Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation.
In this article, we show that high resolution melting analysis (HRM) is a sensitive and specific method for the detection of methylation. Methylated DNA and unmethylated DNA acquire different sequences after bisulphite treatment resulting in PCR products with markedly different melting profiles. We used PCR to amplify both methylated and unmethylated sequences and assessed HRM for the determination of the methylation status of the MGMT promoter region. Reconstruction experiments showed that MGMT methylation could be detected at levels as low as 0.1%. Moreover, MS-HRM allows for estimation of the methylation level by comparing the melting profiles of unknown PCR products to the melting profiles of PCR products derived from standards with a known unmethylated to methylated template ratio. We used MS-HRM for the analysis of eight cell lines of known methylation status and a panel of colorectal cancer specimens. The simplicity and high reproducibility of the MS-HRM protocol makes MS-HRM the method of choice for methylation assessment in many diagnostic and research applications.
Wojdacz
Tomasz K
TK
Molecular Pathology Research Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia. wojdacz@humgen.au.dk
Dobrovic
Alexander
A
D014408:Biomarkers, Tumor / Q000235:genetics*; D002460:Cell Line; D015179:Colorectal Neoplasms / Q000175:diagnosis; D004247:DNA / Q000737:chemistry; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics; D025202:Molecular Diagnostic Techniques; D009691:Nucleic Acid Denaturation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics; D013696:Temperature; D025521:Tumor Suppressor Proteins / Q000235:genetics*
C090121:BNIP3 protein, human; D014408:Biomarkers, Tumor; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D025521:Tumor Suppressor Proteins; D004247:DNA; D015254:DNA Modification Methylases; C515491:MGMT protein, human; D045643:DNA Repair Enzymes