Beranek D T, Weis C C, Swenson D H
Department of Health and Human Services, Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079, USA.
Carcinogenesis. 1980 Jul;1(7):595-606. doi: 10.1093/carcin/1.7.595.
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 methanesulfonate, [3H]ethyl methanesulfonate, N-[3H]-methyl-N-nitrosourea, and N-[14C]ethyl-N-nitrosourea, respectively. The methods described provide reproducible and quantitative methods of analysis for all the known methylated or ethylated products in a single DNA sample.
已开发出高效常规降解和分离乙基化及甲基化DNA的方法。烷基化DNA通过中性热法水解，生成3-和7-烷基嘌呤以及O2-烷基胞嘧啶。部分脱嘌呤DNA通过在0.1N HCl中沉淀与碱基分离。DNA沉淀的部分进一步通过0.1N HCl水解生成嘌呤碱基，或通过酶水解生成核苷和磷酸三酯。化学和酶消化产物通过高压液相色谱系统组合进行分离，以定量估算甲基化或乙基化DNA产生的以下产物：1-、3-和7-烷基腺嘌呤、O2-烷基胞嘧啶、3-、O6-和7-烷基鸟嘌呤以及O2-、3-和O4-烷基胸腺嘧啶。未检测到N6-烷基腺嘌呤、1-烷基鸟嘌呤和N2-烷基鸟嘌呤，检测到了3-烷基胞嘧啶但未进行定量。根据胸苷酰（3'-5'）胸苷回收的烷基磷酸三酯量估算磷酸三酯。使用这些方法，分别能够解释与[14C]甲磺酸甲酯、[3H]甲磺酸乙酯、N-[3H]-甲基-N-亚硝基脲和N-[14C]乙基-N-亚硝基脲反应的DNA中结合烷基的98%、81%、98%和92%。所述方法为单个DNA样品中所有已知的甲基化或乙基化产物提供了可重复的定量分析方法。
