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ARS Home » Northeast Area » Boston, Massachusetts » Jean Mayer Human Nutrition Research Center On Aging » Research » Publications at this Location » Publication #134124
Title: A NEW METHOD TO ASSESS GENOMIC DNA METHYLATION USIN HIGH-PERFORMANCE LIQUID CHROMATOGAPHY

Author
item FRISO, SIMONETTA - HNRCA
item CHOI, SANG-WOON - HNRCA
item DOLNIKOWSKI, GREGORY G - HNRCA
item SELHUB, JACOB - HNRCA

Submitted to: Analytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/1/2002
Publication Date: 9/1/2002
Citation: FRISO, S., CHOI, S., DOLNIKOWSKI, G., SELHUB, J. A NEW METHOD TO ASSESS GENOMIC DNA METHYLATION USIN HIGH-PERFORMANCE LIQUID CHROMATOGAPHY. ANALYTICAL CHEMISTRY. 2002.74(17):4526-31.

Interpretive Summary: DNA, a building block of genes, is regulated by a system called DNA methylation. This system is extremely important to regulate cell growth and it has been studied to understand aberrant condition of cell growth such as cancer, one of the most unfavorable disease for which much still needs to be understood. Unfortunately, the methods currently available to measure DNA methylation suffer of high imprecision and therefore we decided to develop a method that could provide more reliability. We describe this new method in the present manuscript.

Technical Abstract: Eukaryotic DNA is methylated at some cytosine residues and this epigenetic feature performs critical functions. We developed a method for quantitative determination of 5-methyl-2'-deoxycytidine in human DNA using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). The DNA was enzymatically hydrolyzed by sequential digestion with three enzymes. DNA hydrolyzates were subsequently separated by reverse-phase high-performance liquid chromatography in isocratic mode. The four major DNA bases and 5-methyl-2'-deoxycytidine were resolved and eluted in 13 minutes. Identification of 2'-deoxycytidine and 5-methyl-2'-deoxycytidine was obtained by combined diode array UV spectra analysis and mass spectra of chromatographic peaks. The isotopomers **15N3 2'-deoxycytidine and methyl-D3, ring-6-D1 5-methyl-2'-deoxycytidine were used as internal standards. Ions of m/z 126 and 130 were used to detect 5-methyl-2'-deoxycytidine and its isotopomer, and ions of m/z 112 and 115 were used to detect 2'-deoxycytidine and its stable isotopomer, respectively. The DNA methylation status was calculated based on the amount of 5-methyl-2'-deoxycytidine per mcg DNA with percent relative standard deviations (%RSD) for method precision of 7.1 (within-day) and 5.7 (day-to-day). This method also allows the measurement of 5-methyl-2'-deoxycytidine expressed as a percentage of total deoxycytidine residues in genomic DNA with %RSD for method precision of 1.9 (within-day) and 1.7 (day-to-day). This LC/MS method for quantitative determination of genomic DNA methylation status is rapid, sensitive, selective and precise.