HIGH-PERFORMANCE MEMBRANE CHROMATOGRAPHY OF SUPERCOILED PLASMID DNA

Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynuc leotides on these stationary phases, taking a 7.2-kb predominantly sup ercoiled plasmid as example. Gradient and isocratic elution was studie d. In contrast to protein high-performance membrane chromatography (HP MC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in th e starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corr esponded to the three-albeit isolated-bands in the agarose gel, Presum ably the three fractions were supercoiled, nicked, and open circular p lasmid DNA. Linearization of the plasmid lowered the adsorption energy , and the linearized plasmid eluted earlier than the nonlinearized one . The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 x 52 mn , 10-mu m porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 x 35 mm), The results and characteristic difference s observed in these experiments were interpreted in the light of the n ewly developed theory of HPMC.