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Methylation-Sensitive High Resolution Melting (MS-HRM)

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DNA Methylation Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1708))

Abstract

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1–1% methylated alleles in an unmethylated background.

Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.

The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

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References

  1. Frommer M, McDonald LE, Millar DS et al (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 89:1827–1831

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  3. Wojdacz TK, 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

    Article  PubMed  PubMed Central  Google Scholar 

  4. Warnecke PM, Stirzaker C, Melki JR et al (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA. Nucleic Acids Res 25:4422–4426

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wojdacz TK, Hansen LL (2006) Techniques used in studies of age-related DNA methylation changes. Ann N Y Acad Sci 1067:479–487

    Article  CAS  PubMed  Google Scholar 

  6. Wojdacz TK, Borgbo T, Hansen LL (2009) Primer design versus PCR bias in methylation independent PCR amplifications. Epigenetics 4:231–234

    Article  CAS  PubMed  Google Scholar 

  7. Wojdacz TK, Hansen LL, Dobrovic A (2008) A new approach to primer design for the control of PCR bias in methylation studies. BMC Res Notes 1:54

    Article  PubMed  PubMed Central  Google Scholar 

  8. Xiao Z, Li B, Wang G et al (2014) Validation of methylation-sensitive high-resolution melting (MS-HRM) for the detection of stool DNA methylation in colorectal neoplasms. Clin Chim Acta 431:154–163

    Article  CAS  PubMed  Google Scholar 

  9. Wojdacz TK, Dobrovic A, Hansen LL (2008) Methylation-sensitive high-resolution melting. Nat Protoc 3:1903–1908

    Article  CAS  PubMed  Google Scholar 

  10. Tse MY, Ashbury JE, Zwingerman N et al (2011) A refined, rapid and reproducible high resolution melt (HRM)-based method suitable for quantification of global LINE-1 repetitive element methylation. BMC Res Notes 4:565

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wojdacz TK, Windelov JA, Thestrup BB et al (2014) Identification and characterization of locus specific methylation patterns within novel loci undergoing hypermethylation during breast cancer pathogenesis. Breast Cancer Res 16:R17

    Article  PubMed  PubMed Central  Google Scholar 

  12. Kristensen LS, Wojdacz TK, Thestrup BB et al (2009) Quality assessment of DNA derived from up to 30 years old formalin fixed paraffin embedded (FFPE) tissue for PCR-based methylation analysis using SMART-MSP and MS-HRM. BMC Cancer 9:453

    Article  PubMed  PubMed Central  Google Scholar 

  13. Oster B, Thorsen K, Lamy P et al (2011) Identification and validation of highly frequent CpG island hypermethylation in colorectal adenomas and carcinomas. Int J Cancer 129:2855–2866

    Article  CAS  PubMed  Google Scholar 

  14. Reinert T, Modin C, Castano FM et al (2011) Comprehensive genome methylation analysis in bladder cancer: identification and validation of novel methylated genes and application of these as urinary tumor markers. Clin Cancer Res 17:5582–5592

    Article  CAS  PubMed  Google Scholar 

  15. Snell C, Krypuy M, Wong EM et al (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

    Article  PubMed  PubMed Central  Google Scholar 

  16. Liu W, Guan M, Su B et al (2010) Rapid determination of AKAP12 promoter methylation levels in peripheral blood using methylation-sensitive high resolution melting (MS-HRM) analysis: application in colorectal cancer. Clin Chim Acta 411:940–946

    Article  CAS  PubMed  Google Scholar 

  17. Wojdacz TK, Thestrup BB, Cold S et al (2011) No difference in the frequency of locus-specific methylation in the peripheral blood DNA of women diagnosed with breast cancer and age-matched controls. Future Oncol 7:1451–1455

    Article  CAS  PubMed  Google Scholar 

  18. Wojdacz TK, Thestrup BB, Overgaard J et al (2011) Methylation of cancer related genes in tumor and peripheral blood DNA from the same breast cancer patient as two independent events. Diagn Pathol 6:116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Li X, Wang Y, Zhang Z et al (2013) Correlation of and methylation levels between peripheral blood leukocytes and colorectal tissue DNA samples in colorectal cancer patients. Oncol Lett 6:1370–1376

    CAS  PubMed  PubMed Central  Google Scholar 

  20. White HE, Hall VJ, Cross NC (2007) Methylation-sensitive high-resolution melting-curve analysis of the SNRPN gene as a diagnostic screen for Prader-Willi and Angelman syndromes. Clin Chem 53:1960–1962

    Article  CAS  PubMed  Google Scholar 

  21. Candiloro IL, Mikeska T, Hokland P et al (2008) Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene. Epigenetics Chromatin 1:7

    Article  PubMed  PubMed Central  Google Scholar 

  22. Candiloro IL, Mikeska T, Dobrovic A (2011) Assessing combined methylation-sensitive high resolution melting and pyrosequencing for the analysis of heterogeneous DNA methylation. Epigenetics 6:500–507

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hernandez HG, Tse MY, Pang SC et al (2013) Optimizing methodologies for PCR-based DNA methylation analysis. BioTechniques 55:181–197

    Article  CAS  PubMed  Google Scholar 

  24. Wojdacz TK, Moller TH, Thestrup BB et al (2010) Limitations and advantages of MS-HRM and bisulfite sequencing for single locus methylation studies. Expert Rev Mol Diagn 10:575–580

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

The authors thank T.K. Wojdacz and I.L. Daugaard for their critical review of the manuscript.

Competing interests

LLH is an inventor on a patent and co-founder of the company MethylDetect on the aspects of the MS-HRM technology.

Author information

Authors and Affiliations

  1. Institute of Biomedicine, Aarhus University, Bartholins Allé 6, Aarhus C, 8000, Denmark

    Dianna Hussmann & Lise Lotte Hansen

Authors
  1. Dianna Hussmann
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  2. Lise Lotte Hansen
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Corresponding author

Correspondence to Lise Lotte Hansen .

Editor information

Editors and Affiliations

  1. Laboratory for Epigenetics and Environment, Centre National de Recherche en Génomique Humaine, CEA—Institut de Biologie Francois Jacob, Evry, France

    Jörg Tost

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Hussmann, D., Hansen, L.L. (2018). Methylation-Sensitive High Resolution Melting (MS-HRM). In: Tost, J. (eds) DNA Methylation Protocols. Methods in Molecular Biology, vol 1708. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7481-8_28

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  • DOI: https://doi.org/10.1007/978-1-4939-7481-8_28

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7479-5

  • Online ISBN: 978-1-4939-7481-8

  • eBook Packages: Springer Protocols

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