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Combined High-Performance Liquid Chromatography/³²P-Postlabeling Assay of N⁷-Methyldeoxyguanosine

Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892 [P. G. S., A. C. P., A. W., C. C. H.], and Molecular Genetics Laboratory, The Children's Hospital, Denver, Colorado 80218-1088 [V. L. W.]

A highly sensitive and specific assay for the detection of N⁷-methyl-2'-deoxyguanosine (N7methyldG) has been developed by combining high-performance liquid chromatography, ³²P-postlabeling, and nucleotide chromatography. Separation of normal nucleotides and adducts by high-performance liquid chromatography and then combining a portion of 2'-deoxyguanosine to the N7methyldG allows for quantitation using an internal standard. The directly determined molar ratio is not subject to errors in digestion, variable ATP-specific activity, or assumptions in relative adduct-labeling efficiency. The detection limit was one N7methyldG adduct in 10⁷ unmodified 2'-deoxyguanosine bases. N7methyldG adducts have been detected in 5 human lung samples in which O⁶-methyl-2'-deoxyguanosine adducts had been previously determined. The mean ratio of N7methyldG to O⁶-methyl-2'-deoxyguanosine was determined to be approximately 10. The current assay complements the high-performance liquid chromatography/³²P-postlabeling assay for O⁶-methyl-2'-deoxyguanosine and increases the detection sensitivity of DNA methylated by exogenous alkylating agents.

¹ Present address: Carcinogenesis Department, Christie Hospital and Holt Radium Institute, Manchester, M20 9BX, England.

² To whom requests for reprints should be addressed, at Laboratory of Human Carcinogenesis, Division of Cancer Etiology, National Cancer Institute, Building 37, Room 2C05, 9000 Rockville Pike, Bethesda, MD 20892.

Received 4/ 9/90. Accepted 7/17/90.

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