Advertisement

DNA-Methylation Analysis by the Bisulfite-Assisted Genomic Sequencing Method

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 200)

Abstract

The postreplicative methylation of DNA at the C5 position of cytosines is found in a broad spectrum of organisms ranging from prokaryotes to human (1). In prokaryotes the major role of cytosine C5 methylation (like adenine N6 and cytosine N4 methylation) is to protect the genome against DNA degrading nucleases (restriction/modification), whereas in many eukaryotes cytosine C5 methylation (found within CpG dinucleotides) plays a pivotal role in the control of gene expression, inactivation of repetitive sequences, stability of chromosomes, and in cell transformation leading to development of cancer. The growing evidence that the cytosine methylation is also crucial in embryonic development of mammals regulating genomic imprinting, X inactivation and cell differentiation (2) has caused a demand for effective methods that would detect this modification with high sensitivity and reliability.

Keywords

Polymerase Chain Reaction Product Cytosine Methylation Agarose Bead Cytosine Residue Sodium Metabisulphite 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Jost, J. P. and Saluz, H. P., eds. (1993) DNA Methylation: Molecular Biology and Biological Significance. Birkhauser Verlag, Basel, Switzerland.Google Scholar
  2. 2.
    Li, E. B. C. and Jaenisch, R. (1992) Targeted mutation of the DNA methyltrans-ferase gene results in embryonic lethality. Cell 69, 915–926.PubMedCrossRefGoogle Scholar
  3. 3.
    Southern, E. M. (1975) Detection of specific sequences among DNA-fragments separated by gel electrophoresis. J. Mol. Biol. 98,503–517.PubMedCrossRefGoogle Scholar
  4. 4.
    Singer, S. J., Robinson, M. D., Bellve, A. R., Simon, M. I., and Riggs, A. D. (1990) Measurement by quantitative PCR of changes in HPRT, PGK-1, PGK-2, APRT, MTase, and Zfy gene transcripts during mouse spermatogenesis. Nucleic Acids Res. 18, 1255–1259.CrossRefGoogle Scholar
  5. 5.
    Kafri, T., Ariel, M., Brandeis, M., Shemer, R., Urven, L., McCarrey, J., et al. (1992) Developmental pattern of gene specific DNA methylation of the mouse embryo and germ line. Genes Dev. 6, 705–714.PubMedCrossRefGoogle Scholar
  6. 6.
    Brandeis, M., Kafri, T., Ariel, M., Chaillet, J. R., McCarrey, J., Razin, A. and Cedar, H. (1993) The ontogeny of allele-specific methylation associated with imprinted genes in the mouse. EMBO J. 12, 3669–3677.PubMedGoogle Scholar
  7. 7.
    Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 65,499–560.PubMedCrossRefGoogle Scholar
  8. 8.
    Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc. Natl. Acad. Sci. USA 81,1991–1995.PubMedCrossRefGoogle Scholar
  9. 9.
    Pfeifer, G. P., Steigerwald, S. D., Mueller, P. R., Wold, B. and Riggs, A. D. (1989) Genomic sequencing and methylation analysis by ligation mediated PCR. Science 246, 810–813.PubMedCrossRefGoogle Scholar
  10. 10.
    Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W., 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.PubMedCrossRefGoogle Scholar
  11. 11.
    Clark, S. J., Harrison, J., Paul, C. L., and Frommer, M. (1994) High sensitivity mapping of methylated cytosines. Nucleic Acids Res. 22,2990–2997.PubMedCrossRefGoogle Scholar
  12. 12.
    Feil, R., Walter, J., Allen, N.D., and Kelsey, G. (1994) Developmental control of allelic methylation in the imprinted mouse Igf2 and H19 genes. Development 120, 2933–2943.Google Scholar
  13. 13.
    Raizis, A. M., Schmitt, F., and Jost, J. P. (1994) A bisulphite method of 5-methyl-cytosine mapping that minimises template degradation. Anal. Biochem. 226, 161–166.CrossRefGoogle Scholar
  14. 14.
    Olek, A., Oswald, J., and Walter, J. (1996) A modified and improved method for bisulphite based cytosine methylation analysis.Nucleic Acids Res. 24, 5064–5066.PubMedCrossRefGoogle Scholar
  15. 15.
    Paulin, R., Grigg, G. W., Davey, M. W., and Piper, A. A. (1998) Urea improves efficiency of bisulphite-mediated sequencing of 5<<-methylcytosine in genomic DNA. Nucleic Acids Res. 26,5009–5010.PubMedCrossRefGoogle Scholar
  16. 16.
    Gonzalgo, M. and Jones, P. A. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res. 25,2529–2531.PubMedCrossRefGoogle Scholar
  17. 17.
    Paul, C. L. and Clark, S. J. (1996) Cytosine methylation: quantitation by automated genomic sequencing and GENESCAN analysis. BioTechniques 21, 126–133.PubMedGoogle Scholar
  18. 17a.
    Sambrook, G., Fritsch, E. F., and Maniatis, T. (1988) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Google Scholar
  19. 18.
    Hayatsu, H., Wataya, Y., Kai, K., and Iida, S. (1970) Reaction of sodium bisulphite with uracil, cytosine, and their derivatives. Biochemistry 9,2858–2864.PubMedCrossRefGoogle Scholar
  20. 19.
    Shapiro, R., Braverman, B., Louis, J. B., and Servis, R. E. (1973) Nucleic acid reactivity and conformation: reaction of cytosine and uracil with sodium bisulfite. J. Biol. Chem. 248,.4060–4064.PubMedGoogle Scholar
  21. 20.
    McLaren, A., Gonos, E. S., Carr, T., and Goddard, J. P. (1993) The conformation of tRNA genes. Chemical modification studies. FEBS Lett. 13, 177–180.CrossRefGoogle Scholar
  22. 21.
    Goodchild, J., Fellner, P., and Porter, A. G. (1975) The determination of secondary structure in the polyC tract of encephalomyocarditis virus RNA with sodium bisulphite. Nucleic Acids Res. 2,797–805.CrossRefGoogle Scholar
  23. 22.
    Kelly, J. M., Goddard, J. P., and Maden, E. H. (1978) Evidence on the conformation of HeLa-cell 5.8S ribosomal ribonucleic acid from the reaction of specific cytidine residues with sodium bisulphite. Biochem. J. 173, 521–532.PubMedGoogle Scholar
  24. 23.
    Wang, R. Y.-H., Gehrke, C. W., and Ehrlich, M. (1980) Comparison of bisulfite modification of 5-methyldeoxycytidine and deoxycytidine residues. Nucleic Acids Res. 8,4777–4790.PubMedCrossRefGoogle Scholar
  25. 24.
    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 bisulphite-treated DNA. Nucleic Acids Res. 25, 4422–4426.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  1. 1.Max-Planck-Institute for Molecular GeneticsBerlinGermany

Personalised recommendations