Simultaneous determination of genomic DNA methylation and uracil misincorporation. 2009

Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
Unit EGEAL and Laboratory of Nutritional Genomics, Institut Polytechnique LaSalle Beauvais, Agrohealth, Beauvais, France. abalo.chango@isab.fr

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.

UI MeSH Term Description Entries
D008745 Methylation Addition of methyl groups. In histo-chemistry methylation is used to esterify carboxyl groups and remove sulfate groups by treating tissue sections with hot methanol in the presence of hydrochloric acid. (From Stedman, 25th ed) Methylations
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D002460 Cell Line Established cell cultures that have the potential to propagate indefinitely. Cell Lines,Line, Cell,Lines, Cell
D002851 Chromatography, High Pressure Liquid Liquid chromatographic techniques which feature high inlet pressures, high sensitivity, and high speed. Chromatography, High Performance Liquid,Chromatography, High Speed Liquid,Chromatography, Liquid, High Pressure,HPLC,High Performance Liquid Chromatography,High-Performance Liquid Chromatography,UPLC,Ultra Performance Liquid Chromatography,Chromatography, High-Performance Liquid,High-Performance Liquid Chromatographies,Liquid Chromatography, High-Performance
D003856 Deoxyuracil Nucleotides Uracil nucleotides which contain deoxyribose as the sugar moiety. Deoxyuridine Phosphates,Nucleotides, Deoxyuracil,Phosphates, Deoxyuridine
D004247 DNA A deoxyribonucleotide polymer that is the primary genetic material of all cells. Eukaryotic and prokaryotic organisms normally contain DNA in a double-stranded state, yet several important biological processes transiently involve single-stranded regions. DNA, which consists of a polysugar-phosphate backbone possessing projections of purines (adenine and guanine) and pyrimidines (thymine and cytosine), forms a double helix that is held together by hydrogen bonds between these purines and pyrimidines (adenine to thymine and guanine to cytosine). DNA, Double-Stranded,Deoxyribonucleic Acid,ds-DNA,DNA, Double Stranded,Double-Stranded DNA,ds DNA
D004305 Dose-Response Relationship, Drug The relationship between the dose of an administered drug and the response of the organism to the drug. Dose Response Relationship, Drug,Dose-Response Relationships, Drug,Drug Dose-Response Relationship,Drug Dose-Response Relationships,Relationship, Drug Dose-Response,Relationships, Drug Dose-Response
D014498 Uracil One of four nucleotide bases in the nucleic acid RNA.
D044503 5-Methylcytosine A methylated nucleotide base found in eukaryotic DNA. In ANIMALS, the DNA METHYLATION of CYTOSINE to form 5-methylcytosine is found primarily in the palindromic sequence CpG. In PLANTS, the methylated sequence is CpNpGp, where N can be any base. 5-Methylcytosine Monohydrochloride,5 Methylcytosine,5 Methylcytosine Monohydrochloride,Monohydrochloride, 5-Methylcytosine
D021241 Spectrometry, Mass, Electrospray Ionization A mass spectrometry technique used for analysis of nonvolatile compounds such as proteins and macromolecules. The technique involves preparing electrically charged droplets from analyte molecules dissolved in solvent. The electrically charged droplets enter a vacuum chamber where the solvent is evaporated. Evaporation of solvent reduces the droplet size, thereby increasing the coulombic repulsion within the droplet. As the charged droplets get smaller, the excess charge within them causes them to disintegrate and release analyte molecules. The volatilized analyte molecules are then analyzed by mass spectrometry. ESI Mass Spectrometry,Electrospray Ionization Mass Spectrometry,Mass Spectrometry, ESI,Spectrometry, ESI Mass

Related Publications

Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
April 1980, Proceedings of the National Academy of Sciences of the United States of America,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
January 2010, The American journal of clinical nutrition,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
January 2009, Gastroenterology,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
September 2010, Mutagenesis,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
September 1997, Carcinogenesis,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
March 1993, European journal of haematology,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
February 1990, The International journal of neuroscience,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
March 1986, Biochemical and biophysical research communications,
Abalo Chango, and Afif M Abdel Nour, and Celine Niquet, and Frederic J Tessier
March 1978, Cell,
Copied contents to your clipboard!