Hydrophilic poly-N-substituted acrylamide matrices for microchannel electrophoresis of DNA and proteins: Matrix design, synthesis and characterization

Capillary electrophoresis (CE) has become the dominant technique for DNA sequencing, which relies upon the electrophoretic separation of DNA fragments in entangled, uncrosslinked polymer solutions. Although a number of polymers have been employed for DNA sequencing in microscale electrophoresis systems, the ideal polymer matrix has not yet been found. This research addresses the fundamental question of the impact of polymer chemical and physical properties on matrix performance for DNA sequencing by CE, as part of a systematic search for the ideal matrix. Homopolymers of acrylamide (AAM) and N,N-dimethylacrylamide (DMA) and copolymers of DMA and N,N-diethylacrylamide were synthesized by free-radical polymerization. A systematic investigation of polymer chain characteristics, rheological properties and the DNA sequencing performance of polymer matrices was performed. The study revealed the importance of polymer hydrophilicity for high-performance DNA sequencing matrices, through formation of robust, entangled polymer networks. Based on this finding, a polymer matrix was synthesized from the novel monomer, N-hydroxyethylacrylamide (HEA), for DNA sequencing applications. HEA was found to be more hydrophilic than AAM and DMA. Poly-N-hydroxyethylacrylamide (PHEA) exhibits good adsorptive capillary-coating ability, efficiently suppressing electroosmotic flow (EOF). Under DNA sequencing conditions, adsorbed PHEA coatings suppress EOF for over 600 hours of electrophoresis. DNA sequencing read lengths produced by PHEA matrices improve with increasing polymer concentration and decreasing electric field strength. When used both as a separation matrix and as an adsorbed coating in bare capillaries, the PHEA matrix can resolve over 620 bases of contiguous DNA sequence within 3 hours. In addition to DNA separation, the unique combination of hydrophilicity and capillary coating ability of PHEA makes it an attractive coating for CE of proteins. In a systematic comparative study, the adsorbed PHEA coating exhibited minimal interactions with proteins, providing highly efficient protein separations with excellent reproducibility on par with a covalent polyacrylamide coating. Hydrophobic interactions between proteins and a relatively hydrophobic poly(DMA) coating, on the other hand, adversely affected separation reproducibility and efficiency. The long-term stability of the adsorbed PHEA layer, demonstrated by EOF suppression and reproducibility of separation performance, proves the high quality of this coating.