Isolation of inverted repeat sequences, including IS1, IS2, and IS3, in Escherichia coli plasmids.

Abstract

A method is described for isolation of inverted repeat DNA sequences that occur in E. coli plasmids. The isolation procedures involved denaturation of intact plasmid DNA, a rapid (30 s) renaturation of inverted repeat sequences in the genome, digestion of the single-stranded portion by S1 nuclease to recover duplex DNA, and detection and purification of the duplexes using 1.4% agarose gel electrophoresis. If a plasmid DNA carried inverted repeats of 1 type or 2 different types of special DNA sequences, these procedures showed either 1 or 2 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, indicating that the inverted repeat sequences must be in the same strand. Several repeated sequences, including IS[insertion sequence]1, IS2 and IS3, from derivatives of F and R plasmids were isolated by this method.