Methylation-sensitive High Resolution Melting for the Rapid Analysis of DNA Methylation
Thomas Mikeska and Alexander Dobrovic
from: Epigenetics: A Reference Manual (Edited by: Jeffrey M. Craig and Nicholas C. Wong). Caister Academic Press, U.K. (2011)
Methylation-sensitive high resolution melting (MS-HRM) is an inexpensive and robust closed tube screening methodology that enables rapid analysis of locus specific DNA methylation for multiple samples. MS-HRM is based on the differential melting behaviour of PCR amplification products derived from methylated and unmethylated templates after bisulfite treatment. The melting profiles of an unknown sample are compared to the melting profiles of standards with known DNA methylation levels. MS-HRM has the advantage of allowing ready distinction between homogenous and heterogeneous DNA methylation. Estimation of DNA methylation to quite low levels in a semi-quantitative manner is possible for homogeneously methylated templates where all the CpG sites are methylated. However for heterogeneous methylation, the formation of multiple heteroduplexes makes quantitation difficult. Digital MS-HRM utilises limiting dilution to amplify single templates enabling DNA methylation analysis at a single allele resolution and enables the quantitative analysis of both homogenous and heterogeneous methylation. The PCR products either generated by MS-HRM or digital MS-HRM can subsequently undergo Sanger DNA sequencing or pyrosequencing to investigate DNA methylation at individual CpG positions read more ...