Restriction Digestion and Real-Time PCR (qAMP)

  • Christopher C. Oakes
  • Sophie La Salle
  • Jacquetta M. Trasler
  • Bernard Robaire
Part of the Methods in Molecular Biology book series (MIMB, volume 507)


DNA methylation in mammals has been shown to play many important roles in diverse biological phenomena. Here we describe a simple and straightforward method that quantitatively measures site-specific levels of DNA methylation in a quick and cost-effective manner. The quantitative analysis of DNA methylation using real-time PCR (qAMP) technique involves the digestion of genomic DNA using methylation-sensitive and methylation-dependent restriction enzymes followed by real-time PCR. This approach generates accurate and reproducible results without the requirement for prior treatment of the DNA with sodium bisulfite.


DNA methylation analysis method restriction enzyme real-time PCR 


  1. 1.
    Goll, M. G., Bestor, T. H. (2005) Eukaryotic cytosine methyltransferases. Annu Rev Biochem 74, 481–514.CrossRefPubMedGoogle Scholar
  2. 2.
    Liu, L., Wylie, R. C., Andrews, L. G., et al. (2003) Aging, cancer and nutrition: the DNA methylation connection. Mech Ageing Dev 124, 989–898.CrossRefPubMedGoogle Scholar
  3. 3.
    Ting, A. H., McGarvey, K. M., Baylin, S. B. (2006) The cancer epigenome – components and functional correlates. Genes Dev 20, 3215–3231.CrossRefPubMedGoogle Scholar
  4. 4.
    Clark, S. J., Harrison, J., Paul, C. L., et al. (1984) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22, 2990–2997.Google Scholar
  5. 5.
    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
  6. 6.
    Grunau, C., Clark, S. J., Rosenthal, A. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29, e65.CrossRefPubMedGoogle Scholar
  7. 7.
    Munson, K., Clark, J., Lamparska-Kupsik, K., et al. (2007) Recovery of bisulfite-converted genomic sequences in the methylation-sensitive QPCR. Nucleic Acids Res 35, 2893–2903.CrossRefPubMedGoogle Scholar
  8. 8.
    Oakes, C. C., La Salle, S., Robaire, B., et al. (2006) Assessment of a quantitative, non-bisulfite-based method for the analysis of DNA methylation using real-time PCR (qMAP). Epigenetics 1, 146–152.CrossRefPubMedGoogle Scholar
  9. 9.
    Oakes, C. C., Kelly, T. L., Robaire, B., et al. (2007) Adverse effects of 5-aza-2′-deoxycytidine on spermatogenesis include reduced sperm function and selective inhibition of de novo DNA methylation. J Pharmacol Exp Ther 322, 1171–1180.CrossRefPubMedGoogle Scholar
  10. 10.
    Oakes, C. C., La Salle, S., Smiraglia, D. J., et al. (2007) Developmental acquisition of genome-wide DNA methylation occurs prior to meiosis in male germ cells. Dev Biol 307, 368–379.CrossRefPubMedGoogle Scholar
  11. 11.
    Oakes, C. C., La Salle, S., Smiraglia, D. J., et al. (2007) A unique configuration of genome-wide DNA methylation patterns in the testis. Proc Natl Acad Sci USA 104, 228–233.CrossRefPubMedGoogle Scholar
  12. 12.
    La Salle, S., Oakes, C. C., Neaga, O. R., et al. (2007) Loss of spermatogonia and wide-spread DNA methylation defects in newborn male mice deficient in DNMT3L. BMC Dev Biol 7, 104CrossRefPubMedGoogle Scholar
  13. 13.
    Stewart, F. J., Panne, D., Bickle, T. A., et al. (2000) Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme. J Mol Biol 298, 611–622.CrossRefPubMedGoogle Scholar
  14. 14.
    Imamura, T., Kerjean, A., Heams, T. et al. (2005) Dynamic CpG and non-CpG methylation of the Peg1/Mest gene in the mouse oocyte and preimplantation embryo. J Biol Chem 280, 20171–20175.CrossRefPubMedGoogle Scholar
  15. 15.
    Ramsahoye, B. H., Biniszkiewicz, D., Lyko, F., et al. (2000) Non-CpG methylation is prevalent in embryonic stem cells and may be mediated by DNA methyltransferase 3a. Proc Natl Acad Sci USA 97, 5237–5242.CrossRefPubMedGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Christopher C. Oakes
    • 1
  • Sophie La Salle
    • 1
  • Jacquetta M. Trasler
    • 2
  • Bernard Robaire
    • 3
  1. 1.Department of Pharmacology and Therapeutics and The Montreal Children’s Hospital Research InstituteMcGill UniversityQuebecCanada
  2. 2.Department of Pharmacology and Therapeutics, Department of Pediatrics, Department of Human Genetics and The Montreal Children’s Hospital Research InstituteMcGill UniversityQuebecCanada
  3. 3.Department of Pharmacology and Therapeutics and Department of Obstetrics and GynecologyMcGill UniversityQuebecCanada

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