Study and optimization of capillary electrophoresis DNA sequencing system performance

For nearly three decades, capillary electrophoresis (CE) DNA sequencing technology has been remaining the most successful and powerful tool for DNA analysis. Vast majority of the sequence data collected during the Human Genome Project (HGP) was obtained using advanced high-throughput CE instruments.;The success of the HGP is largely the result of early investments in the development of cost-effective DNA sequencing methods. The main objective of our research is exploring the possibility of developing CE-based sequencing systems that could reduce the cost of DNA sequencing by several orders of magnitude. The primary justification for these efforts is the idea that the technology could become so affordable that sequencing the full genomes of individual patients would be warranted from a health-care perspective.;First, a systematical approach to the sequencing system analysis, allowing formulation of the optimization strategies as well as providing a platform for comparison of available sequencing instruments, is developed. Exact relationships between all the major characteristics of a CE-based sequencing system are established for the first time and primary limitations and design guidelines discussed. As an example, the developed approach is applied to the characterization of the Stony Brook Sequencer built in our laboratory.;Second, a comprehensive study of the electrophoretic process in fused silica capillaries followed by the optimization of the electrophoretic conditions is performed. This study is aimed at determination of the best possible operational protocols and includes investigation of the formation and propagation of an anomalous resistive region in the capillary during electrophoresis. A practical solution for the compensation of this negative effect and a simple method for its monitoring are reported.;Finally, a novel approach to the design of capillary array electrophoresis systems capable of operating with nano-liter scale samples in conjunction with a novel sample injection method is proposed to significantly increase the sequencing system's throughput. This study includes modeling and computer simulation of the field-flow injection cell of our design as well as experimental evaluation of the method.