Solid-Phase DNA Sequencing Reactions Performed in Micro-Capillary Reactors and Solid-Phase Reversible Immobilization in Microfluidic Chips for Purification of Dye-Labeled DNA Sequencing Fragments

• Main Author: Xu, Yichuan
• Other Authors: Steve A. Soper
• Format: Others
• Language: en
• Published: LSU 2003
• Subjects: Chemistry
• Online Access: http://etd.lsu.edu/docs/available/etd-0221103-161521/

The research presented in this dissertation involves micro-capillary reactors for solid phase DNA sequencing, the identification of dye terminator sequencing fragments with time-resolved methods, and purification of dye-labeled DNA fragments using solid- phase reversible immobilization in microfluidic chips. Solid-phase micro-reactors have been prepared for DNA sequencing applications using slab gel electrophoresis. A PCR product was immobilized to the interior wall of a fused-silica capillary tube via a biotin-streptavidin linkage. Solid-phase sequencing was carried out in micro-capillary reactors using a four-lane, single color dye primer chemistry strategy. The read length was found to be 589 bases. The complementary DNA fragments generated in the small volume (~62 nL) reactor were directly injected into the gel-filled capillary for size separation with detection accomplished using near-IR laser-induced fluorescence. A set of terminators labeled with near-IR heavy-atom modified tricarbocyanine dyes were investigated for a terminator sequencing protocol in conjunction with slab gel electrophoresis. This protocol gave 605 bp read lengths. A one color, two lifetime format of DNA sequencing was implemented. A pixel-by-pixel analysis was employed to identify each of the bases in the run. The resulting read accuracy for the two-dye capillary run was 90.6%. The use of photoactivated polycarbonate (PC) for purification of dye-labeled terminator sequencing fragments using solid-phase reversible immobilization (SPRI) was investigated. SPRI cleanup of dye-terminator sequencing fragments using a photoactivated PC microchannel and slab gel electrophoresis produce a read length of 620 bases with a calling accuracy of 98.9%. The PC-SPRI cleanup format was also integrated to a capillary gel electrophoresis system. In this case, the immobilization microchannel contained microposts to increase the loading level of DNAs to improve signal intensity without the need for pre-concentration.