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 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.
Raucci
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Menarini Ricerche Spa, Pomezia-RM, Italy. graucci@sudbiotec.it
Maggi
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Parente
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D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004270:DNA, Circular / Q000737:chemistry; D004278:DNA, Superhelical / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods; D007536:Isomerism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation
D004270:DNA, Circular; D004278:DNA, Superhelical; D004247:DNA