Anal. Chem. 1997, 69, 1192-1196
Capillary Electrophoresis of Supercoiled and Linear DNA in Dilute Hydroxyethyl Cellulose Solution Richard W. Hammond,† Hidehiro Oana,†,‡ Jeffrey J. Schwinefus,† Jeffrey Bonadio,§ Robert J. Levy,| and Michael D. Morris*,†
Department of Chemistry, Department of Pathology, School of Medicine, and Department of Pediatrics and Communicable Diseases, School of Medicine, University of Michigan, Ann Arbor, Michigan 48109
Capillary electrophoresis in dilute hydroxyethyl cellulose is shown to separate supercoiled DNA in the size range 2000-16 000 base pairs. The plasmids migrate more slowly than linear ds-DNA of the same sizes. Plasmid bandwidths are larger than observed for ds-DNA, allowing identification of the type of DNA by bandwidth. The differing dependence of mobility on chain length can be explained by assuming that a plasmid migrates as an elastic rod, while ds-DNA migrates as a wormlike chain. Capillary electrophoretic (CE) separation of both double- and single-stranded linear deoxyribonucleic acid (DNA) has been widely investigated.1-3 Most CE separations of linear DNA are performed in entangled solutions of linear polymers such as polyacrylamide (PAA), hydroxyethyl cellulose (HEC), or poly(ethylene oxide).1-3 These widely employed CE media bear some resemblance to gels. The loose network of entangled polymers contains openings through which nucleic acids can migrate. Relative to gels, CE separations of linear DNA in polymer solutions have shorter analysis times and higher resolution. Although the separations work well, migration times can still be long because the solutions have high viscosities. In this paper, we investigate the prospect for analytically useful separations of plasmids. In nature, plasmids are bacterial DNA sequences that replicate independently of the rest of the genome and therefore have a degree of independence from host cells.4 Many bacterial plasmids carry genes for antibiotic resistance. In DNA cloning, a DNA fragment that contains a gene of interest is inserted into the purified DNA genome of a self-replicating genetic element such as a plasmid. Plasmids used for this purpose are referred to as cloning vectors or expression vectors. Generally, plasmid vectors used for gene cloning are small circular molecules of doublestranded DNA derived from larger plasmids that occur naturally in bacterial cells.
Following its purification in vitro, plasmid DNA predominantly exists in a supercoiled conformation.5 There are indications that the gel electrophoretic behavior of supercoiled DNA is different from that of linear DNA. Hightower and Santi6 reported separations using orthogonal field agarose gel electrophoresis (OFAGE) in which the mobilities of plasmids ranging from 20 to 60 thousand base pairs (kbp) actually increase with size. Wang and Lai7 report separations for plasmids up to 300 kbp using conventional gel electrophoresis with mobility inversely proportional to size (that is, no band inversion). We were surprised to find only one previous report pertaining to CE of circular double-stranded DNA, especially given that this natural form of DNA represents an essential tool of recombinant DNA technology and consequently, biotechnology. In this report Nackerdien and co-workers,8 using PAA as a separating media, demonstrated that a 3890-bp plasmid migrates more rapidly than its nicked form but more slowly than the linear form. The separation was completed in 45-50 min. In general,6,7 small (
