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Methods Mol Biol. 2018;1708:551-571. doi: 10.1007/978-1-4939-7481-8_28.

Methylation-Sensitive High Resolution Melting (MS-HRM).

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Institute of Biomedicine, Aarhus University, Bartholins Allé 6, Aarhus C, 8000, Denmark.
Institute of Biomedicine, Aarhus University, Bartholins Allé 6, Aarhus C, 8000, Denmark.


Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.


DNA methylation; Heterogeneous methylation; MS-HRM; PCR bias; Primer design

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