A novel electrophoretic mechanism and separation parameter for selective nucleic acid concentration based on synchronous coefficient of drag alteration (SCODA)

• Main Author: Pel, Joel
• Format: Others
• Language: English
• Published: University of British Columbia 2009
• Online Access: http://hdl.handle.net/2429/13402

Molecular manipulation and separation techniques form the building blocks for much of fundamental science, yet many separation challenges still remain, in fields as diverse as forensics and metagenomics. This thesis presents SCODA (Synchronous Coefficient of Drag Alteration), a novel and general molecular separation and concentration technique aimed at addressing such challenges. SCODA takes advantage of physical molecular properties associated with the non‐linear response of long, charged polymers to electrophoretic fields, which define a novel parameter for DNA separation. The SCODA method is based on superposition of synchronous, time-varying electrophoretic fields, which can generate net drift of charged molecules even when the time-averaged molecule displacement generated by each field individually is zero. Such drift can only occur for molecules, such as DNA, whose motive response to electrophoretic fields is non-linear. This thesis presents the development of SCODA for extraction of DNA, and outlines the design of the instrumentation required to achieve the SCODA effect. We then demonstrate the selectivity, efficiency, and sensitivity of the technique. Contaminant rejection is also quantified for humic acids and proteins, with SCODA displaying excellent performance compared to existing technologies. Additionally, the ability of this technology to extract high molecular weight DNA is demonstrated, as is its inherent fragment length selection capability. Finally, we demonstrate two applications of this method to metagenomics projects where existing technologies performed poorly or failed altogether. The first is the extraction of high molecular weight DNA from soil, which is limited in length to fragments smaller than 50 kb with current direct extraction methods. SCODA was able to recover DNA an order of magnitude larger than this. The second application is DNA extraction from highly contaminated samples originating in the Athabasca tar sands, where existing technology had failed to recover any usable DNA. SCODA was able to recover sufficient DNA to enable the discovery of 200 putatively novel organisms.