Dubelman S, Shapiro R
Nucleic Acids Res. 1977 Jun;4(6):1815-27. doi: 10.1093/nar/4.6.1815.
A procedure is reported for the isolation of cross-linked nucleosides from nitrous acid-treated calf thymus DNA. Cross-linked DNA was hydrolyzed enzymatically with deoxyribonuclease I and snake venom phosphodiesterase and fractionated on a DEAE-Sephadex column. After desalting, the fractions were characterized by ultraviolet spectroscopy, anion exchange high pressure liquid chromatography, gel filtration, and two dimensional thin layer chromatography. A cross-linked dinucleotide, and a series of oligonucleotides were isolated. The oligomers, which had resisted digestion by the above enzyme system, were digested to the nucleoside level by a spleen phosphodiesterase-alkaline phosphatase combination. A second cross-linked product was isolated from this mixture. The cross-linked nucleosides were less than 0.17% of the total nucleotides of the DNA. The methods developed here are recommended for the isolation of products from DNA treated with other cross-linking agents.
本文报道了一种从亚硝酸处理的小牛胸腺DNA中分离交联核苷的方法。交联DNA用脱氧核糖核酸酶I和蛇毒磷酸二酯酶进行酶解，并在DEAE-葡聚糖凝胶柱上进行分离。脱盐后，通过紫外光谱、阴离子交换高压液相色谱、凝胶过滤和二维薄层色谱对各馏分进行表征。分离出一种交联二核苷酸和一系列寡核苷酸。这些抗上述酶系统消化的寡聚物，通过脾磷酸二酯酶-碱性磷酸酶联合作用消化至核苷水平。从该混合物中分离出第二种交联产物。交联核苷占DNA总核苷酸的比例不到0.17%。本文开发的方法推荐用于从用其他交联剂处理的DNA中分离产物。
