I'm developing a MethyLight assay to verify a specific CpG site in a promoter region of my interest gene in rats. Unfortunately, I don't have a lot of choices to design the probe and primers, since I have a specific target. Besides that, after the treatment with bisulfite, the DNA lost the complexity because of the cytosines unmethylated turning in thymine, leading to a lack of cytosine in the sequence. Thus, the Tm of my primers and probes are low (45°C for primers and 55°C for probes). Is possible the qPCR still work with these parameters?
Hi. It is ok that you have a difference between probe and primers Tm and as a result Ta. But such low Ta leads to decreasing of reaction specifity. In general the system can work but be carefull with the specifity. For me, the first step is gradient (temperature - in two step PCR it is annealing-elongation step /oligos - from 100 to 1000 nM, including probe/magnesium - 1-5 mM, but more probably 1-2 mM). As a result you can manage your specifity and choose the right reaction conditions.
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