TaqMan probes specificity - I need to amplify and quantify specifically homologous genes (Jun/18/2008 )

Hi,
First, sorry for my english because I'm french.
I would like to know, if a Taqman MGB probe, which presents a mismatch (1/15 i.e) can be detected during a RQ-PCR.
Indeed, I want to amplify some genes with 85-95% of homology. I have already designed specific primers which seem to be OK.
My positive samples PCR products have the expected sequence, but I want to make sure that possible unspecific products can't be cted during the reaction with other samples.
Thanks.

According to Applied Biosystems, the Taqman MGB probes won't work with a 1 bp mismatch. I had a bit of a discussion with them about this last year because we are studying the expression of a family of genes that also have a large amount of homology. I was worried about false positives, but if your primers are good and there is a 1 bp mismatch in the Taqman probe you should be fine. Remember that MGB probes are what people use to detect SNPs, so I guess they really are completely sensitive to a one bp difference.

Ginger

Also, remember that for a Taqman probe to give a fluorescent signal, it has to be degraded by a DNA polymerase. If your primers are specific, even if the Taqman probes could anneal to to a non-specific target, they won't generate a fluorescent signal because they'd need to have one of your primers binding nearby.

Ginger

Thanks.
I'm still doubtful of the fact that my probe couldn't anneal to unspecified PCR products. Indeed, there are 14 homologous genes -85-95%- and I'm not absolutely sure of the specificity of my primers.