DIRECT CHEMICAL METHOD FOR SEQUENCING RNA
DIRECT CHEMICAL METHOD FOR SEQUENCING RNA
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DOI：
10.1073/pnas.76.4.1760
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
PEATTIE, DA
中科院分区：
文献类型：
--
作者：
PEATTIE, DA
四种不同的碱基特异性化学反应产生了一种直接测序末端标记为32P的[酿酒酵母]RNA的方法。在对每种RNA碱基进行部分特异性修饰后，胺催化的链断裂产生标记片段，其长度决定了序列中每个核苷酸的位置。硫酸二甲酯修饰鸟苷。焦碳酸二乙酯主要攻击腺苷。联氨攻击尿嘧啶和胞嘧啶，但盐抑制与尿嘧啶的反应。在所有情况下，苯胺诱导随后的链断裂。标记的片段在聚丙烯酰胺凝胶上进行电泳分离，然后进行放射自显影，确定RNA序列。在3”末端标记的RNA为每个嘌呤和嘧啶产生干净的切割模式，并允许从标记的末端确定整个RNA序列到100-200个碱基。
Four different base-specific chemical reactions generate a means of directly sequencing [Saccharomyces cerevisiae] RNA terminally labeled with 32P. After a partial specific modification of each kind of RNA base, an amine-catalyzed strand scission generates labeled fragments whose lengths determine the position of each nucleotide in the sequence. Dimethyl sulfate modifies guanosine. Diethyl pyrocarbonate attacks primarily adenosine. Hydrazine attacks uridine and cytidine, but salt suppresses the reaction with uridine. In all cases, aniline induces a subsequent strand scission. The electrophoretic fractionation of the labeled fragments on apolyacrylamide gel, followed by autoradiography, determines the RNA sequence. RNA labeled at the 3'' end yields clean cleavage patterns for each purine and pyrimidine and allows a determination of the entire RNA sequence out to 100-200 bases from the labeled terminus.