Methylation-Sensitive High-Resolution Melting (MS-HRM) is based on a PCR methylation detection method.
The number of cytosines (C) in a PCR product, amplified from a sodium bisulfite modified template, depends on the methylation status of cytosines in the un-modified template.
Sodium bisulfite preserves the methylated cytosines and modifies non-methylated cytosines into uracil, which subsequently is substituted with thymine during PCR.
Thus, if the locus is methylated, a C rich PCR product is obtained after PCR amplification(see A below), and a T rich PCR product if the locus was non-methylated(see B below).
These PCR products will display different melting profiles (see also:What is DNA melting?) when subjected to Methylation-Sensitive High-Resolution Melting (MS-HRM) analyses.
The PCR product amplified from the non-methylated version of a specific locus will have relatively low melting temperature and melts earlier in the temperature gradient(Figure 1 – red curve)than the PCR product amplified from the methylated version of the same locus, which will melt at relatively higher temperature(Figure 1 – blue curve).
In summary, MS-HRM is a PCR methylation detection method, dependant on bisulfite modification, and enables cost effective, fast, and highly sensitive assessment of locus specific methylation status.
A: PCR product obtained from the methylated version of the locus:
B: PCR product obtained from the non-methylated version of the locus: