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Methylation-sensitive high-resolution melting

Nature Protocols volume 3, pages 19031908 (2008) | Download Citation

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Abstract

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.

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Acknowledgements

We thank the Lundbeck, Toyota and Harboe foundations (grants to T.K.W. and L.L.H.), the National Health and Medical Research Council of Australia and US Army Medical Research and Materiel Command (grants to A.D.) for the support of the research leading to this publication.

Author information

Affiliations

  1. Institute of Human Genetics, University of Aarhus, The Bartholin Building, Wilhelm Meyers Allé, Bygn. 1242, DK-8000 Aarhus C, Denmark.

    • Tomasz K Wojdacz
    •  & Lise Lotte Hansen
  2. Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Becket Street, Victoria 8006, Australia.

    • Tomasz K Wojdacz
    •  & Alexander Dobrovic

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Contributions

A.D. and L.L.H. contributed equally to this work.

Competing interests

The authors are co-inventors on patent applications on aspects of the MS-HRM methodology. The patents have been filed for by University of Aarhus and Peter MacCallum Cancer Centre.

Corresponding author

Correspondence to Tomasz K Wojdacz.

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DOI

https://doi.org/10.1038/nprot.2008.191

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