PCR with degenerate primer

Wolfgang Schechinger wgschech at med.uni-tuebingen.de
Thu Feb 5 15:56:08 EST 1998


If your cDNA is of eukaryotic origin and there is the possibility 
that there's another gene coding for a protein downstream, there 
probably is a kozak consensus element around the next start-ATG.

You'll have to deal with a sequence like this: one GCCGCCATGG. 
Sometimes they differ slightly, but at least the Gs should be 
conserved as far as I know. You could synthesize a set of degenerate 
primers using this knowledege, of course for the opposite strand. I 
would recommend reading Mrs Kozak's paper on this issue before 
constructing the primers. You might find it in the net asking e.g. 
HotBot using the words "kozak" and "consensus".

Another approach could be cloning your cDNA fragments into any 
plasmid and doing PCR with your N-terminal primer and another primer 
that corresponds to a piece of sequence from your plasmid; often 
this can be the T7 or Sp6 promotor (if your plasmid has one of them 
or both).

Good luck!

Wolfgang



> Hi:
> 
> I am trying to fish out the cDNA of which 20aa N-terminal seq is
> known. I have linkered cDNA as template. So in my PCR, one primer
> anneals to the linker, the other one is a set of nested degenerate
> primers designed based on aa seq. But I couldn't get any specific
> bands out of my PCR. Anybody has done similar experiments with
> success?  Please give me some briliant suggestions. -- qin ling
> 
> 
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Wolfgang Schechinger         
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
http://www.medizin.uni-tuebingen.de/~wgschech/research.htm
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