Capillary electrophoresis of supercoiled DNA molecules: Parameters governing the resolution of topoisomers and their separation from open forms

We describe the separation of covalently closed and open circular DNA forms with capillary electrophoresis, This technique is expected to be applied i n the research of novel anticancer molecules targeting the activity of topo isomerase I. The separation of a plasmid mixture containing fully supercoil ed 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 po lymers. 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 analys is, We identified three main parameters that influence the separation: (I) Low concentrations of MgCl2 in the separation buffer are required for a goo d resolution of topoisomers, (II) Cellulose derivatives can be used as siev ing polymers; in our hands, HPMC and HEC worked best. (III) High molecular mass forms of sieving polymers allow the best separations.