A GENERAL-METHOD FOR DETECTING AND SIZING LARGE PLASMIDS

We have devised a method for detecting and estimating the sizes of lar ge bacterial plasmids in the presence of genomic DNA by pulsed-field g el electrophoresis (PFGE). Bacteria harboring plasmids were embedded i n agarose and lysed using a rapid protocol. Plugs were incubated with S1 nuclease and subjected to PFGE in agarose gels. S1 nuclease convert ed supercoiled plasmids into full-length linear molecules. Large plasm ids migrated as discrete bands that were readily observed after ethidi um staining. Their sizes were reliably estimated by comparison with li near DNA markers. Without S1 digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, m aking size determinations problematic. S1-PFGE detected megaplasmids u p to 609 kilobases (kb) in six genera of bacteria (Agrobacterium, Esch erichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus). The procedure gave size values consistent with previous estimates for cha racterized megaplasmids. Eight new plasmids between 102 and 316 kb wer e discovered in Klebsiella and Staphylococcus. S1-PFGE avoids the diff iculties of plasmid isolation, eliminates the preparation of probes, a nd does not require knowledge of restriction enzyme cleavage sites. It detects multiple large plasmids up to the limits of PFGE and can be u sed to screen for megaplasmids in many strains simultaneously. (C) 199 5 Academic Press, Inc.