PCR primers

Dr. Duncan Clark duncan at genesys.demon.co.uk
Tue Mar 17 14:45:21 EST 1998


In article <6ek5et$nfa$1 at pulp.ucs.ualberta.ca>, Richard L
<lm11 at gpu.srv.ualberta.ca> writes
>Dear PCR experts,
>
>I want to clone a gene from some bacreria by using PCR. The only clue
>about primers is short-sequence concensus conserved in other bacteria. For
>example, a short sequence like this: 
>
>Seq1: ggc att cag ctc gct cct gcg
>       G   I   Q   L   A   P   A  
>Seq2: ggg att gtt tta gca cct gcg
>       G   I   V   L   A   P   A 
>Seq3: tgg att aac ttg ggg cca ata
>       W   I   N   L   G   P   I
>Seq4: tgg att aac ttg ggg cca ata
>       W   I   N   L   G   P   I
>          ***     ***     ***     
> 
>How can I get a 17-mer primer from this comparison?
>

By using the codon usage for your bug (if you don't know it go to
Genbank, find every gene you can for your bug and make up your own
table) you can makes a best guess for certain codons ie can you get away
with TTG for L rather than CTC and TTG. I would end your primer at the
3' end with the CC in the proline as that will be common to all. Don't
make one oligo too redundant. You are better off having a couple of
oligos with reduced redundancy. You cannot guarantee that L will be TTG.
Depending on your bug it could be TTA, TTG, CTA, CTC, CTG or CTT. You
could make one oligo using CTN in that position and another oligo with
TT(A/G) etc. This is where codon usage tables really help.

Good luck.

Duncan

-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
TEl/FAX 01252376288
http://www.dnamp.com
http://www.genesys.demon.co.uk



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