Help: make primers from amino acid sequence

Rosemary T. Hoffman rth at netdoor.com
Mon Jul 15 23:00:36 EST 1996


This isn't exactly the answer you wanted but this is how to proceed.

1) Find a codon usage table for your organism.  If none exists, look at
sequences of known proteins and determine which codons are preferred.

2) Select codons based on this usage.  Try to find regions that contain
several amino acids which are encoded by only one or two different codons.
Design a minimally degenerate primer this way.

for example: if your amino acid sequence was X-Y-Z and X was encoded by
either ACT or ACA in this organism; Y by CCG, CGG, or GGG; and Z by TTT

your primer would be  AC(T/A)(C/G)(C/G)GTTT

When the primer is synthesized, a mixture of 50% T and 50% A is used for
the 3rd position, for example, and 50% C and 50% G for the 4th, etc.  You
will obtain a mixture of oligos starting with 
5'- ACTC.................... -3'
5'- ACTG....
5'- ACAC.....
5'- ACAG.....
etc.

The idea is that as you increase stringency, the one closest to the native
sequence will hybridize while those that are poorer matches won't.

Not a perfect solution, but sometimes the best solution available.

Rosemary




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