Gupta R C, Randerath K
Nucleic Acids Res. 1979 Aug 10;6(11):3443-58. doi: 10.1093/nar/6.11.3443.
A rapid, simple, and highly sensitive method for sequence analysis of RNA was developed, which consists of the following steps: (i) controlled hydrolysis of the RNA by brief heating in water; (ii) (32P)-labeling of 5'-hydroxyl groups of the fragments produced in (i); (iii) resolution of labeled fragments by size on polyacrylamide gels giving the familiar "ladder"; (iv) contact transfer ("print") of the ladder from the gel to a PEI-cellulose thin layer; (v) in situ treatment of the ladder with RNase T2 resulting in the release of 5'-(32P)-labeled nucleoside-3',5' diphosphates; (vi) contact transfer and thin-layer separation of (32P)-labeled nucleotides on PEI-cellulose in ammonium sulfate and ammonium formate solvents; (vii) autoradiography. The chromatographic behavior of the 4 major and 18 modified nucleotides was determined. The positions of major and modified nucleotides in the sequence can be read directly from the separation patterns displayed on X-ray film. As this is the only sequencing method presently available that allows one to display and identify directly the positions in the RNA chain of major and modified nucleotides, no additional procedures are required to analyze the latter.
开发了一种快速、简单且高度灵敏的RNA序列分析方法，该方法包括以下步骤：（i）通过在水中短暂加热对RNA进行可控水解；（ii）对步骤（i）中产生的片段的5'-羟基进行（32P）标记；（iii）通过聚丙烯酰胺凝胶按大小分离标记片段，得到常见的“梯状条带”；（iv）将梯状条带从凝胶接触转移（“印迹”）到聚乙烯亚胺纤维素薄层上；（v）用RNase T2对梯状条带进行原位处理，导致释放出5'-（32P）标记的核苷-3',5'-二磷酸；（vi）在硫酸铵和甲酸铵溶剂中，对聚乙烯亚胺纤维素上的（32P）标记核苷酸进行接触转移和薄层分离；（vii）放射自显影。测定了4种主要核苷酸和18种修饰核苷酸的色谱行为。主要核苷酸和修饰核苷酸在序列中的位置可直接从X射线胶片上显示的分离模式中读出。由于这是目前唯一一种能够直接显示和鉴定主要核苷酸和修饰核苷酸在RNA链中位置的测序方法，因此无需额外的程序来分析后者。
