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Nucleic Acids Research Advance Access published online on August 20, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn534
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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DNA methylation determination by liquid chromatography–tandem mass spectrometry using novel biosynthetic [U-15N]deoxycytidine and [U-15N]methyldeoxycytidine internal standards

Eoin P. Quinlivan1,* and Jesse F. Gregory, III2

1Biomedical Mass Spectrometry Laboratory, General Clinical Research Center and 2Food Science and Human Nutrition Department, University of Florida, Gainesville, FL, USA

*To whom correspondence should be addressed. Tel: +1 352 381 6925; Fax: +1 352 846 0990; Email: epq{at}ufl.edu

Received April 24, 2008. Revised July 24, 2008. Accepted August 4, 2008.

Methylation of the promoter CpG regions regulates gene transcription by inhibiting transcription factor binding. Deoxycytidine methylation may regulate cell differentiation, while aberrations in the process may be involved in cancer etiology and the development of birth defects (e.g. neural tube defects). Similarly, nutritional deficiency and certain nutragenomic interactions are associated with DNA hypomethylation. While LC-MS has been used previously to measure percentage genomic deoxycytidine methylation, a lack of a secure source of internal standards and the need for laborious and time-consuming DNA digestion protocols constitute distinct limitations. Here we report a simple and inexpensive protocol for the biosynthesis of internal standards from readily available precursors. Using these biosynthetic stable-isotopic [U-15N]-labeled internal standards, coupled with an improved DNA digestion protocol developed in our lab, we have developed a low-cost, high-throughput (>500 samples in 4 days) assay for measuring deoxycytidine methylation in genomic DNA. Inter- and intraassay variation for the assay (%RSD, n = 6) was <2.5%.


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