Wilson V L, Smith R A, Autrup H, Krokan H, Musci D E, Le N N, Longoria J, Ziska D, Harris C C
Anal Biochem. 1986 Feb 1;152(2):275-84. doi: 10.1016/0003-2697(86)90409-4.
A simple and sensitive method for the quantitation of 5-methyldeoxycytidine in DNA has been developed by the adaptation of the Randerath 32P-postlabeling technique. Nucleic acids were digested to 3'-monophosphate nucleotides, which were converted to 32P-labeled 3',5'-bisphosphate nucleotides, the 3'-phosphate was cleaved by the action of nuclease P1, and the resultant 5'-[32P]-monophosphate nucleotides were separated by two-dimensional thin-layer chromatography. Less than 1 microgram of DNA was required for the precise quantitation of 5-methyldeoxycytidine content to a detectable limit of 0.01% of the total cytidine residues methylated. The genomic 5-methyldeoxycytidine content may thus be quantitated in tissue samples, small or selective cell populations, senescing or terminally differentiating cells, or DNA from any source. We report here, for the first time, the genomic 5-methyldeoxycytidine content of normal human bronchial epithelial and normal human pulmonary mesothelial cells. The chromatographic separation of all of the normal and some of the rare monophosphate deoxyribonucleotides and ribonucleotides has been characterized. Thus, 5-bromodeoxyuridine and the RNA contamination of DNA or the DNA contamination of RNA can also be quantitated during the same analysis.
通过对兰德拉斯32P后标记技术进行改进，开发出了一种简单且灵敏的定量DNA中5 - 甲基脱氧胞苷的方法。核酸被消化成3'-单磷酸核苷酸，然后转化为32P标记的3',5'-双磷酸核苷酸，3'-磷酸在核酸酶P1的作用下被切割，所得的5'-[32P]-单磷酸核苷酸通过二维薄层色谱法进行分离。精确测定5 - 甲基脱氧胞苷含量至可检测极限（占总甲基化胞苷残基的0.01%）所需的DNA量少于1微克。因此，基因组5 - 甲基脱氧胞苷含量可在组织样本、小的或选择性细胞群体、衰老或终末分化细胞或任何来源的DNA中进行定量。我们在此首次报告正常人支气管上皮细胞和正常人肺间皮细胞的基因组5 - 甲基脱氧胞苷含量。已对所有正常的和一些稀有的单磷酸脱氧核糖核苷酸及核糖核苷酸的色谱分离特性进行了表征。因此，在同一分析过程中也可对5 - 溴脱氧尿苷以及DNA的RNA污染或RNA的DNA污染进行定量。
