I haven't used methprimer for quite sometime now as I tend to design primers by eye and microsoft word.
However, methprimer express from ThermoFisher is still a free download and worth downloading and using, the primers selected are close to what I pick manually and are likely to work.
Because you are amplifying from bisulfite converted genome, there is a lot of degneracy induced and the complexity of the genome is reduced, all C's become U's and therefore T's after PCR. So it is no surprise your primer can hit many locations, it is the primer combination that will give amplification by PCR that you are interested in, therefore you want to get a specific product of expected size. That would be your readout....
Thanks for your quick reply. Then I think I do not have to worry those matches. And Could I ask some more questions?
1. Based on your experience, what is the optimal product length? Is 200+bp OK?
2. If I have already have one target loci of the CpG, can I design a primer that only contain this CpG, and if there is difference then I could say that it is this loci that matters. For example, the DNA sequence below is the bisulfite modification DNA, the red is Forward and the Orange is Reverse, and the highlight yellow is the one that I interested. But I read an article and it suggests that more CpG is better in the product. But if so, how we can say which CpG that matter.
3. When the CpG can not be avoided in the primers, the methprimer software of the ThermoFisher will mismatch the C into R. Still take the example above: the Reverse sequence after bisulfite, contains 2 CpGs, and then the software design the Reverse primer like this: ACTTAACTCTCRACRATATCTACCTAA. How it works after the mismatch the C into R?
Thanks a lot!!!