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Methylation-sensitive high-resolution melting

Nature Protocols volume 3, pages 1903–1908 (2008)Cite this article

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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References

  1. Bird, A.P. & Wolffe, A.P. Methylation-induced repression—belts, braces, and chromatin. Cell 99, 451–454 (1999).

    Article  CAS  PubMed  Google Scholar 

  2. Jones, P.A. & Laird, P.W. Cancer epigenetics comes of age. Nat. Genet. 21, 163–167 (1999).

    Article  CAS  PubMed  Google Scholar 

  3. Esteller, M., Corn, P.G., Baylin, S.B. & Herman, J.G. A gene hypermethylation profile of human cancer. Cancer Res. 61, 3225–3229 (2001).

    CAS  PubMed  Google Scholar 

  4. Costello, J.F. et al. Aberrant CpG-island methylation has non-random and tumour-type-specific patterns. Nat. Genet. 24, 132–138 (2000).

    Article  CAS  PubMed  Google Scholar 

  5. Palmisano, W.A. et al. Predicting lung cancer by detecting aberrant promoter methylation in sputum. Cancer Res. 60, 5954–5958 (2000).

    CAS  PubMed  Google Scholar 

  6. Wojdacz, T.K., Dobrovic, A. & Algar, E.M. Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting. Hum. Mutat. 29, 1255–1260 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Feinberg, A.P. Epigenetics at the epicenter of modern medicine. JAMA 299, 1345–1350 (2008).

    Article  CAS  PubMed  Google Scholar 

  8. Wojdacz, T.K. & Hansen, L.L. Techniques used in studies of age-related DNA methylation changes. Ann. N.Y. Acad. Sci. 1067, 479–487 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Dobrovic, A. Methods for Analyses of DNA Methylation. Molecular Diagnostics for the Clinical Laboratorian. (Humana Press, Totowa, NJ, 11, 2005).

    Google Scholar 

  10. Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D. & Baylin, S.B. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc. Natl. Acad. Sci. USA 93, 9821–9826 (1996).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Eads, C.A. et al. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 28, E32 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Frommer, M. et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl. Acad. Sci. USA 89, 1827–1831 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Clark, S.J., Harrison, J., Paul, C.L. & Frommer, M. High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22, 2990–2997 (1994).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Eads, C.A. & Laird, P.W. Combined bisulfite restriction analysis (COBRA). Methods Mol. Biol. 200, 71–85 (2002).

    CAS  PubMed  Google Scholar 

  15. Bianco, T., Hussey, D. & Dobrovic, A. Methylation-sensitive, single-strand conformation analysis (MS-SSCA): a rapid method to screen for and analyze methylation. Hum. Mutat. 14, 289–293 (1999).

    Article  CAS  PubMed  Google Scholar 

  16. Worm, J., Aggerholm, A. & Guldberg, P. In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin. Chem. 47, 1183–1189 (2001).

    CAS  PubMed  Google Scholar 

  17. Wojdacz, T.K. & Dobrovic, A. Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res. 35, e41 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

  18. Warnecke, P.M. et al. Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res. 25, 4422–4426 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wojdacz, T.K. & Hansen, L.L. Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques 41, 274, 276, 278 (2006).

    Article  CAS  PubMed  Google Scholar 

  20. Shen, L., Guo, Y., Chen, X., Ahmed, S. & Issa, J.P. Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. Biotechniques 42, 48, 50, 52 passim (2007).

    Article  CAS  PubMed  Google Scholar 

  21. Wittwer, C.T., Reed, G.H., Gundry, C.N., Vandersteen, J.G. & Pryor, R.J. High-resolution genotyping by amplicon melting analysis using LCGreen. Clin. Chem. 49, 853–860 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Wojdacz, T.K., Hansen, L.L. & Dobrovic, A. A new approach to primer design for the control of PCR bias in methylation studies. BMC Res. Notes 1, 54 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  23. Gudnason, H., Dufva, M., Bang, D.D. & Wolff, A. Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 35, e127 (2007).

    Article  PubMed  PubMed Central  Google Scholar 

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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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Authors and Affiliations

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

Authors
  1. Tomasz K Wojdacz
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  2. Alexander Dobrovic
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  3. Lise Lotte Hansen
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Contributions

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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