A PCR problem
dspinella at chugaibio.com
Thu Dec 4 10:16:37 EST 1997
Olivier Cochet wrote:
> Hi everybody,
> Perhaps an abvious solution to this question ?
> I have a cDNA mixture containing the gene of interest (A) and an unwanted
> gene (B), highly homologous, excepted for an internal part. My 5' and 3'
> primers amplify A and B genes (300 bp). I want to get rid off gene B by
> designing an internal primer (anti sense) specific of B but degenerated at
> the 3' end in such a way that no amplification would be possible.
> So, in my PCR mixture, I will have 3 primers.
> Questions are:
> - Does the elongation, starting from the 3' outer primer, will be blocked
> by the internal primer annealed to gene B ? In other words, may this
> internal primer be displaced by the outer primer ?
> - Is there any way of labeling the 3' end of the internal primer to ensure
> zero amplification ? (other than degenracy)
> - Does anybody has a reference of such a protocol ?
> Please, mail me at: Olivier.cochet at curie.fr
> Any information would be welcome.
> Olivier Cochet - Institut Curie Olivier.Cochet
> Biotechnologie des anticorps @Kurie.fr
> 26 rue d'Ulm - 75248 Paris - France Repl. K by c
I don't think your idea will work. Your fear is correct: your central
(B-specific) primer will be displaced by Taq polymerase as it extends
the external primer -- this is the way the "Taq Man" quantitative assay
works. You can prevent extension of any primer by adding a 3' dideoxy
nucleotide, but again, it won't help in the application that you are
attempting as it will be displaced (even though it won't extend).
Is there a restriction enzyme site in the sequence unique to B such that
you can pre-digest your cDNA and digest the B template only? That would
prevent subsequent amplification of B in the mixture. Otherwise you'll
simply have to design a primer within the unique sequence of B to ensure
specificity of amplification.
Hope that helps. Cheers.
-- Dom Spinella
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