Tabler M, Günther I, Kern R, Sänger H L
Max-Planck-Institut für Biochemie, Planegg-Martinsried, F.R.G.
J Virol Methods. 1989 Feb;23(2):111-26. doi: 10.1016/0166-0934(89)90125-0.
A microscale procedure for the isolation and purification of viroid RNA from one gram of viroid-infected leaf tissue and for its subsequent sequencing at the cDNA level is described using potato spindle tuber viroid (PSTV) as model system. Total nucleic acids are phenol-extracted and salt-fractionated with 2 M LiCl. The viroid-containing fraction is then subjected to bidirectional polyacrylamide gel electrophoresis. This removes all co-fractionated cellular RNAs from the circular viroid RNA which is finally recovered from the gel in pure form by isotachophoresis. Thus, from one gram of PSTV-infected tomato leaf tissue, about 100-250 ng of circular PSTV RNA can be obtained and used as template for several DNA primer-directed reverse transcription reactions. From the primer-extended overlapping cDNAs the entire sequence of the viroid progeny synthesized in an individual plant or plant leaf can thus be established by Maxam-Gilbert sequencing. This renders the procedure especially suited for the routine analysis of the in vivo fate of viroid mutants constructed in vitro.
本文描述了一种从1克感染类病毒的叶片组织中分离和纯化类病毒RNA，并随后在cDNA水平进行测序的微量方法，该方法以马铃薯纺锤块茎类病毒（PSTV）作为模型系统。总核酸用苯酚提取并用2M LiCl进行盐分级分离。然后将含类病毒的级分进行双向聚丙烯酰胺凝胶电泳。这从环状类病毒RNA中去除了所有共分级的细胞RNA，最终通过等速电泳从凝胶中以纯形式回收环状类病毒RNA。因此，从1克感染PSTV的番茄叶片组织中，可以获得约100-250纳克的环状PSTV RNA，并用作几个DNA引物定向逆转录反应的模板。通过Maxam-Gilbert测序，可以从引物延伸的重叠cDNA中确定在单个植物或植物叶片中合成的类病毒后代的完整序列。这使得该方法特别适合于对体外构建的类病毒突变体的体内命运进行常规分析。
