The effects of spermine on plasmid DNA transmission through ultrafiltration membranes

Open Access
Author:
Bolten, Rachel Marie
Area of Honors:
Chemical Engineering
Degree:
Bachelor of Science
Document Type:
Thesis
Thesis Supervisors:
  • Andrew Zydney, Thesis Supervisor
  • Michael Janik, Honors Advisor
Keywords:
  • plasmid DNA
  • ultrafiltration
  • DNA purification
  • spermine
Abstract:
Although recent studies have demonstrated that ultrafiltration can be an effective method of plasmid DNA purification, the selectivity between the different plasmid isoforms is often below that needed for large-scale production of gene therapy agents. The objectives of this thesis were to evaluate the effects of spermine, a positively-charged polyamine, on the transmission of supercoiled, open-circular, and linear isoforms of plasmid DNA through ultrafiltration membranes and to determine whether the addition of spermine could facilitate isoform separation. Experiments were performed with Biomax 300 kDa ultrafiltration membranes. The linear and open-circular isoforms of the 9.8 kbp (kilo base pair) MDY plasmid were prepared by enzymatic digestion of the supercoiled plasmid. The addition of low concentrations of spermine (≤ 10 µM) increased transmission for all three plasmid isoforms, with the largest increase obtained with the supercoiled isoform. The use of spermine concentrations above 10 µM caused a significant reduction in plasmid transmission, likely due to the inhibition of plasmid elongation. The largest selectivity for the purification of the supercoiled isoform from the open-circular isoform was observed with the addition of 2 µM spermine in a 10 mM NaCl TE buffer. These results suggest that polyamines can be used to control the transmission of plasmid DNA during ultrafiltration and may be able to facilitate the separation of the different DNA isoforms due to the difference effects on isoform transmission.