Abstract
As DNA methylation analysis enters the mainstream of biomedical research, there is increasing interest in methodologies that can be used for rapid analysis of a wide variety of biological and clinical samples. The methods that have been commonly used for methylation analysis are usually time-consuming and require multiple steps. The methylation-sensitive high-resolution melting (MS-HRM) assay is an in-tube assay which was developed to assess promoter methylation in sodium bisulphite-modified DNA and is particularly useful for assessing methylation in short fragments of DNA derived from formalin-fixed paraffin-embedded biopsies.
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References
Rice, J.C., Massey-Brown, K.S., and Futscher, B.W. (1998) Aberrant methylation of the BRCA1 CpG island promoter is associated with decreased BRCA1 mRNA in sporadic breast cancer cells. Oncogene 17, 1807–12.
Herman, J.G., Latif, F., Weng, Y., Lerman, M.I., Zbar, B., Liu, S., Samid, D., Duan, D.S., Gnarra, J.R., Linehan, W.M., et al. (1994) Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. Proc Natl Acad Sci U S A 91, 9700–4.
Kristensen, L.S. and Hansen, L.L. (2009) PCR-based methods for detecting single-locus DNA methylation biomarkers in cancer diagnostics, prognostics, and response to treatment. Clin Chem 55, 1471–83.
Dobrovic, A. (2005) Methods for Analysis of DNA Methylation, in Molecular Diagnostics for the Clinical Laboratorian (Coleman, W. B., and Tsongalis, G. J., Eds.) 2 ed., Humana Press, Totowa, NJ.
Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L., and Paul, C.L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci U S A 89, 1827–31.
Herman, J.G., Graff, J.R., Myohanen, S., Nelkin, B.D., and Baylin, S.B. (1996) Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci U S A 93, 9821–6.
Eads, C.A., Danenberg, K.D., Kawakami, K., Saltz, L.B., Blake, C., Shibata, D., Danenberg, P.V., and Laird, P.W. (2000) MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res 28, E32.
Trinh, B.N., Long, T.I., and Laird, P.W. (2001) DNA methylation analysis by MethyLight technology. Methods 25, 456–62.
Worm, J., Aggerholm, A., and Guldberg, P. (2001) In-tube DNA methylation profiling by fluorescence melting curve analysis. Clin Chem 47, 1183–9.
Cottrell, S.E., Distler, J., Goodman, N.S., Mooney, S.H., Kluth, A., Olek, A., Schwope, I., Tetzner, R., Ziebarth, H., and Berlin, K. (2004) A real-time PCR assay for DNA-methylation using methylation-specific blockers. Nucleic Acids Res 32, e10.
Wojdacz, T.K. and Dobrovic, A. (2007) Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Nucleic Acids Res 35, e41.
Wittwer, C.T., Reed, G.H., Gundry, C.N., Vandersteen, J.G., and Pryor, R.J. (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Clin Chem 49, 853–60.
Snell, C., Krypuy, M., Wong, E.M., Loughrey, M.B., and Dobrovic, A. (2008) BRCA1 promoter methylation in peripheral blood DNA of mutation negative familial breast cancer patients with a BRCA1 tumour phenotype. Breast Cancer Res 10, R12.
Candiloro, I.L., Mikeska, T., Hokland, P., and Dobrovic, A. (2008) Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene. Epigenetics Chromatin 1, 7.
Wojdacz, T.K. and Hansen, L.L. (2006) Reversal of PCR bias for improved sensitivity of the DNA methylation melting curve assay. Biotechniques 41, 274–8.
Wojdacz, T.K., Hansen, L.L., and Dobrovic, A. (2008) A new approach to primer design for the control of PCR bias in methylation studies. BMC Res Notes 1, 54.
Warnecke, P.M., Stirzaker, C., Song, J., Grunau, C., Melki, J.R., and Clark, S.J. (2002) Identification and resolution of artifacts in bisulfite sequencing. Methods 27, 101–7.
Acknowledgements
EMW was supported by the Dora Lush Biomedical Postgraduate Scholarship from the National Health and Medical Research Council of Australia. Development and continued studies of MS-HRM were made possible by grants to AD from the National Health and Medical Research Council of Australia, the US Department of Defense Breast Cancer Research Program under award number W81XWH-06-1-0670, the Cancer Council of Victoria and the National Breast Cancer Foundation of Australia. Views and opinions of, and endorsements by the authors do not reflect those of the US Army or the Department of Defense.
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Wong, E.M., Dobrovic, A. (2011). Assessing Gene-Specific Methylation Using HRM-Based Analysis. In: Park, D. (eds) PCR Protocols. Methods in Molecular Biology, vol 687. Humana Press. https://doi.org/10.1007/978-1-60761-944-4_14
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DOI: https://doi.org/10.1007/978-1-60761-944-4_14
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