Date of Award
Doctor of Philosophy
Edward S. Yeung
The purpose of this research is to develop a laser-induced fluorescence detection system for DNA sequencing with multiplexed capillary electrophoresis in accordance with the requirements of simplicity, high sensitivity, high throughput, and low cost;The detection and separation powers of capillary electrophoresis (CE) were first demonstrated with an indirect laser-induced fluorescence detection system capable of quantifying sodium and potassium in individual human erythrocytes. Contamination problems in such a highly sensitive CE system were investigated;The charge injection device (CID) is optimized for CE application by proper software development and optical arrangement to achieve both high sensitivity and high sampling rate. With the CID operated at room temperature, the detection limit of 1 x 10[superscript]-11 M fluoroscein can be easily achieved. The sampling rate can be as high as 4 Hz while the signal to noise ratio (S/N) is still sufficient for base calling. Exposure time gradient is applied to improve the S/N for large DNA fragments in a Sanger reaction product separated by CE;A 100-capillary array can be illuminated by a tilted laser beam that is focused with a 10-cm focal length cylindrical lens. The excitation configuration facilitates efficient incorporation of laser power into each capillary in the array while tolerating some mismatch of refractive indices. Analysis of DNA sequencing data suggests that crosstalk should be under 2.5%. Immersing the capillaries in water or ethylene glycol helps to reduce crosstalk down to 1%;A tilted glass filter is used to split images of capillaries to achieve a simple one-laser two-color detection system for base-calling while high optical throughput is maintained. Both peak-height ratio coding and ratiogram coding are applied to call bases up to 340 base pair (bp) with 99% accuracy;Combined with high speed separation, a raw sequencing rate of 3000 bp/min is achievable with the 100-capillary system. The system can be readily scaled up to 1000 capillaries to achieve a raw sequencing rate of 40 million bp per day. Sequencing the whole human genome will only take two months.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Li, Qingbo, "Laser-based detection system for high-throughput DNA sequencing with multiplexed capillary electrophoresis " (1995). Retrospective Theses and Dissertations. 11068.