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Abstract:
Over the last few years plasmid DNA has gained more and more importance as a non-viral vector for gene therapy or as DNA vaccine. Given that plasmid DNA is less effective at transfecting cells than viral vectors methods to produce large quantities of supercoiled plasmid DNA in a short time have become topics of research. In most protocols however downstream processing still is a major bottleneck, especially the time-consuming loading of chromatographic columns. In this work we describe a fast method for capturing plasmid DNA from bacterial lysate which sidesteps this problem. For large volumes of feedstocks column loading becomes very time-consuming as the flow rate is limited by back pressure.
Since adsorption of nucleic acids on anion-exchange adsorbers is very fast process time can be shortened dramatically by using batch adsorption. Furthermore, if the ratio of lysate and stationary volume exceeds a certain point differences in adsorption behaviour between plasmid DNA and RNA can be observed and used for purification. In order to further minimise process time all washing and desorption steps are also carried out in batch mode. Here the advantages of using a custom built funnel with metal filter become obvious as it greatly simplifies the resuspension of the stationary phase and allows on-filter flushing.
