Development of sieving matrices for DNA separations by capillary electrophoresis and microchip electrophoresis

A model based on polymer molecular architecture and topology for the separation matrix has been developed. It can be divided into four categories, as discussed in the following. We have experimentally tested each feature presented in the model. In brief, our findings can be summarized as follows.; a. In order to slow down the chain dynamics, we introduced graft copolymers (PDMA-g-PMMA) with short grafted chains and graft densities appropriate for the mesh size of the polymer network. Self-assembly could be triggered by changing the solvent quality. The association could effectively increase the polymer molecular weight to a range beyond the synthetic limits. Meanwhile in a polymer network formed by entanglements of graft copolymer chains, the cross-linking points would be less mobile due to side chain obstructions or associations. The decrease in the slippage of those entanglements stabilizes the mesh formation and therefore enhances the resolution. The graft copolymers showed better separation properties than those of homopolymers of comparable size range.; b. Recent success in preparing quasi-interpenetrating networks as high performance DNA sequencing matrices within our research group has led us to set up a model to help design the polymers for the dsDNA fragments analysis (30-300 bp) by microchip electrophoresis. Polyacrylamide (PAM) and poly(N,N-dimethylacrylamide) (PDMA) quasi-interpenetrating network has been synthesized. The central idea in creating an interpenetrating polymer network (IPN) is the ability to use two different types of polymers that are miscible but do not quite like each other that the polymer chains tend to be extended by avoiding each other. Due to the increase in polymer solution concentration, the incompatibility between PAM and PDMA has also been increased. Then, the PAM/PDMA quasi-interpenetrating network solution turned turbid because of microphase separation. The mixture solution of PAM and PDMA was studied instead of quasi-interpenetrating network for the dsDNA separation. After the filtration of the turbid solution in a microcentrifuge, the lower layer which is a PAM-rich layer due to the molecular redistribution was found to have the strong sieving ability for the small DNA fragments separation.; c. To solve the phase separation problem from less compatible polymers at high concentrations, random copolymers of PAM and PDMA were synthesized to test the combined advantages of high sieving ability of PAM and the dynamic coating ability of PDMA for the small DNA fragments separation by microchip electrophoresis. An empirical model estimating the relationship among polymer molecular weight, polymer concentration and zero-shear solution viscosity has been developed. The rational design of the sieving matrix based on the viscosity requirement and target dsDNA fragment size range is viable.; d. Genotyping analysis on micro-fabricated chips is 100-fold faster than capillary electrophoresis. However, there is no separation medium available for oligonucleotide fragments in the range of 10-40 base in the commercialized instruments, such as the Agilent Bioanalyzer 2100. We present an approach, using triblock copolymers ExPyE x and ExByEx with E, P, B, and subscript denoting oxyethylene, oxypropylene, oxybutylene, and segment length, respectively. The triblock copolymers are amphiphilic and the solubility of two flanking E-blocks in water is much better than that of the central block (P-block or B-block). The temperature-dependent solubility of the central block makes the triblock copolymer very easy to form micelles by self-assembly. At high concentrations, the micelles are densely packed, leading to the formation of a gel-like quasi-lattice ordered structure. Depending on their relative segment lengths, the triblock copolymer will form, for example, face-centered cubic structure or body-centered cubic structure. There are at least four qualitatively different domains in the gel-like ordered structure,...