Degenerate Primers

JOHNSON_T johnson_t at am.pclsv3.sandoz.com
Thu Sep 23 11:33:31 EST 1993


I have found clones from lambda libraries 
using degenerate oligonucleotide primers 
using PCR.  It is not a straight-forward 
process. Here are some tips that helped me. 
First, the codon usage tables can be found 
in GCG for many different species.  There 
is also an updated table found in the 
back of Nucleic Acid Research (I think 
this is the right journal). If you look at 
a range of species from chikens to humans 
you will find very small differences in 
codon preference.  However, this changes 
when looking at lower species such as 
bacteria and fungi.  Moreover, this is only 
important if you are going to design 
guessmers and not totally degenerate 
oligos.

Second, I chose the largest amino acid 
sequence regardless of the total degeneracy 
of the resulting oligos.  I designed either 
single primers for the ends of this 
particular region or nested primers to 
reamplify the first PCR.  I believe it is 
essential to know the size of the expected 
PCR product because you will get other 
non-specific bands from degenerate priming. 
If you try to PCR the entire gene or 
regions between peptide sequences you will 
not know the size of the product and can 
only make a guess as to which band might be 
your clone.

Third, the primers need to be as long as 
possible regardless of the degeneracy 
produced.  I was using primers between 25 
and 30 bases.  When I subcloned the PCR 
band of expected molecular size I found 
several different clones.  Interestingly, 
they were all primed by different 
degenerate primers but all had the same 
correct intraprimer sequence.  I hope this 
helps you out!!

p.s. I tried to send this reply, however 
your address was not recognized. Therefore 
I am sending to the newsgroup hoping that 
you will find it there.
_________________________________________

Todd M. Johnson
Sandoz Pharmaceuticals
Johnson_T at AM.PCLSV3.SAN.COM
_________________________________________




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