Critical study on the development and design of an automated multicapillary electrophoresis instrument with collection of mutant DNA fractions using Constant Denaturant Capillary Electrophoresis (CDCE)

摘要

Constant Denaturant Capillary Electrophoresis (CDCE) is a separation tool based on the cooperative melting equilibrium principle that is used to detect mutations as low as of 106. This technique has already demonstrated invaluable clinical applications in correlated preventative prognosis, medical evaluations, and interventions. Accordingly, there is a high demand to utilize CDCE as a cost-effective, high-throughput screening and separation technique to detect mutations in large DNA pooled samples. The aim of this thesis is twofold: to describe DNA separation theories and technologies, as well as CDCE separation theory and applications; and to describe and analyze the design of and modifications applied to an integrated automated multicapillary instrument with collection of mutant fractions by using CDCE to meet the stringent requirements for detecting low-frequency mutations in pooled samples from large populations. The modified SCE2410 24-capillary DNA Sequencer, HTMS Model (High-throughput Mutational Spectrometer) by Q. Li et al. has been identified as the instrument that best meets these requirements. This thesis will analyze this integrated HTMS instrumental design and modifications involving the multicapillary cartridge, the optical detection device, six independently controlled solid-state thermal heaters for the thermostat array in the CDCE temperature control system, and automated matrix replacement and fraction collection. The overall HTMS system design has led to results of high optical sensitivity (1 x 10-12M fluorescence in detection limits), precise and stable temperature control (± 0.010C), and automated sample delivery, injection, matrix replacement, and fraction collection.