Simultaneous determination of genomic DNA methylation and uracil misincorporation.
OBJECTIVE
To develop a method for the simultaneous measurement of 5-methylcytosine (5-metC) and 2'-deoxyuridine monophosphate (dU).


METHODS
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.


RESULTS
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.


CONCLUSIONS
Our results confirm that the HPLC/ESI-MS method reported earlier for measuring 5-metC allows measurement of uracil misincorporation into DNA.
Chango
Abalo
A
Unit EGEAL and Laboratory of Nutritional Genomics, Institut Polytechnique LaSalle Beauvais, Agrohealth, Beauvais, France. abalo.chango@isab.fr
Abdel Nour
Afif M
AM
Niquet
Celine
C
Tessier
Frederic J
FJ
D044503:5-Methylcytosine / Q000737:chemistry*; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D008745:Methylation; D009693:Nucleic Acid Hybridization; D021241:Spectrometry, Mass, Electrospray Ionization; D014498:Uracil / Q000737:chemistry*
D003856:Deoxyuracil Nucleotides; D014498:Uracil; D044503:5-Methylcytosine; D004247:DNA; C007267:2'-deoxyuridylic acid