Analysis and purification of plasmid DNA by reversed-phase high-performance liquid chromatography

Large nucleic acids can be separated by reversed-phase high-performance liquid chromatography. Under our experimental conditions, the retention time depends not on the chain length but rather on the base composition and the secondary structure of the molecule. Because of the torsional strain caused by the supercoiling of the plasmid, more of its bases are accessible for interaction with the hydrophobic stationary phase. This increases the retention time of the supercoiled DNA compared to the relaxed or linear DNA. We have exploited these properties to analyze the quality of plasmid preparations. The method is more sensitive to contaminants than common electrophoretic techniques. Furthermore, we describe a convenient and rapid procedure for purifying plasmid DNA. The highly pure plasmid is biologically more active for most of the enzymatic reactions commonly used in genetic engineering.