Ligation and supercoiling of plasmids containing single strand interruptions

Abstract We have developed a simple procedure for determining if a plasmid DNA contains single strand interruptions that can be sealed by DNA ligase and thus contain single strand interruptions with 5'-phosphate and 3'-hydroxyl groups. In this procedure, plasmid DNAs with ligatable nicks were ligated by T4 DNA ligase, supercoiled by E. coli DNA gyrase and analyzed by agarose gel electrophoresis. Our results show that after sealing nicked circular DNA with DNA ligase, supercoiling by DNA gyrase produces a rapidly migrating DNA species that can easily be distinguished from nicked circular DNA by agarose gel electrophoresis.