核糖核酸的酶消化方法直接顺序分析

应用硷基特异性核酸内切酶进行RNA的顺序分析。以~(32)P标记RNA分子的5′或3′末端的四种硷基能被酶试剂部分裂解。放射性的水解产物经胶电泳按其大小而分离。放射自显影后它的核苷酸顺序从带谱上可以直接读出。核糖核酸酶(RNasc)T_1(G特异性)、U_2(A特异性)、Phy M(U+A特异性)和B.cereus(C+U特异性)是最常用于顺序RNA的四种酶。

DIRECT ENZYMATIC SEQUENCING OF RNA

A method for sequencing RNA is described using base-specific endonucleases. RNA molecules labelled with 32P at 5' or 3' end can be partially cleaved at each of the four bases using enzymatic agents. The resutanl radioactive products are separated by high resolution polyacrylamide slab gel electrophoresis by sixe. An autoradiograph of such a gel displays a series of bands where each band correspond to a discrete nucieotide fragment, and the nucleotide sequence is read directly from this banding pattern. RNase T1 (guanine specific), U2 ( adenine specific), phy M(uracil and adenine specific) and B.cereus ( cytosine and uracil specific) are the most useful bccau.se of relative fidelity of cleavage at the appropriate bases.