A comprehensive quantitative analysis of methylated and ethylated DNA using high pressure liquid chromatography

Abstract

Methods were developed for the efficient routine degradation and fractionation of ethylated and methylated DNA. Alkylated DNA was hydrolyzed by a neutral thermal method to yield 3- and 7- alkylpurines and O²-alkylcytosines. The partially apurinic DNA was separated from the bases by precipitation in 0.1 N HCl. Portions of the DNA precipitate were further hydrolyzed either by 0.1 N HCl to yield purine bases, or by enzymes to yield nucleosides and phosphotriesters. The chemical and enzymic digests were fractionated by a combination of high pressure liquid chromatography systems to yield quantitative estimates of the following products from methylated or ethylated DNA: 1-, 3-, and 7-alkyladenines, O²-alkylcytosines, 3-, O⁶-, and 7- alkylguanines and O²-, 3-, and O⁴-alkylthymines. N⁶-Alkyladenines, 1-alkylguanines and N²-alkylguanines were not detected and the 3- alkylcytosines were detected but not quantified. Phosphotriesters were estimated from the amounts of recovered alkyl phosphotriesters of thymidylyl (3'-5') thymidine. Using these methods, it was possible to account for 98, 81, 98, and 92% of the DNA bound alkyl groups obtained from DNA reacted with [¹⁴C]methyl methane-sulfonate, [³H]ethyl methanesulfonate, N-[³H]-methyl-N-nitrosourea, and N-[¹⁴C]ethyl-N-nitro-sourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.