Ohtsubo H, Ohtsubo E
Proc Natl Acad Sci U S A. 1976 Jul;73(7):2316-20. doi: 10.1073/pnas.73.7.2316.
A method is described for isolation of inverted repeat DNA sequences that occur in E. coli plasmids. The procedures of the isolation involved: (a) denaturation of intact plasmid DNA, (b) a rapid, 30 sec, renaturation of inverted-repeat sequences in the genome, (c) digestion of the single-stranded portion by S1 nuclease to recover duplex DNA, and (d) detection and purification of the duplexes using 1.4% agarose gel electrophoresis. If a plasmid DNA carried inverted repeats of either one type or two different types of special DNA sequences, these procedures enabled us to observe either one or two characteristic DNA bands, respectively, in the agarose gels. If a plasmid DNA did not carry any inverted repeats, or if the plasmid DNA only carried direct repeat sequences, no characteristic DNA bands were recovered. Cleavage of the spacer DNA between inverted repeat sequences generated no gel bands. This indicated that the inverted repeat sequences must be in the same strand. Using this method, we isolated and purified several repeated sequences, including IS1, IS2, and IS3, from derivatives of F and R plasmids.
本文描述了一种从大肠杆菌质粒中分离反向重复DNA序列的方法。分离步骤包括：（a）完整质粒DNA的变性；（b）基因组中反向重复序列快速复性30秒；（c）用S1核酸酶消化单链部分以回收双链DNA；（d）使用1.4%琼脂糖凝胶电泳检测和纯化双链体。如果质粒DNA携带一种或两种不同类型特殊DNA序列的反向重复序列，这些步骤能使我们分别在琼脂糖凝胶中观察到一条或两条特征性DNA条带。如果质粒DNA不携带任何反向重复序列，或者仅携带正向重复序列，则不会回收特征性DNA条带。切割反向重复序列之间的间隔DNA不会产生凝胶条带。这表明反向重复序列必须在同一条链上。使用该方法，我们从F质粒和R质粒的衍生物中分离并纯化了几个重复序列，包括IS1、IS2和IS3。
