Taqman qpcr sensitivity - (Jan/30/2008 )
I am looking to design a qPCR assay using a taqman probe....
firstly - the PCR is a touchdown, due to complicated regions, will this have an effect on the binding of the taqman probe?
seondly- will tis be sensitive enough to detect a gene duplication?
the Tm of the taqman probe should be clearly above the Tm of the primers (~10°C). the probe anneals during the cooling from the 95°C denaturation step to the annealing step, so in my opinion different annealing temperatures (in a normal range) should not affect binding of the probe.
a copy-number difference of 2-fold should be easily detected:
"Real-time reproducibility. Amplification of the RNase P gene from human
genomic DNA. Samples were run in replicates of 144 using the fluorogenic 5’
nuclease assay. The system can distinguish between two samples containing
5,000 and 10,000 template copies with a confidence level of 99.7%." (ABI, 7900HT)
i hope this is what you wanted to know.