Abstract
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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Goll, M. G., Bestor, T. H. (2005) Eukaryotic cytosine methyltransferases. Annu Rev Biochem 74, 481–514.
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.
Ting, A. H., McGarvey, K. M., Baylin, S. B. (2006) The cancer epigenome – components and functional correlates. Genes Dev 20, 3215–3231.
Clark, S. J., Harrison, J., Paul, C. L., et al. (1984) High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22, 2990–2997.
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.
Grunau, C., Clark, S. J., Rosenthal, A. (2001) Bisulfite genomic sequencing: systematic investigation of critical experimental parameters. Nucleic Acids Res 29, e65.
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.
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.
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.
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.
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.
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, 104
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Oakes, C.C., La Salle, S., Trasler, J.M., Robaire, B. (2009). Restriction Digestion and Real-Time PCR (qAMP). In: Tost, J. (eds) DNA Methylation. Methods in Molecular Biology, vol 507. Humana Press. https://doi.org/10.1007/978-1-59745-522-0_20
Download citation
DOI: https://doi.org/10.1007/978-1-59745-522-0_20
Publisher Name: Humana Press
Print ISBN: 978-1-934115-61-9
Online ISBN: 978-1-59745-522-0
eBook Packages: Springer Protocols