A new sieving matrix for DNA sequencing, genotyping and mutation detection and high-throughput genotyping with a 96-capillary array system

Abstract

<p>Capillary electrophoresis has been widely accepted as a fast separation technique in DNA analysis. In this dissertation, a new sieving matrix is described for DNA analysis, especially DNA sequencing, genetic typing and mutation detection. A high-throughput 96 capillary array electrophoresis system was also demonstrated for simultaneous multiple genotyping;We first evaluated the influence of different capillary coatings on the performance of DNA sequencing. A bare capillary was compared with a DB-wax, an FC-coated and a poly(vinylpyrrolidone) (PVP) dynamically coated capillary with PEO as sieving matrix. It was found that covalently-coated capillaries had no better performance than bare capillaries while PVP coating provided excellent and reproducible results;We also developed a new sieving matrix for DNA separation based on commercially available poly(vinylpyrrolidone). This sieving matrix has a very low viscosity and an excellent self-coating effect. Successful separations were achieved in uncoated capillaries. Sequencing of M13mp18 showed good resolution up to 500 bases in treated PVP solution;Temperature gradient capillary electrophoresis and PVP solution was applied to mutation detection. A heteroduplex sample and a homoduplex reference were injected during a pair of continuous runs. A temperature gradient of 10°C with a ramp of 0.7°C/min was swept throughout the capillary. Detection was accomplished by laser induced fluorescence detection. Mutation detection was performed by comparing the pattern changes between the homoduplex and the heteroduplex samples. High throughput, high detection rate and easy operation were achieved in this system;We further demonstrated fast and reliable genotyping based on CTTv STR system by multiple-capillary array electrophoresis. The PCR products from individuals were mixed with pooled allelic ladder as an absolute standard and coinjected from a 96-vial tray. Simultaneous one-color laser-induced fluorescence detection was achieved by using a CCD camera. The allele peaks for the unknown sample were identified by comparing the normalized peak intensities of the mixtures to those of the pooled ladder by using a straightforward algorithm. An extremely high level of confidence in matching the bands was indicated with negligible cross-talk (<0.89%) between adjacent capillaries.</p>