Medical Principles and Practice 2009-01-01

Simultaneous determination of genomic DNA methylation and uracil misincorporation.

Abalo Chango, Afif M Abdel Nour, Celine Niquet, Frederic J Tessier

Index: Med. Princ. Pract. 18(2) , 81-4, (2009)

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Abstract

To develop a method for the simultaneous measurement of 5-methylcytosine (5-metC) and 2'-deoxyuridine monophosphate (dU).Genomic DNA was extracted from the HepG2 cell line grown in experimental complete medium or in folate-depleted medium. Samples were treated with RNAse A and RNAse T1 to avoid any RNA contamination. High-performance liquid chromatography (HPLC)/electrospray ionization mass spectrometric (ESI-MS) method was used to separate nucleotides after enzymatic hydrolysis of DNA with nuclease P1, phosphodiesterase I and alkaline phosphatase.Using this sensitive new methodology, we were able to quantify simultaneously the concentration of DNA-5-metC and DNA-uracil in DNA. The linear correlation coefficient (R(2)) between the MS signal and the concentration of 5-metC in a range of 0.5-5 microM or dU in a range of 10-100 microM was 0.9954 and 0.9999, respectively. The coefficient of variation was 16.94 and 14.77%, respectively. The applicability of this assay is demonstrated by detection of a decrease in 5-metC% and elevation of dU/thymidylate (dT) into genomic DNA extracted from the HepG2 cell line grown in a folate-depleted medium.Our results confirm that the HPLC/ESI-MS method reported earlier for measuring 5-metC allows measurement of uracil misincorporation into DNA.Copyright 2009 S. Karger AG, Basel.