Bio-COBRA: Absolute Quantification of DNA Methylation in Electrofluidics Chips

  • Romulo Martin Brena
  • Christoph Plass
Part of the Methods in Molecular Biology book series (MIMB, volume 507)


DNA methylation is the best-studied epigenetic modification, and in mammals it describes the conversion of cytosine to 5-methylcytosine in the context of CpG dinucleotides. In recent years, it has become evident that epigenetic mechanisms are severely disrupted in human neoplasia, and evidence suggests that alterations of DNA methylation patterns may be an integral mechanism in the etiology of other diseases such as bipolar disorder and schizophrenia. The main effect of altered DNA methylation is the disruption of normal patterns of gene expression through genomic instability and hypermethylation of CpG islands, which together could lead to uncontrolled cell proliferation. DNA methylation can be reversed through pharmacological intervention via the systemic administration of DNA methylation inhibitors. Thus, the ability to accurately quantify DNA methylation levels in genomic sequences is a prerequisite to assess not only treatment efficacy, but also the effect of the DNA methylation inhibitors on bystander tissues. Several methods are currently available for the analysis of DNA methylation. Nonetheless, accurate and reproducible quantification of DNA methylation remains challenging. Here, we describe Bio-COBRA, a modified protocol for combined bisulfite restriction analysis (COBRA) that incorporates an electrophoresis step in microfluidics chips. Microfluidics technology involves the handling of small amounts of liquid in miniaturized systems. Bio-COBRA provides a platform for the rapid and quantitative assessment of DNA methylation patterns in large sample sets. Its sensitivity and reproducibility also make it an excellent tool for the analysis of DNA methylation in clinical samples.


Bio-COBRA electrofluidics chips quantification DNA methylation DNA methylation inhibitor dynamic range, 2100 Bioanalyzer 



We would like to thank Herbert Auer and Dr. Karl Kornacker, both of whom were involved in the development and testing of this protocol.


  1. 1.
    Jones, P. A., Laird, P. W. (1999) Cancer epigenetics comes of age. Nat Genet 21, 163–167.CrossRefPubMedGoogle Scholar
  2. 2.
    Jones, P. A., Baylin, S. B. (2002) The fundamental role of epigenetic events in cancer. Nat Rev Genet 3, 415–428.CrossRefPubMedGoogle Scholar
  3. 3.
    Clark, S. J., Harrison, J., Frommer, M. (1995) CpNpG methylation in mammalian cells. Nat Genet 10, 20–27.CrossRefPubMedGoogle Scholar
  4. 4.
    Franchina, M., Kay, P. H. (2000) Evidence that cytosine residues within 5′-CCTGG-3′ pentanucleotides can be methylated in human DNA independently of the methylating system that modifies 5′-CG-3′ dinucleotides. DNA Cell Biol 19, 521–526.CrossRefPubMedGoogle Scholar
  5. 5.
    Jabbari, K., Bernardi, G. (2004) Cytosine methylation and CpG, TpG (CpA) and TpA frequencies. Gene 333, 143–149.CrossRefPubMedGoogle Scholar
  6. 6.
    Gardiner-Garden, M., Frommer, M. (1987) CpG islands in vertebrate genomes. J Mol Biol 196, 261–282.CrossRefPubMedGoogle Scholar
  7. 7.
    Larsen, F., Gundersen, G., Lopez, R., et al. (1992) CpG islands as gene markers in the human genome. Genomics 13, 1095–1107.CrossRefPubMedGoogle Scholar
  8. 8.
    Szyf, M., Pakneshan, P., Rabbani, S. A. (2004) DNA demethylation and cancer: therapeutic implications. Cancer Lett 211, 133–143.CrossRefPubMedGoogle Scholar
  9. 9.
    Baylin, S. B., Herman, J. G., Graff, J. R., et al. (1998) Alterations in DNA methylation: a fundamental aspect of neoplasia. Adv. Cancer Res. 72, 141–196.CrossRefPubMedGoogle Scholar
  10. 10.
    Bastian, P. J., Palapattu, G. S., Lin, X., et al. (2005) Preoperative serum DNA GSTP1 CpG island hypermethylation and the risk of early prostate-specific antigen recurrence following radical prostatectomy. Clin Cancer Res 11, 4037–4043.CrossRefPubMedGoogle Scholar
  11. 11.
    Friedrich, M. G., Chandrasoma, S., Siegmund, K. D., et al. (2005) Prognostic relevance of methylation markers in patients with non-muscle invasive bladder carcinoma. Eur J Cancer 41, 2769–2778.CrossRefPubMedGoogle Scholar
  12. 12.
    Levenson, V. V. (2004) DNA methylation biomarkers of cancer: moving toward clinical application. Pharmacogenomics 5, 699–707.CrossRefPubMedGoogle Scholar
  13. 13.
    Brock, M. V., Gou, M., Akiyama, Y., et al. (2003) Prognostic importance of promoter hypermethylation of multiple genes in esophageal adenocarcinoma. Clin Cancer Res 9, 2912–2919.PubMedGoogle Scholar
  14. 14.
    Usadel, H., Brabender, J., Danenberg, K. D., et al. (2002) Quantitative adenomatous polyposis coli promoter methylation analysis in tumor tissue, serum, and plasma DNA of patients with lung cancer. Cancer Res 62, 371–375.PubMedGoogle Scholar
  15. 15.
    Maruyama, R., Toyooka, S., Toyooka, K. O., et al. (2001) Aberrant promoter methylation profile of bladder cancer and its relationship to clinicopathological features. Cancer Res 61, 8659–8663.PubMedGoogle Scholar
  16. 16.
    Maruyama, R., Toyooka, S., Toyooka, K. O., et al. (2002) Aberrant promoter methylation profile of prostate cancers and its relationship to clinicopathological features. Clin Cancer Res 8, 514–519.PubMedGoogle Scholar
  17. 17.
    Fraga, M. F., Ballestar, E., Paz, M. F., et al. (2005) Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci USA 102, 10604–10609.CrossRefPubMedGoogle Scholar
  18. 18.
    Li, L. C., Dahiya, R. (2002) MethPrimer: designing primers for methylation PCRs. Bioinformatics 18, 1427–1431.CrossRefPubMedGoogle Scholar
  19. 19.
    Tusnady, G. E., Simon, I., Varadi, A., et al. (2005) BiSearch: primer-design and search tool for PCR on bisulfite-treated genomes. Nucleic Acids Res 33, e9.CrossRefPubMedGoogle Scholar
  20. 20.
    Brena, R. M., Huang, T. H., Plass, C. (2006) Quantitative assessment of DNA methylation: potential applications for disease diagnosis, classification, and prognosis in clinical settings. J Mol Med 84, 1–13.CrossRefGoogle Scholar
  21. 21.
    Brena, R. M., Auer, H., Kornacker, K., et al. (2006) Accurate quantification of DNA methylation using combined bisulfite restriction analysis coupled with the Agilent 2100 Bioanalyzer platform. Nucleic Acids Res 34, e17.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Romulo Martin Brena
    • 1
  • Christoph Plass
    • 2
  1. 1.Division of Human Cancer GeneticsThe Ohio State UniversityColumbusUSA
  2. 2.Division C010, German Cancer Research Center (DKFZ)Toxicology and Cancer Risk FactorsHeidelbergGermany

Personalised recommendations