The number of cytosines (C) in the PCR product amplified from a bisulfite modified template depends on methylation status of cytosines within that template before bisulfite modification. In principle, after PCR amplification a C rich PCR product is obtained if the amplified locus was methylated (see A below) and a T rich PCR product if the locus was not methylated (see B below). These PCR products will display different melting profiles (see also: What is DNA melting?) when subjected to MS-HRM analyses. The PCR product amplified form non-methylated version of specific locus will have relatively low melting temperature and melt 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).
Methylation-Sensitive High-Resolution Melting in based on this principle and enables cost effective, fast and highly sensitive assessment of methylation status of a specific locus.
A: PCR product obtained from the methylated version of the locus: