Skip to main content

Advertisement

SpringerLink
Log in

Detection of genomic DNA methylation with denaturing high performance liquid chromatography

  • Research Article
  • Published:
Human Cell Aims and scope Submit manuscript

Abstract

DNA methylation contributes to the epigenetic control of gene expression. Variations in the methylation status can result in the silencing of genes. DNA methyltransferase converts cytosine to 5-methyl cytosine in CpG islands located in the promoter regions of genes. When CpG islands are hypermethylated, the gene is repressed/silenced, and similarly when it is hypomethylated, transcription can take place and the gene is expressed. The classical methods to detect DNA methylation require labor-intensive and time-consuming steps. As a result of large-scale expression profiling studies, high-throughput techniques are needed to screen for alterations in the methylation patterns. Denaturing high performance liquid chromatography (DHPLC) is a reliable, highly sensitive technique for mutation discovery. In the present study we examined the suitability of DHPLC technology to detect alterations in methylation pattern of the promoter regions of several genes. We report reliable and reproducible results in distinguishing methylated and unmethylated promoter regions of human PCDHGB6, c-MYC, MGMT1, CDKN2A/p16, and ATM genes. These DHPLC profiles were independently confirmed with bisulfite genomic sequencing. In conclusion, DHPLC technology serves as a rapid screening tool to monitor the genomic DNA methylation and could be used to increase the throughput efficiency of methylation analysis.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Bedford MT, van Helden PD. A method to analyze allele-specific methylation. Biotechniques 1990; 9: 744–8.

    CAS  PubMed  Google Scholar 

  2. Frommer M, McDonald LE, Millar DS et al. A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc Natl Acad Sci USA 1992; 89: 1827–31.

    Article  CAS  PubMed  Google Scholar 

  3. Herman JG, Graff JR, Myohanen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA 1996; 93: 9821–6.

    Article  CAS  PubMed  Google Scholar 

  4. Garcia-Manero G, Daniel J, Smith TL et al. DNA methylation of multiple promoter-associated CpG islands in adult acute lymphocytic leukemia. Clin Cancer Res 2002; 8: 2217–24.

    CAS  PubMed  Google Scholar 

  5. Kinoshita-Kikuta E, Kinoshita E, Koike T. A mobility shift detection method for DNA methylation analysis using phosphate affinity polyacrylamide gel electrophoresis. Anal Biochem 2008; 378: 102–104.

    Article  CAS  PubMed  Google Scholar 

  6. Uhlmann K, Brinckmann A, Toliat MR, Ritter H, Nurnberg P. Evaluation of a potential epigenetic biomarker by quantitative methyl-single nucleotide polymorphism analysis. Electrophoresis 2002; 23: 4072–9.

    Article  CAS  PubMed  Google Scholar 

  7. Kato D, Sekioka N, Ueda A et al. A nanocarbon film electrode as a platform for exploring DNA methylation. J Am Chem Soc 2008; 130: 3716–17.

    Article  CAS  PubMed  Google Scholar 

  8. Kholod N, Boniver J, Delvenne P. A new dimethyl sulfoxide-based method for gene promoter methylation detection. J Mol Diagn 2007; 9: 574–81.

    Article  CAS  PubMed  Google Scholar 

  9. Kristinsson R, Lewis SE, Danielson PB. Comparative analysis of the HV1 and HV2 regions of human mitochondrial DNA by denaturing high-performance liquid chromatography. J Forensic Sci 2009; 54: 28–36.

    Article  CAS  PubMed  Google Scholar 

  10. Underhill PA, Passarino G, Lin AA et al. The phylogeography of Y chromosome binary haplotypes and the origins of modern human populations. Ann Hum Genet 2001; 65: 43–62.

    Article  CAS  PubMed  Google Scholar 

  11. Nectoux J, Bahi-Buisson N, Guellec I et al. The p.Val66Met polymorphism in the BDNF gene protects against early seizures in Rett syndrome. Neurology 2008; 70: 2145–51.

    Article  CAS  PubMed  Google Scholar 

  12. Huber CG. Micropellicular stationary phases for high-performance liquid chromatography of double-stranded DNA. J Chromatogr A 1998; 806: 3–30.

    Article  CAS  PubMed  Google Scholar 

  13. Xiao W, Oefner PJ. Denaturing high-performance liquid chromatography: a review. Hum Mutat 2001; 17: 439–74.

    Article  CAS  PubMed  Google Scholar 

  14. Hattori M, Torii C, Yagihashi T et al. Diagnosis of Russell-Silver syndrome by the combined bisulfite restriction analysis-denaturing high-performance liquid chromatography assay. Genet Test Mol Biomarkers 2009; 13: 623–30.

    Article  CAS  PubMed  Google Scholar 

  15. Matin MM, Baumer A, Hornby DP. An analytical method for the detection of methylation differences at specific chromosomal loci using primer extension and ion pair reverse phase HPLC. Hum Mutat 2002; 20: 305–11.

    Article  CAS  PubMed  Google Scholar 

  16. Tusnady GE, Simon I, Varadi A, Aranyi T. BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes. Nucleic Acids Res 2005; 33: e9.

    Article  PubMed  Google Scholar 

  17. Delcuve GP, Rastegar M, Davie JR. Epigenetic control. J Cell Physiol 2009; 219: 243–50.

    Article  CAS  PubMed  Google Scholar 

  18. Tost J. DNA methylation: an introduction to the biology and the disease-associated changes of a promising biomarker. Methods Mol Biol 2009; 507: 3–20.

    Article  CAS  PubMed  Google Scholar 

  19. Walsh CP, Xu GL. Cytosine methylation and DNA repair. Curr Top Microbiol Immunol 2006; 301: 283–315.

    Article  CAS  PubMed  Google Scholar 

  20. Chang JW, Huang TH, Wang YC. Emerging methods for analysis of the cancer methylome. Pharmacogenomics 2008; 9: 1869–78.

    Article  CAS  PubMed  Google Scholar 

  21. Xiong Z, Laird PW. COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 1997; 25: 2532–4.

    Article  CAS  PubMed  Google Scholar 

  22. Gonzalgo ML, Liang G. Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation. Nat Protoc 2007; 2: 1931–6.

    Article  CAS  PubMed  Google Scholar 

  23. Weisenberger DJ, Trinh BN, Campan M et al. DNA methylation analysis by digital bisulfite genomic sequencing and digital MethyLight. Nucleic Acids Res 2008; 36: 4689–98.

    Article  CAS  PubMed  Google Scholar 

  24. Benhattar J, Clement G. Methylation-sensitive single-strand conformation analysis: a rapid method to screen for and analyze DNA methylation. Methods Mol Biol 2004; 287: 181–93.

    CAS  PubMed  Google Scholar 

  25. Ho SM, Tang WY. Techniques used in studies of epigenome dysregulation due to aberrant DNA methylation: an emphasis on fetal-based adult diseases. Reprod Toxicol 2007; 23: 267–82.

    Article  CAS  PubMed  Google Scholar 

  26. Hayatsu H. The bisulfite genomic sequencing used in the analysis of epigenetic states, a technique in the emerging environmental genotoxicology research. Mutat Res 2008; 659: 77–82.

    Article  CAS  PubMed  Google Scholar 

  27. Wang Y, Hayakawa J, Long F et al. “Promoter array” studies identify cohorts of genes directly regulated by methylation, copy number change, or transcription factor binding in human cancer cells. Ann N Y Acad Sci 2005; 1058: 162–85.

    Article  CAS  PubMed  Google Scholar 

  28. Baumer A, Wiedemann U, Hergersberg M, Schinzel A. A novel MSP/DHPLC method for the investigation of the methylation status of imprinted genes enables the molecular detection of low cell mosaicisms. Hum Mutat 2001; 17: 423–30.

    Article  CAS  PubMed  Google Scholar 

  29. Miyamoto K, Fukutomi T, Akashi-Tanaka S et al. Identification of 20 genes aberrantly methylated in human breast cancers. Int J Cancer 2005; 116: 407–14.

    Article  CAS  PubMed  Google Scholar 

  30. Gartel AL. A new mode of transcriptional repression by c-myc: methylation. Oncogene 2006; 25: 1989–90.

    Article  CAS  PubMed  Google Scholar 

  31. Wu CH, van Riggelen J, Yetil A, Fan AC, Bachireddy P, Felsher DW. Cellular senescence is an important mechanism of tumor regression upon c-Myc inactivation. Proc Natl Acad Sci USA 2007; 104: 13028–33.

    Article  CAS  PubMed  Google Scholar 

  32. Yamada H, Vijayachandra K, Penner C, Glick A. Increased sensitivity of transforming growth factor (TGF) beta 1 null cells to alkylating agents reveals a novel link between TGFbeta signaling and O(6)-methylguanine methyltransferase promoter hypermethylation. J Biol Chem 2001; 276: 19052–8.

    Article  CAS  PubMed  Google Scholar 

  33. Esteller M, Hamilton SR, Burger PC, Baylin SB, Herman JG. Inactivation of the DNA repair gene O6-methylguanine-DNA methyltransferase by promoter hypermethylation is a common event in primary human neoplasia. Cancer Res 1999; 59: 793–7.

    CAS  PubMed  Google Scholar 

  34. Danam RP, Howell SR, Brent TP, Harris LC. Epigenetic regulation of O6-methylguanine-DNA methyltransferase gene expression by histone acetylation and methyl-CpG binding proteins. Mol Cancer Ther 2005; 4: 61–9.

    CAS  PubMed  Google Scholar 

  35. Merlo A, Herman JG, Mao L et al. 5′ CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat Med 1995; 1: 686–92.

    Article  CAS  PubMed  Google Scholar 

  36. Taylor AM, Byrd PJ. Molecular pathology of ataxia telangiectasia. J Clin Pathol 2005; 58: 1009–15.

    Article  CAS  PubMed  Google Scholar 

  37. Kim WJ, Vo QN, Shrivastav M, Lataxes TA, Brown KD. Aberrant methylation of the ATM promoter correlates with increased radiosensitivity in a human colorectal tumor cell line. Oncogene 2002; 21: 3864–71.

    Article  CAS  PubMed  Google Scholar 

  38. Ai L, Vo QN, Zuo C et al. Ataxia-telangiectasia-mutated (ATM) gene in head and neck squamous cell carcinoma: promoter hypermethylation with clinical correlation in 100 cases. Cancer Epidemiol Biomarkers Prev 2004; 13: 150–6.

    Article  CAS  PubMed  Google Scholar 

  39. Roy K, Wang L, Makrigiorgos GM, Price BD. Methylation of the ATM promoter in glioma cells alters ionizing radiation sensitivity. Biochem Biophys Res Commun 2006; 344: 821–6.

    Article  CAS  PubMed  Google Scholar 

  40. Gumy-Pause F, Wacker P, Maillet P, Betts DR, Sappino AP. ATM alterations in childhood non-Hodgkin lymphoma. Cancer Genet Cytogenet 2006; 166: 101–11.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Medical Laboratory and Radiation Sciences, University of Vermont, 302 Rowell Building, 05405, Burlington, VT, USA

    Romaica A. Omaruddin & M. Ahmad Chaudhry

Authors
  1. Romaica A. Omaruddin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M. Ahmad Chaudhry
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M. Ahmad Chaudhry.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Omaruddin, R.A., Chaudhry, M.A. Detection of genomic DNA methylation with denaturing high performance liquid chromatography. Hum Cell 23, 41–49 (2010). https://doi.org/10.1111/j.1749-0774.2010.00084.x

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1111/j.1749-0774.2010.00084.x

Key words

Search

Navigation