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Methylation-sensitive high-resolution melting

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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Figure 1: The effect of the PCR annealing temperature on the sensitivity of the ATM MS-HRM assay.
Figure 2: Derivative curve-based analysis of HRM results of RARβ2 gene.
Figure 3: Normalized HRM curve-based estimation of methylation levels for GSTP1 gene.

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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.

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

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Correspondence to Tomasz K Wojdacz.

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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.

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Wojdacz, T., Dobrovic, A. & Hansen, L. Methylation-sensitive high-resolution melting. Nat Protoc 3, 1903–1908 (2008). https://doi.org/10.1038/nprot.2008.191

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