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Nucleic Acids Research, 1980, Vol. 8, No. 20 4763-4776
© 1980

CHEMISTRY

Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA*

Kenneth C. Kuo, Roy A. McCune, Charles W. Gehrke, Rosemarie Midgett and Melanie Ehrlich

Department of Biochemistry, Experiment Station Chemical Laboratories, University of Missouri Columbia, MO 65211 Department of Biochemistry, Tulane Medical School New Orleans, LA 70112, USA

Received August 4, 1980.

We have developed a method to accurately determine (< 3% RSD) the complete major and modified base composition of a few micrograms of unlabeled DNA. The DNA samples were quantitatively hydrolyzed with DNase 1 Nuclease P1, and bacterial alkaline phosphatase. The resulting deoxyribonucleosides were directly separated in 70 min by reversed-phase high performance liquid chromatography with detection by ultraviolet absorption at 254 nm and 280 nm (RP-HPLC). The highly sensitive and selective dual wavelength quantitation greatly enhances the precision and accuracy of the chromatographic analysis. Contamination of DNA preparations with RNA does not interfere with the DNA analysis due to the high resolution of the chromatography. We have used this method for the quantitation of m5dCyd in 5 µg of calf thymus and salmon sperm DNA in which the m5dCyd comprises only 1 to 2% of the total bases. This method should be a useful research tool in studies on various DNAs and DNA subfractions and should help to elucidate the functions of methylation of DNA.

*Contribution from Missouri Agricultural Experiment Station, Journal Series No. .Approved by the Director


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