Salt-induced Changes in Transmission of Linear, Open-circular and Supercoiled Plasmid DNA

Open Access
Author:
Currie, David P
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Andrew Zydney, Thesis Supervisor
  • Michael John Janik, Honors Advisor
Keywords:
  • ultrafiltration
  • plasmid
  • DNA
  • transmission
  • salt
  • linear
  • supercoiled
  • open-circular
Abstract:
Previous studies have demonstrated that ultrafiltration can be used to effectively purify plasmid DNA, potentially providing an opportunity for the large-scale production of DNA-based vaccines and gene therapy agents. However, the selectivity for the separation of the different DNA isoforms has typically been inadequate. The objectives of this thesis were to: (1) determine the effect of varying salt concentrations on the transmission of the linear, supercoiled, and open-circular isoforms of plasmid DNA through different ultrafiltration membranes, (2) compare the effects of monovalent (NaCl) and divalent (MgCl2) salts on plasmid transmission, and (3) examine the possibility of using salt concentration to improve the selectivity of isoform separation. Experiments were performed with UltraCel composite regenerated cellulose membranes having 100 and 1000 kDa nominal molecular weight cutoff along with 100kDa Biomax polyethersulfone membranes. Data were obtained using a 3 kilobase pair plasmid in Tris-EDTA buffer with varying NaCl and MgCl2 salt concentrations. Limited experiments were also performed using a 16.8 kbp plasmid. Sieving coefficients were evaluated using a PicoGreen fluorescence assay. The results show a strong increase in plasmid transmission with increasing salt concentration for all three isoforms. In addition, the critical flux for plasmid transmission decreased with increasing salt concentration for both the monovalent and divalent salts. MgCl2 has a much larger effect on plasmid transmission than NaCl, consistent with the greater increase in ionic strength and the possibility of intramolecular salt bridges. The effect of salt on transmission of the linear plasmid was less pronounced than that on either the open-circular or supercoiled plasmids, which could potentially be exploited for enhanced separations. These results provide valuable insights into the effects of salt concentration on plasmid ultrafiltration and on the possible optimization of plasmid isoform separations.