Skip to main content

Profiling DNA Methylation by Bisulfite Genomic Sequencing

Problems and Solutions

  • Protocol
Epigenetics Protocols

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 287))

Abstract

The surge of interest in DNA methylation during the last two decades has triggered an urgent need for an effective method to detect the methylation status of the cytosines in the genome. Bisulfite genomic sequencing is the most attractive choice so far for many laboratories. Various protocols have been established, but difficulties are often encountered, particularly by individuals who have limited experience in this field. This analysis presents a simple protocol that has consistently worked well in our laboratory. Discussions of potential technical problems and corresponding solutions are also included to facilitate the reproducibility of this protocol.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Cirradi, M., Izzo, A., Badaracco, G., and Landsberger, N. (2002) Molecular mechanisms of gene silencing mediated by DNA methylation. Mol. Cell. Biol. 22, 3157–3173.

    Article  Google Scholar 

  2. Havlis, J. and Trbusek, M. (2002) 5-Methylcytosine as a marker for the monitoring of DNA methylation. J. Chromatogr. B 781, 373–392.

    Article  CAS  Google Scholar 

  3. Stirzaker, C, Millar, D. S., Paul, C. L., Warnecke, P. M., Harrison, J., Vincent, P. C, Frommer, M., and Clark, S. J. (1997) Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res. 57, 2229–2237.

    PubMed  CAS  Google Scholar 

  4. Fraga, M. F. and Esteller, M. (2002) DNA methylation, a profile of methods and applications. BioTechniques 33, 632–649.

    PubMed  CAS  Google Scholar 

  5. Grigg, G and Clark, S. (1994) Sequencing 5-methylcytosine residues in genomic DNA. BioEssays 16, 431–436.

    Article  PubMed  CAS  Google Scholar 

  6. Rein, T., DePamphilis, M. L., and Zorbas, H. (1998) Identifying 5-mthylcytosine and related modifications in DNA genomes. Nucleic Acids Res. 26, 2255–2264.

    Article  PubMed  CAS  Google Scholar 

  7. Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA 81, 1991–1995.

    Article  PubMed  CAS  Google Scholar 

  8. Tommasi, S., LeBon, J. M., Riggs, A. D., and Singer-Sam, J. (1993) Methylation analysis by genomic sequencing of 5′ region of mouse Pgk-1 gene and a cautionary note concerning the method. Somat. Cell Mol. Genet. 19, 529–541.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  10. Frommer, M., McDonald, L. E., Millar, D. S., et al. (1992) 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.

    Article  PubMed  CAS  Google Scholar 

  11. Wang, R. Y. H., Gerhke, C. W., and Ehrlich, M. (1980) Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 8, 4777–4790.

    Article  PubMed  CAS  Google Scholar 

  12. Grigg, G. W. (1996) Sequencing 5-methylcytosine residues by the bisulphate method. DNA Sequence J. Sequencing Mapping 6, 189–198.

    Article  CAS  Google Scholar 

  13. Hajkova, P., El-Maarri, O., Engemann, S., Oswald, J., Olek, A., and Walter, J. (2002) DNA-methylation analysis by the bisulfite-assisted genomic sequencing method, in DNA Methylation Protocols (Mills, K. I. and Ramsahoye, B. H., eds). Humana, Totowa, NJ, pp. 143–154.

    Chapter  Google Scholar 

  14. Grunau, C, Clark, S. J., and Rosenthal, A. (2001) Bisulfite genomic sequencing, systematic investigation of critical experimental parameters. Nucleic Acids Res. 29, 1–7.

    Article  Google Scholar 

  15. Paulin, R., Grigg, G W., Davey, M. W., and Piper, A. A. (1998) Urea improves efficiency of bisulphate-mediated sequencing of 5′-methylcytosine in genomic DNA. Nucleic Acids Res. 26, 5009–5010.

    Article  PubMed  CAS  Google Scholar 

  16. Feil, R., Charlton, J., Bird, A. P., Walter, J., and Reik, W. (1994) Methylation analysis on individual chromosomes: improved protocol for bisulphate genomic sequencing. Nucleic Acids Res. 22, 695–696.

    Article  PubMed  CAS  Google Scholar 

  17. McDonald, L. E. and Kay, G. F. (1997) Methylation analysis using bisulfite genomic sequencing: application to small numbers of intact cells. BioTechniques 22, 272,273.

    PubMed  CAS  Google Scholar 

  18. Raizis, A. M., Schmitt F., and Jost, J. P. (1995) A bisulfite method of 5-methylcytosine mapping that minimizes template degradation. Anal. Biochem. 226, 161–166.

    Article  PubMed  CAS  Google Scholar 

  19. Voss, K. O., Roos, P., Nonay, R. L., and Dovichi, N. J. (1998) Combating PCR bias in bisulfite-based cytosine methylation analysis: betaine-modified cytosine deamination PCR. Anal. Chem. 70, 3818–3823.

    Article  CAS  Google Scholar 

  20. Warnecke, P. M., Stirzaker, C, Melki, J. R., Millar, D. S., Paul, C. L., and Clark, S. J. (1997) Detection and measurement of PCR bias in quantitative methylation analysis of bisulphate-treated DNA. Nucleic Acids Res. 25, 4422–4426.

    Article  PubMed  CAS  Google Scholar 

  21. 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, 101–107.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2004 Humana Press Inc., Totowa, NJ

About this protocol

Cite this protocol

Liu, L., Wylie, R.C., Hansen, N.J., Andrews, L.G., Tollefsbol, T.O. (2004). Profiling DNA Methylation by Bisulfite Genomic Sequencing. In: Tollefsbol, T.O. (eds) Epigenetics Protocols. Methods in Molecular Biology™, vol 287. Humana Press. https://doi.org/10.1385/1-59259-828-5:169

Download citation

  • DOI: https://doi.org/10.1385/1-59259-828-5:169

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-336-7

  • Online ISBN: 978-1-59259-828-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics