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DNA Methylation Analysis by Bisulfite Conversion, Cloning, and Sequencing of Individual Clones

  • Yingying Zhang
  • Christian Rohde
  • Sascha Tierling
  • Heinrich Stamerjohanns
  • Richard Reinhardt
  • Jörn Walter
  • Albert Jeltsch
Part of the Methods in Molecular Biology book series (MIMB, volume 507)

Abstract

DNA methylation is an essential epigenetic modification in the human genome. For the investigation of DNA methylation patterns, bisulfite conversion and DNA sequencing is a method of choice, because it provides detailed information on the methylation pattern of individual DNA molecules at single CG site resolution. The method is based on the deamination of cytosine residues to uracils in the presence of NaOH and sodium bisulfite. Since methylcytosine is not converted under these conditions, the original methylation state of the DNA can be analyzed by sequencing of the converted DNA. After the conversion reaction, the DNA sequence under investigation is amplified by polymerase chain reaction (PCR) with primers specific for one strand of the bisulfite-converted DNA. The PCR product is cloned and individual clones are sequenced. Here, we describe an advanced protocol for bisulfite conversion, protocols for cloning, and tools for primer design (Methprimer, Bisearch). In addition, we present tools for the web display of primary data and data analysis (BiQ Analyzer, BDPC) and describe the setup of a sequencing and analysis pipeline for medium to high throughput.

Keywords

DNA methylation bisulfite sequencing cloning 

Notes

Acknowledgments

This work has been supported by the BMBF NGFN2 program. Technical support by S. Becker is gratefully acknowledged.

References

  1. 1.
    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.CrossRefPubMedGoogle Scholar
  2. 2.
    Grunau, C., Clark, S. J., Rosenthal, A. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29, e65.CrossRefPubMedGoogle Scholar
  3. 3.
    Bock, C., Reither, S., Mikeska T., et al. (2005) BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing. Bioinformatics 21, 4067–4068.CrossRefPubMedGoogle Scholar
  4. 4.
    Tusnády, G. E., Simon, I., Váradi, A., et al. (2005) BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes. Nucleic Acids Res 33, e9.CrossRefPubMedGoogle Scholar
  5. 5.
    Li, L. C., Dahiya, R. (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18, 1427–1431.CrossRefPubMedGoogle Scholar
  6. 6.
    Yamada, Y., Watanabe, H., Miura, F., et al. (2004) A comprehensive analysis of allelic methylation status of CpG islands on human Chromosome 21q. Genome Res 14, 247–266.CrossRefPubMedGoogle Scholar
  7. 7.
    Takai, D., Jones, P. A. (2002) Comprehensive analysis of CpG islands in human chromosomes 21 and 22. Proc Natl Acad Sci USA 99, 3740–3745.CrossRefPubMedGoogle Scholar
  8. 8.
    Handas, V., Jeltsch, A. (2004) Anomalous mobility of polymerase chain reaction products after bisulfite treatment of DNA. Anal Biochem 333, 196–198.CrossRefGoogle Scholar
  9. 9.
    Rohde, C., Zhang, Y., Jurkowski, T. P., Stamerjohanns, H., Reinhardt, R. Jeltsch, A. (2008) Bisulfite sequencing Data Presentation and Compilation (BDPC) web server - a useful tool for DNA methylation analysis. Nucl. Acids Res. 36, e34.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Yingying Zhang
    • 1
  • Christian Rohde
    • 1
  • Sascha Tierling
    • 2
  • Heinrich Stamerjohanns
    • 1
  • Richard Reinhardt
    • 3
  • Jörn Walter
    • 2
  • Albert Jeltsch
    • 1
  1. 1.School of Engineering and ScienceJacobs University BremenBremenGermany
  2. 2.Institut für Genetik, FB BiowissenschaftenUniversität des SaarlandesSaarbrückenGermany
  3. 3.Max Planck Institute for Molecular GeneticsBerlinGermany

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