A Novel Thermo-Gelling Matrix for Microchannel DNA Sequencing (23110
Northwestern investigators have created novel "thermo-gelling" polymer networks for use as DNA sequencing matrices for high-throughput microchannel electrophoresis in capillary arrays. Capillary array electrophoresis with these networks provides efficient resolution of both small and large DNA sequencing fragments and longer sequence read lengths with high accuracy.
ADVANTAGE: The matrices are easily loaded into electrophoresis capillary arrays at room temperature but thermogel above 35ºC to form transparent hydrogels providing DNA sequencing with enhanced resolution, longer sequencing read lengths and accuracy in shorter time than non-thermo-responsive polymer matrices. The reversible thermo-gelation enables replacement of the sieving matrix between runs, eliminating sample carry-over between analyses and associated experimental problems. Polymer matrix wall-coating properties also suppress capillary electro-osmotic flow.
SUMMARY: Size-based electrophoretic separation of DNA is critical in genomic research. Miniaturized DNA electrophoresis within fused-silica capillaries greatly increases analysis speed, reduces reagent consumption, and facilitates automation and quantitation. To date, the best sequencing performance has been obtained with ultra-high-molar-mass (> 10 million g/mole), linear polyacrylamide (LPA). However, the high-molar-mass polymer solutions used in microchannel electrophoresis, for improved sequencing resolution and read length, have very high low-shear viscosity, necessitating high-pressure loading into microchannels. High-pressure matrix replacement contributes significantly to the construction and maintenance costs of microchannel electrophoresis instruments. Moreover, the low pressure tolerance of most microfluidic devices prohibits use of such high resolution polymer systems. LPA matrices also require capillary wall modification to suppress electro-osmotic flow for reproducible separations.
The development of polymeric matrices with "switchable viscosities" enables the decoupling of capillary loading and DNA separation properties, and promises the use of glass/plastic microchips for high-throughput DNA sequencing. This invention provides a novel class of thermo-gelling polymers offering a DNA sequencing matrix with easy capillary loading, excellent sieving performance and "dynamic" (adsorptive) wall-coating. A unique combination of N-ethoxyethylacrylamide (pNEEA) and N-methoxyethylacrylamide (NMEA) monomers was employed to generate a thermo-responsive copolymer matrix (~2.0 MDa, 1.45 PDI) that exhibits minimal viscosity ~600 cP at 35˚C, that rapidly increases to ~20,000 cP between 35 – 45˚C in a DNA sequencing buffer medium. Electrophoretic analysis of a MegaBACE DNA sequencing standard with the matrix gave a 600-base read at 98.5% base-calling accuracy at 44˚C in 100 minutes. The copolymer exhibits good adsorptive wall-coating ability with a 100-fold reduction in electro-osmotic mobility versus that of an uncoated capillary. The demonstrated properties of these polymer matrices promise excellent performance in capillary electrophoresis and microfluidic device applications.
Annelise Barron and Cheuk Wai
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