DNA sequencing, genotyping, and analysis by end-labeled free-solution electrophoresis (ELFSE).

摘要

Separation of DNA molecules according to size is a critical process in modern molecular biology, including high-impact applications such as DNA sequencing and genotyping. These demanding separations are typically performed by capillary electrophoresis in sieving matrices consisting of entangled solutions of linear polymers. Although this technology has been very successful, the viscous polymer solutions present fundamental and practical barriers to further increases in the throughput of sequencing operations.; End-Labeled Free-Solution Electrophoresis (ELFSE) represents an alternative strategy for size-based separation of DNA. In this technique, each DNA molecule in a mixture is conjugated to a polymeric "drag-tag" that modifies the free-draining properties of the DNA, enabling electrophoretic separation without a polymer matrix. Rapid separations with long read lengths are theoretically possible using ELFSE. Since no viscous sieving matrix is necessary, ELFSE should simplify the transition of DNA sequencing separations onto integrated microfluidic devices currently under development.; Despite the great promise of the technique, experimental progress has been modest, with the primary obstacle being the availability of drag-tags that are sufficiently large and monodisperse for high-resolution separation of a wide range of sizes of DNA. This dissertation presents key experimental advances in the implementation of ELFSE. New drag-tags have been tested, including monodisperse synthetic polymers, as well as repetitive polypeptides or "protein polymers" engineered specifically for ELFSE. These drag-tags have been employed for DNA sequencing, and although the read lengths are not yet competitive with matrix-based sequencing, the success demonstrated here is an important step toward rapid, long read-length sequencing by ELFSE.; In addition to DNA sequencing, several novel approaches are reported for ELFSE, including a highly multiplexed genotyping assay based on a single-base extension reaction. ELFSE separations have also been performed with drag-tags at both ends of the DNA chain, an approach that offers enhanced hydrodynamic drag and extends the range of separations possible with ELFSE. Other aspects of ELFSE that have been explored include techniques for high-yielding conjugation reactions between drag-tags to DNA, and a novel approach for coating microchannel walls enabling high-resolution separations.