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Combined Bisulfite Restriction Analysis (COBRA)

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

Abstract

Most molecular biological techniques used to analyze specific loci in complex genomic DNA involve some form of sequence-specific amplification, whether it is biological amplification by cloning in Escherichia coli, direct amplification by polymerase chain reaction (PCR), or signal amplification by hybridization with a probe that can be visualized. Since DNA methylation is added postreplicatively by a dedicated maintenance DNA methyltransferase that is not present in either E. coli or in the PCR reaction, the methylation information is lost during molecular cloning or PCR amplification. Molecular hybridization does not discriminate between methylated and unmethylated DNA, since the methyl group on the cytosine does not participate in base pairing. The lack of a facile way to amplify the methylation information in complex genomic DNA has been a significant impediment to DNA methylation research.

Keywords

Polymerase Chain Reaction Polymerase Chain Reaction Product Polymerase Chain Reaction Amplification Polymerase Chain Reaction Reaction Sodium Bisulfite 
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.
    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
  2. 2.
    Rein, T., DePamphilis, M. L., and Zorbas, H. (1998) Identifying 5-methylcytosine and related modifications in DNA genomes. Nucleic Acids Res. 26, 2255–2264.PubMedCrossRefGoogle Scholar
  3. 3.
    Herman, J. G., Graff, J. R., Myöhänen, S., Nelkin, B. D., and Baylin, S. B. (1996)Methylation-specific PCR: a novel PCR assay for methylation status of CpG Islands. Proc. Natl. Acad. Sci. USA 93, 9821–9826.PubMedCrossRefGoogle Scholar
  4. 4.
    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
  5. 5.
    Xiong, Z. and Laird, P. W. (1997) COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res. 25,2532–2534.PubMedCrossRefGoogle Scholar
  6. 6.
    Sadri, R. and Hornsby, P. J. (1996) Rapid analysis of DNA methylation using new restriction enzyme sites created by bisulfite modification. Nucleic Acids Res. 24,5058–5059.PubMedCrossRefGoogle Scholar
  7. 7.
    Wang, R. Y., 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
  8. 8.
    Hayatsu, H. (1976) Bisulfite modification of nucleic acids and their constituents. Prog. Nucleic Acid Res. Mol. Biol. 16,75–124.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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
  11. 11.
    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.Google Scholar
  12. 12.
    Church, G. M. and Gilbert, W. (1984)Genomic sequencing. Proc. Natl. Acad. Sci. USA 81,1991–1995.PubMedCrossRefGoogle Scholar
  13. 13.
    Laird, P. W., Zijderveld, A., Linders, K., Rudnicki, M. A., Jaenisch, R., and Berns, A. (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Res. 19,4293.PubMedCrossRefGoogle Scholar
  14. 14.
    Shibata, D. (1992) The polymerase chain reaction and the molecular genetic analysis of tissue biopsies, in Diagnostic Molecular Pathology: A Practical Approach, (Herrington, C. S. and McGee, J. O. D. eds.), IRL Press, Oxford, UK, pp. 85–111.Google Scholar

Copyright information

© Humana Press Inc. 2002

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUSC Norris Comprehensive Cancer CenterLos Angeles
  2. 2.Department of Surgery and Biochemistry and Molecular BiologyUSC Norris Comprehensive Cancer CenterLos Angeles

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