title: Separation of plasmid DNA topoisomers by multimodal chromatography.
abstract: The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal.
authors.forename: A Rita
authors.initials: AR
authors.lastname: Silva-Santos
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal.
authors.forename: Cláudia P A
authors.initials: CP
authors.lastname: Alves
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal. Electronic address: miguelprazeres@tecnico.ulisboa.pt.
authors.forename: Duarte Miguel F
authors.initials: DM
authors.lastname: Prazeres
authors.affiliation: iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, 1049-001 Lisboa, Portugal. Electronic address: a.azevedo@tecnico.ulisboa.pt.
authors.forename: Ana M
authors.initials: AM
authors.lastname: Azevedo
mesh_terms: D002845:Chromatography / Q000379:methods*; D004270:DNA, Circular / Q000737:chemistry* / Q000302:isolation & purification*; D008024:Ligands; D015394:Molecular Structure; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification*; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D004270:DNA, Circular; D008024:Ligands; D012965:Sodium Chloride
keywords: DNA; Multimodal chromatography; Plasmid DNA; Supercoiling; Topoisomers