Nucleic Acids Research Advance Access published online on February 8, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm013
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Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation
1Molecular Pathology Research Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett Street, Melbourne, Victoria 8006, Australia, 2Institute of Human Genetics, University of Aarhus, The Bartholin Building, DK-8000 Aarhus C, Denmark and 3Department of Pathology, University of Melbourne, Parkville, Victoria 3010, Australia
*To whom correspondence should be addressed. Tel: +61 3 9656 1807; Fax: +61 3 9656 1460; Email: alexander.dobrovic{at}petermac.org Correspondence may also be addressed to Tomasz K.Wojdacz. Email: wojdacz{at}humgen.au.dk
Received October 29, 2006. Revised December 28, 2006. Accepted December 31, 2006.
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.
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