PCR with degenerate primer
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
> 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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