Singh R P, Michniewicz J J, Narang S A
Can J Biochem. 1976 Jul;54(7):600-8. doi: 10.1139/o76-089.
The existence of three infectious forms of potato spindle tuber viroid (PSTV) RNA from Scopolia sinensis was demonstrated by fractionation with high salt, by reverse phase and high pressure liquid chromatography, and by polyacrylamide gel electrophoresis. Purification of fraction II was achieved by the following steps: extraction of nucleic acid with phenol, precipitation of the RNA with cetyltrimethylammonium bromide, fractionation of the RNA with lithium chloride and isopropanol, and finally gel electrophoresis. A procedure using reverse phase chromatography was developed to obtain 70-90% recovery of RNA from polyacrylamide gels. Purified PSTV fraction II RNA was digested with ribonuclease A and T and labelled with [gamma-32P[ATP using polynucleotide kinase. The labelled digests were separated by the electrophoresis-homochromatography procedures of Sanger. About 20 and 30 spots were obtained with ribonuclease A and T, respectively.
通过高盐分级分离、反相和高压液相色谱以及聚丙烯酰胺凝胶电泳，证实了来自东莨菪的马铃薯纺锤块茎类病毒（PSTV）RNA存在三种感染性形式。II级分的纯化通过以下步骤实现：用苯酚提取核酸，用十六烷基三甲基溴化铵沉淀RNA，用氯化锂和异丙醇对RNA进行分级分离，最后进行凝胶电泳。开发了一种使用反相色谱的方法，从聚丙烯酰胺凝胶中获得70 - 90%的RNA回收率。纯化的PSTV II级分RNA用核糖核酸酶A和T消化，并用多核苷酸激酶用[γ-32P]ATP进行标记。标记的消化产物通过桑格的电泳-同系色谱法进行分离。用核糖核酸酶A和T分别得到约20个和30个斑点。
