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qPCR detection for single nucleotide substitution - (Apr/28/2006 )

Greetings, I'm searching for some advice on qPCR detection for a single nucleotide substitution.

I want to modify a conventional PCR protocol which follows BGL/ii RFLP (single site digest), to a qPCR assay. The substitution involves either T or C as follows:

Template A ...tttcaattaaagaCcttttaggattttat...
Template B ...tttcaattaaagaTcttttaggattttat...

I need to be able to determine which template is present (or even both) in a given sample, as economical as possible, with the Biorad iCycler. I have looked into taqman with minor groove binding from ABI, and as I figure it would cost me $250 for Template A probe (6-FAM, 3'-MGB) and at least that much again for the Template B proble w/MGB (different dye=>$$$?).

Does anyone here have any suggestions for a cost saving method? Must it be a duplex setup?

Thanks for any input you have!!

--Brian

-OlsonB-

I'm not sure that qPCR can setect such subtle things easily, you could try dHPLC if there is one at your research insitute or just plain old sequencing is good, relatively cheap too, compared to qPCR.

-bob1-

QUOTE (OlsonB @ Apr 28 2006, 09:53 AM)
Greetings, I'm searching for some advice on qPCR detection for a single nucleotide substitution.

I want to modify a conventional PCR protocol which follows BGL/ii RFLP (single site digest), to a qPCR assay. The substitution involves either T or C as follows:

Template A ...tttcaattaaagaCcttttaggattttat...
Template B ...tttcaattaaagaTcttttaggattttat...

I need to be able to determine which template is present (or even both) in a given sample, as economical as possible, with the Biorad iCycler. I have looked into taqman with minor groove binding from ABI, and as I figure it would cost me $250 for Template A probe (6-FAM, 3'-MGB) and at least that much again for the Template B proble w/MGB (different dye=>$$$?).

Does anyone here have any suggestions for a cost saving method? Must it be a duplex setup?

Thanks for any input you have!!

--Brian


if you can manage to using a primer with C or T as 3' end of primer, or very near to the 3' end (3=<nt) of primer, such difference can be discriminated in SYBR green based qRT-PCR.

-rshi-

I was under the impression that molecular beacons could be used to discriminate single nucleotide differences in this sort of situation, but have no personal experience with using them.

-Serratia-

As rshi suggests, if you make a primer which has the uncertain base at the 3' end, then it will extend if there is a match, and not extend if there is a mismatch. As a control, you would want to do two reactions, one with each possible 3' end base primer and the identical template. This is a robust technique, in our experience.

-phage434-

Great thanks for responding! !! I'll try the 3' specificity trick.

-OlsonB-