I am looking for a precise method to determine the level of DNA methylation at a given locus. I am working on material from cows and I wanted to adopt the Methylation-Sensitive High-Resolution Melting (MS-HRM) method for such analysis. However, it is troublesome to obtain 100% methylated DNA for preparing curve points. We are trying to obtain such DNA by amplification of genomic DNA using nucleotides containing a mixture of the methylated cytosine. Unfortunately, so far we cannot obtain a single-strand DNA but we have a lot of unspecific bands. Therefore, I am looking for an alternative method that can be substituted for MS-HRM, which has the same good accuracy. We can't use pyrosequencing because we don't have access to a device, and it would take too long to send samples and analyze them commercially. Someone recommended a method MethyLight (real-time PCR with probes) to determine DNA methylation in a given locus. However, I haven't dealt with it before and I don't know if it would be quite accurate. Maybe one of you knows another method that I can apply to such analyses. This would be very helpful to me and I could speed up the research of my grant.
In my opinion, the ms-HRM technique offers greater accuracy and convenience compared to other methods such as methyl-light, and I personally utilize this technique in my research.
For the use of fully methylated and fully unmethylated DNA as controls, you can commercially purchase these DNAs for human and mouse studies. However, it is also possible to produce fully methylated and fully unmethylated DNA for any organism. Fully methylated DNA can be obtained by treating the organism's DNA with the CpG Methyltransferase enzyme (M.SssI), and fully unmethylated DNA can be obtained using Whole Genome Amplification (WGA) kits.
Additionally, for primer design, you can refer to the article "Methylation-sensitive high-resolution melting" by Dr. Tomasz K Wojdacz and colleagues, published in Nature Protocols.
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