SEPARATION MEDIA FOR DNA CAPILLARY/CHIP ELECTROPHORESIS

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DESCRIPTION (Adapted from the Investigator's Abstract): The specific aim of the renewal proposal deals with further development and optimization of new and better separation media which can overcome many of the problems related to the present-day polyacrylamide solutions or gels: polymer solutions are often too viscous to be filled in capillary tubings; the polymerization in situ approach is not so reproducible; shelf-life for polymer gels is limited. The key scheme is to take advantage of the self-assembly behavior of copolymer colloids. The investigators have achieved the initial goals of designing a polymer network based on the self-assembly behavior of a EPE-type block copolymer (E and P denoted polyoxyethylene and polyoxypropylene, respectively) with the following properties: (1) at 10-15 degrees C, the polymer solution is a low viscosity fluid (less than 30 centipoise) in 1xTBE buffer, for ease of filling and removing the separation medium in narrow-bore capillary tubings; (2) at room temperatures (greater than 20 degrees C), the polymer solution becomes gel-like with good sieving ability (tested down to 14 bases with one base resolution for single stranded (ss) DNA fragments and up to 1 kilobase pairs for ds DNA fragments with one base pair resolutions); and (3) the polymer itself has acted successfully as a dynamic coating material. By correlating the capillary electrophoresis results with structural studies using a variety of physical techniques, such as laser light scattering, small angle X-ray scattering, small angle neutron scattering, NMR, rheometry, viscosimetry and atomic force microscopy, an optimization of the self-assembled polymer network for DNA capillary electrophoresis can be achieved. In addition to the EPE, PEP, EBE, and BEB type triblocks (with B being polyoxybutylene and more hydrophobic than P), it is proposed to use crystallization and coil-collapse as the physical phenomena capable of temperature-dependent reversible cross-linking formation. The crystallization approach is especially attractive, since the supramolecular structure can be more resilient and is not in dynamic equilibrium with single polymer chains as in the self-assembly process.