Background

Plasmid DNA appears in different topologies. Intact plasmids are covalently closed circular (ccc-form) and negatively supercoiled.

The most important topology with respect to an effective gene transfer in vitro as well as in vivo is the ccc-form because of its small size which makes it easier to pass the cell membrane and therefore to transfer it into the cell.

Single strand breaks due to enzymatic activity or mechanical stress lead to a relaxation of the DNA molecule. The resulting form is the so called open circular (oc) topology. Restriction endonucleases digest the DNA at certain positions resulting in the linear form. Mistakes in the cellular replication procedure and homologous replication lead to multimeric plasmid forms. These multimers are concatemers of two or more monomer forms, where also ccc, oc and linear forms can be observed.

The different topologies can be visualized using agarose gel electrophoresis with subsequent staining of the DNA bands with a fluorescent dye. However, the identification of the respective bands is difficult because the mobility of the different structures changes during the electrophoretic procedure. For most plasmids the ccc monomer is the fastest moving form. The oc monomer and ccc dimer very often appear as one band and cannot be distinguished by an agarose gel electrophoresis. Here, the CGE is the only reliable tool.