Capillary Electrophoresis of Supercoiled DNA Molecules:  Parameters Governing the Resolution of Topoisomers and Their Separation from Open Forms

Abstract

We describe the separation of covalently closed and open circular DNA forms with capillary electrophoresis. This technique is expected to be applied in the research of novel anticancer molecules targeting the activity of topoisomerase I. The separation of a plasmid mixture containing fully supercoiled molecules, single topoisomers, and their relaxed and open circular forms was tested in an electric field of 200 V/cm using Tris/borate buffer with the addition of magnesium ions at low concentrations and various sieving polymers. The resulting separation is quite simple to achieve and is clearly comparable to that obtained in agarose gels run at low voltage, but with an improved resolution, a higher quantitativity, and a higher speed of analysis. We identified three main parameters that influence the separation: (I) Low concentrations of MgCl2 in the separation buffer are required for a good resolution of topoisomers. (II) Cellulose derivatives can be used as sieving polymers; in our hands, HPMC and HEC worked best. (III) High molecular mass forms of sieving polymers allow the best separations.

Novel hydroxyethylcellulose‐graft‐poly acrylamide copolymer for separation of double‐stranded DNA fragments by CE

“…The separation medium with these three simultaneous specifications can be used to facilitate the automation of CE and to further enhance its performance [1]. For a given homopolymer, such as linear polyacrylamide (LPA) and its derivatives [2][3][4][5], polyvinylpyrrolidone (PVP) [6], poly(ethylene oxide) (PEO) [7], cellulose and its derivatives [8][9][10], it has difficulty optimizing the three desired specifications. For example, LPA is well known for its sieving properties.…”

A rapid high-resolution method for resolving DNA topoisomers

“…It works by separating ions based on their electrophoretic mobility with the use of an applied voltage and is often performed in sub-millimeter diameter capillaries and micro- and nanofluidic channels. The separation of covalently-closed and open-circular DNA forms with capillary electrophoresis has been described previously [ 16 ], but only plasmid pUC18 was examined and the resolution of only a few topoisomers was reported. In addition, the use of capillary gel electrophoresis with laser-induced fluorescence has been described for the detection of linear, open circle, and supercoiled plasmid DNAs [ 17 ].…”

“…In this case, the resolution of relaxed DNA (from the open circle form) and the separation of individual topoisomers of different linking numbers were not reported. In neither case was the technique suitable for use in a high-throughput format [ 16 , 17 ]. Despite these previous reports, the use of capillary gel electrophoresis has not been widely adopted for analysing topoisomers of circular DNAs and gel electrophoresis remains the predominant analytical method.…”

Capillary gel electrophoresis with laser‐induced fluorescence of plasmid DNA in untreated capillary

“…The use of CGE for the analysis of plasmid isoforms was first reported over ten years ago. Since then, the majority of CGE methods that have been developed have employed the use of coated capillaries to suppress the EOF and eliminate or reduce capillary wall interactions 7–16. Since coated capillaries are typically more expensive and have shorter lifetimes, the use of uncoated capillaries would be ideal for routine analysis.…”