Impact of polymer hydrophobicity on the properties and performance of DNA sequencing matrices for capillary electrophoresis.

To elucidate the impact of matrix chemical and physical properties on DNA sequencing separations by capillary electrophoresis (CE), we have synthesized, characterized and tested a controlled set of different polymer formulations for this application. Homopolymers of acrylamide and N,N-dimethylacrylamide (DMA) and copolymers of DMA and N,N-diethylacrylamide (DEA) were synthesized by free radical polymerization and purified. Polymer molar mass distributions were characterized by tandem gel permeation chromatography - laser light scattering. Polymers with different chemical compositions and similar molar mass distributions were selected and employed at the same concentration so that the variables of comparison between them were hydrophobicity and average coil size in aqueous solution. We find that the low-shear viscosities of 7% w/v polymer solutions decrease by orders of magnitude with increasing polymer hydrophobicity, while hydrophilic polymers exhibit more pronounced reductions in viscosity with increased shear. The performance of the different matrices for DNA sequencing was compared with the same sample under identical CE conditions. The longest read length was produced with linear polyacrylamide (LPA) while linear poly-N,N-dimethylacrylamide (PDMA) gave approximately 100 fewer readable bases. Read lengths with DMA/DEA copolymers were lower, and decreased with increasing DEA content. This study highlights the importance of polymer hydrophilicity for high-performance DNA sequencing matrices, through the formation of robust, highly-entangled polymer networks and the minimization of hydrophobic interactions between polymers and fluorescently-labeled DNA molecules. However, the results also show that more hydrophobic matrices offer much lower viscosities, enabling easier microchannel loading at low applied pressures.

Microchannel DNA sequencing matrices with a thermally controlled "viscosity switch".

Polymers and hydrogels that swell or shrink in response to environmental stimuli such as changes in temperature, pH, or ionic strength are of interest as switchable materials for applications in biotechnology. In this paper, we show that thermoresponsive polymers offer some particular advantages as entangled matrices for DNA sequencing by capillary and microchip electrophoresis. Matrices based on conventional water-soluble polymers demand a compromise in their design for microchannel electrophoresis: whereas highly entangled solutions of high molar mass polymers provide optimal sequencing performance, their highly viscous solutions require application of high pressures to be loaded into electrophoresis microchannels. Here, we demonstrate the reproducible synthesis, precise characterization, and excellent DNA sequencing performance of high molar mass, thermoresponsive polymer matrices that exhibit a reversible, temperature-controlled "viscosity switch" from high-viscosity solutions at 25 degrees C to low-viscosity, microphase-separated colloidal dispersions at a chosen, elevated temperature. The viscosity switch decouples matrix loading and sieving properties, enabling acceleration of microchannel flow by 3 orders of magnitude. DNA sequencing separations yielding read lengths of 463 bases of contiguous sequence in 78 min with 97% base-calling accuracy can be achieved in these matrices. Switchable matrices will be particularly applicable to microfluidic devices with dynamic temperature control, which are likely to provide the next major leap in the efficiency of high-throughput DNA analysis.