Donis-Keller H, Maxam A M, Gilbert W
Nucleic Acids Res. 1977 Aug;4(8):2527-38. doi: 10.1093/nar/4.8.2527.
The positions of adenines, guanines, and pyrimidines can be determined by partial nuclease digestion of a terminally labeles RNA molecule. In urea, at elevated temperatures, RNase T1 generates a pattern reflecting cleavage at guanines while RNase U2 cleaves only at adenine. A limited alkaline hydrolysis provides a continuum of fragments derived from breaks at every phosphodiester bond. The reaction products are electrophoretically fractionated by size in adjacent lanes of a polyacrylamide gel. An autoradiograph of the gel displays the sequence up to 100 nucleotides from the end of the molecule, although uracil cannot as yet be distinguished from cytosine. These techniques form the basis of an RNA sequencing method and are demonstrated on yeast 5.8S ribosomal RNA.
腺嘌呤、鸟嘌呤和嘧啶的位置可通过对末端标记的RNA分子进行部分核酸酶消化来确定。在尿素存在的情况下，高温时，核糖核酸酶T1产生反映鸟嘌呤处切割情况的图谱，而核糖核酸酶U2仅在腺嘌呤处切割。有限的碱性水解可提供源自每个磷酸二酯键断裂的一系列片段。反应产物通过聚丙烯酰胺凝胶相邻泳道按大小进行电泳分离。凝胶的放射自显影片可显示出分子末端长达100个核苷酸的序列，尽管目前尿嘧啶和胞嘧啶无法区分。这些技术构成了一种RNA测序方法的基础，并在酵母5.8S核糖体RNA上得到了验证。
