Thermo-responsive and thermo-gelling polymer networks for DNA sequencing and genotyping by capillary and microchip electrophoresis

Capillary electrophoresis (CE) in the presence of entangled, uncrosslinked polymer solutions is the dominant technology for several important genetic analyses that rely upon the size-based separation of DNA fragments. There is a growing interest in transforming the current CE technology into a microfabricated format for DNA sequencing and genotyping, for higher throughput and lower cost. This research aims at designing high-performance thermo-responsive polymers and copolymers of acrylamide derivatives for DNA sequencing and genotyping and at applying our prior knowledge in CE-based DNA analysis to a microchip-based method. Recent advances in DNA sequencing and genotyping, in miniaturized electrophoresis systems are critically reviewed. Polymers and copolymers of N-ethoxyethylacrylamide and N-methoxyethylacrylamide were synthesized, the polymer physical properties were carefully investigated, and the DNA sequencing performances of these polymer matrices were evaluated. Rheometry results suggest that these polymers undergo a sol-gel transition from viscous solutions to elastic, solid-like materials upon heating. DNA sequencing studies indicate that the physical stability brought about by thermo-gelation enhances single-stranded DNA separation resolution and hence extends the DNA sequencing read length relative to a non-thermogelling control matrix. A different approach to enhancing double-stranded DNA separation resolution was taken in an investigation using blends of two well-studied cellulose derivatives, hydroxyethylcellulose and hydroxypropylcellulose, to create a DNA sieving matrix with a thermally tunable mesh size for dsDNA separation. The thermo-responsive constituent of this polymer blend (hydroxypropylcellulose) allows the mesh size of the sieving matrix to be thermally controlled, to facilitate the optimal separation of DNA fragments over different size ranges, which is not easily attainable with conventional polymer sieving matrices. The approach was extended to blends of thermo-responsive N,N-diethylacrylamide and N,N-dimethylacrylamide copolymers. (Abstract shortened by UMI.)...