Dear Shyam,

I extensively worked in the past with methylation-specific PCR. The program I used to design primers for methylation-specific PCR or bisulfite sequencing is the following one:

https://www.urogene.org/methprimer/

In terms of positive and negative controls (fully methylated and unmethylated DNA), I used the ones from Zymoresearch, and they worked very well. I am sure that also other companies sell similar products nowadays.

Anyway, I think you should move to a quantitative method to detect promoter methylation, as methylation-specific PCR gives a lot of false positives. Thus, I would suggest either COBRA (PubMed ID # 9171110 ) or Methylight (PubMed ID # 10734209).

For the promoter sequences I used the Eukaryotic Promoter Database tool:

https://epd.epfl.ch//index.php

I hope this helps you! ;-)

My best regards,

Diego

For methylation specific primer design you can use Meth primer software where you put your gene promoter sequence derived from promoter retrieve software depending on your gene of interest.