Hi,

Here is a paper that might give you some info about primer design for methylation analysis... Even if this one is described for methylation independant PCR you might find some useful tips.

The first question is :

- what are your methylated and unmethylated controls? 

A common method to obtain such control is to use PBMC DNA and whole genome amplify it to get the 0% and treat it with MssI to get the 100%. However, from my experience I can tell you that you never reach complete 0 or 100% of CH3 this way. So if you perform MSP with a relatively high number of cycles you will always have amplification of both templates.

- To increase the specificity of your MSP primers:

Make sure to have sufficient number of CpG sites in particular in the 3' of the primer sequence (2 or 3 CpGs in the 2nd half of the primer is usually good). You can play with the annealing temperature as you mentioned.

- Don't go too high in terms of Cycles...

As I mentioned, too many cycle will amplify everything.

Good luck

Article A new approach to primer design for the control of PCR bias ...

Thank you.

I am using methylated and unmethylated genomic controls from applied biosystems, but my primers have only one CpG site in their 2nd half. Maybe I should consider deigning new primers.

If your primers have only one CpG site, you should consider designing new primers at a region containing more CpG sites. I think 3 CpG sites need to be present to make a good discrimination between methylated and unmethylated DNA