(301a) Size-Based Separation of Supercoiled Plasmid DNA Using Ultrafiltration

Most previous studies of membrane-based separations have shown no effect of DNA size on plasmid transmission through small pore size ultrafiltration membranes, consistent with the predicted behavior for the elongation of highly flexible polymer chains. However, supercoiled plasmids are known to have a â??branchedâ? structure with the number of branches dependent on the DNA length. This difference in branching could lead to a significant dependence of the transmission on the plasmid size, providing opportunities for size-based separations using ultrafiltration.

Data were obtained with 3.0, 9.8, and 16.8 kbp plasmids using both cellulosic and polyethersulfone ultrafiltration membranes with different nominal molecular weight cutoffs. Initial experiments were performed with purified samples of the supercoiled or linear isoforms, with the results used to identify appropriate conditions for plasmid separation.

Plasmid transmission increased with increasing filtrate flux due to the elongation of the plasmids in the converging flow field. However, the flux dependence was different for each plasmid due to differences in the extent of branching of the twisted supercoiled DNA. This behavior provided a significant selectivity that could be used to separate the 3.0 and 16.8 kbp supercoiled plasmids using small pore size ultrafiltration membranes. In contrast, transmission of the linear (un-branched) plasmids was independent of the plasmid size. These results provide important insights into the use of ultrafiltration for the purification of plasmid DNA.

 

Keywords: Ultrafiltration, DNA, supercoiled, plasmid, membrane separation