PicoSep - A Microfluidic Platform for Single Molecule DNA and RNA Sizing

Project Summary In recent yearsrising importance is being placed on high quality sample preparationparticularly when expensive downstream genomic analysis techniques such as sequencing and genome mapping are involvedHoweverlimited tools exist to verify the quality of DNA RNA beyond UV absorbance measurements to determine purityfluorescence assays to determine yieldand electrophoretic methods to determine sizeNew alternatives to electrophoresis could significantly facilitate sample preparation QCparticularly in applications where improvements in speedsensitivityand performance are necessary or where new metrics could be usefulIn Phase Iwe developed an instrument called PicoSep that integrated inline isotachophoresisITPpreconcentration and single molecule free solution hydrodynamic separation to create one of the most sensitive DNA sizing platforms seen to dateSeparation occurs through a unique hydrodynamic mechanism that enables the separation of both largekband small DNAbpusing only pressure and a bufferfilled microchannel or capillarym IDsizing performance unmatched by any single electrophoresis platformDuring this workwe also discovered a novelelectrode free concentration method called counter flow concentrationCFCUsing CFC or ITPwe can enhance sensitivity up to PCR levelsIn this Phase II proposalwe will develop a high throughput PicoSep instrument and leverage PicoSep s unique properties to facilitate sample preparation in two critical applicationsFirstwe will develop a kit for high molecular weight DNA sizing to replace slow and insensitive pulsed field gel electrophoresis analysis in long read sequencing and genome mappingSecondwe will develop a kit for RNA integrity analysisRNA integrity is the dominant metric used to determine RNA quality and degradationPicoSep s single molecule sensitivity will enable detection of minute amounts of degradation with near zero sample consumptionThese sizing based kits will serve as the initial products while future kits will leverage PicoSep s other single molecule capabilities to determine advanced DNA RNA metrics such as damage from abasic sites and oxidation