosine at every 3rd position
that is 4 fold degenerate you may have to really lengthen your primer. FOr
instance if every amino acid in your sequence was 4-fold degenerate, you'd
have to design a 27-mer with 9 inosines in order for it to act as an
18mer. Granted, this is an extreme case, but still you can see the problem
with using inosines. Inosines lead to the need for longer primers
in order to compensate for the lower melting temperature and/or a drop in
annealing temperature. If you lengthen the primer, you may run into the
problem where you start encountering lots of 6-fold degenerate residues,
which you should try to avoid...especially Serine, which is degenerate at
every codon position. 

The use of a couple of Inosines is not bad, but use it only of you really
need it. Remember, Inosine is more expensive than the other nucleotides :)
and you won't be using it much if you make your own primers. You can
always make 4-fold sites inosines as long as you don't have too many of
them, and make 2-fold sites redundant. But my best advice to you is to
keep the primers short and sweet and inosine-free. Try touchdown PCR
going down to the lowest melting temperature of any of the primers in your
mixture. This higher stringency will lead to less chasing of useless

The most critical issue is in finding good potential sites for primer
design. It's a good idea to scan along the peptide sequence and look for
clusters of 6 amino acids that don't contain 6-fold degenerate residues.
As long as you avoid these, you can be guaranteed that your degenerate
20mer won't be more than 512-fold degenerate, and this is generally
alright for most applications.

Another good tip is to have a forward primer with its 5' nucleotide at a
1st codon position and its 3' nucleotide at 2nd codon position.  The
reverse primer should have a 5' nucleotide at a second position, and a 3'
nucleotide at either a second or 1st codon position. Confusing ? drop me a
line and I can try to confuse you less :)  

Oh, one last tip...find a codon usage table for your'll be
surprised just how much this can help you in designing a degenerate
primer, especially if your peptide sequence is rich in highly degenerate
residues. I have probably given you more here than you really will have to
use, I'm just mentioning some issues just in case you have a particularly
nasty sequence in your hands. Hope everything turns out !


PS. Any programmers out there who would like to help me design a program
for degenerate primer design in evolutionary-type molecular biology ?
please contact me and we can collaborate.

Ed Taboada
Molecular Genetics
University of Ottawa
Ottawa, Canada

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