Methylation-sensitive high-resolution melting.
The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown samples with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR amplification of bisulfite-modified DNA with primers designed to proportionally amplify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.
Wojdacz
Tomasz K
TK
Institute of Human Genetics, University of Aarhus, The Bartholin Building, Wilhelm Meyers Allé, Bygn. 1242, DK-8000 Aarhus C, Denmark. wojdacz@humgen.au.dk
Dobrovic
Alexander
A
Hansen
Lise Lotte
LL
D001482:Base Composition; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction
D004247:DNA