Combined high-performance liquid chromatography/32P-postlabeling assay of N7-methyldeoxyguanosine.
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 10(7) 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.
Shields
P G
PG
Laboratory of Human Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892.
Povey
A C
AC
Wilson
V L
VL
Weston
A
A
Harris
C C
CC
D000328:Adult; D000368:Aged; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis*; D005260:Female; D006801:Humans; D008168:Lung / Q000737:chemistry; D008297:Male; D008875:Middle Aged; D010761:Phosphorus Radioisotopes
D010761:Phosphorus Radioisotopes; C048746:7-methyl-2-deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine