A novel thermogelling matrix for microchannel DNA sequencing based on poly-N-alkoxyalkylacrylamide copolymers.

Abstract

We have developed a novel class of thermogelling polymer networks based on poly-N-alkoxyalkylacrylamides, and demonstrated their use as DNA sequencing matrices for high-throughput microchannel electrophoresis in capillary arrays. Polymers and copolymers of N-ethoxyethylacrylamide (NEEA) and N-methoxyethylacrylamide (NMEA) were synthesized by aqueous-phase free-radical polymerization and characterized by tandem gel permeation chromatography-multi-angle laser light scattering. These copolymer matrices exhibit "re-entrant"-type volume phase transitions, forming entangled networks with high shear viscosity at low (< 20 degrees C) and high (> 35 degrees C) temperatures, and undergoing a "coil-to-globular", lower critical solution temperature (LCST)-like phase transition over an intermediate temperature range (20-35 degrees C). Hence, matrix viscosity is relatively low at room temperature (25 degrees C), and increases rapidly above 35 degrees C. The material properties and phase behavior of these thermogelling polymer networks were studied by steady-shear rheometry. These matrices are easily loaded into capillary arrays at room temperature while existing as viscous fluids, but thermogel above 35 degrees C to form transparent hydrogels via a thermo-associative phase transition. The extent of the intermediate viscosity drop and the final viscosity increase depends on the composition of the copolymers. DNA sequencing by capillary array electrophoresis with four-color laser-induced fluorescence (LIF) detection shows that these thermogelling networks provide enhanced resolution of both small and large DNA sequencing fragments and longer sequencing read lengths, in comparison to appropriate control (closely related, nonthermogelling) polymer networks. In particular, a copolymer comprised of 90% w/w NMEA and 10% w/w NEEA, with a molecular mass of approximately 2 MDa, delivers around 600 bases at 98.5% base-calling accuracy in 100 min of electrophoresis.