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Description
Purpose: To investigate whether centrifugal ultrafiltration (CUF) can be used to isolate and quantify the linear (L) isoform of plasmid DNA, which corresponds to DNA double strand breaks (DSBs) arising from exposure to ionizing radiation. Methods: Plasmid pALD CV77 DNA (Aldevron (Fargo, ND)) was pipetted into 1.5 mL microcentrifuge tubes and irradiated to 0 Gy (control), 25 Gy, and 50 Gy using the CellRad x-ray irradiator (Precision X-ray Irradiation, Inc., Connecticut). After the irradiations, the plasmid DNA was transferred to Nanosep® centrifugal devices (Pall Corporation, Inc) composed of modified polyether sulfone (PES) ultrafiltration membranes to separate the feed/retentate from the filtrate. To perform the centrifugal ultrafiltration, the Nanosep® devices were placed in the Symphony 2417R refrigerated microcentrifuge (Avantor, Inc) and run until the plasma DNA feed solution passed through the ultrafiltration membrane. After centrifuging the samples, the initial feed volume was added back to the retentate as TE buffer for collection, and the filtrate was also collected. Several parameters including DNA concentration, temperature, centrifugal force, NaCl concentration, and buffer (Potassium phosphate (PP) versus Deionized water (DI water)) that affect the transmission of L isoforms and retention of open-circular (OC isoforms) and supercoiled (SC) isoforms, were varied to determine the optimal condition for separating and isolating each plasmid DNA isoform. DNA isoform yields in the retentate, and the filtrate were assessed using agarose gel electrophoresis (AGE). The OC and L isoform gel bands corresponding to DNA single and double strand breaks, respectively, were then imaged and analyzed using CLIQS image quantification software (Totallab Ltd) and the yields were computed. Results: Varying the NaCl concentration from 0 to 100 mM resulted in < 1% transmission of the linear isoform at 25 Gy and 50 Gy. The optimal concentration and temperature for the transmission of the linear isoforms were 2 ng/μL and 24°C. Conclusion: CUF was found to perform well for concentrating the SC plasmid DNA. However, CUF did not perform well for isolating the L isoforms that were generated from irradiating the SC plasmid DNA. Hence, CUF is impractical to use as a tool for accurate DNA DSB quantification.
