Thermo-responsive N,N-dialkylacrylamide copolymer matrices for microchannel DNA sequencing: Synthesis, characterization, and testing by capillary array electrophoresis.

摘要

The implementation of capillary array electrophoresis (CAE) has revolutionized genome sequencing by increasing automation and throughput relative to slab gel electrophoresis. Further increases in throughput are offered by microfabricated genetic analysis devices. However, automated, low-pressure loading of viscous polymeric matrices in chip microchannels remains an unmet challenge. This research has involved the development of thermo-responsive N,N-dialkylacrylamide copolymers exhibiting a reversible, volume phase transition in aqueous solution at a given lower critical solution temperature. The phase transition results in a precipitous drop in the viscosity of the polymer solution at the transition temperature, and can be exploited to allow loading into electrophoresis microchannels under reasonable applied pressure. This thermally controlled viscosity switch enables decoupling of the loading and sieving properties of the entangled polymer solution by simultaneously providing the DNA analysis typically expected from high-viscosity solutions and the rapid, low-pressure loading typically expected from low-viscosity solutions. The copolymers were characterized by a tandem size exclusion chromatography - multi-angle laser light scattering system, UV-Vis spectrophotometry, and temperature-controlled rheometry. The copolymer matrices were also tested by CAE. A copolymer formulation of 42% N,N-diethylacrylamide/58% N,N-dimethylacrylamide produced a sequencing read length of 575 bases in 94 minutes at 98.5% accuracy with a transition temperature of ∼95°C.; We also present an original observation, in which a dramatic narrowing of the polydispersity index (PDI) of high polymers occurs as a consequence of chain scission events in an elongational flow field. In our experiments, semi-dilute aqueous solutions of high-molar mass, polydisperse polymers (PDI ∼ 1.5) were injected under pressure into a capillary. Chain scission events occurring during multiple passes through the capillary cause a marked decrease in PDI, to values as low as 1.12, along with a decrease of molar mass. The phenomenon appears to be entirely physical and independent of the chemical nature of the polymer. Modeling carried out in collaboration with researchers at the University of Ottawa shows the results to be consistent with mid-point scission of polymer chains exceeding a certain critical chain length in the extensional flow.