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
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.
Advisor:Steve A. Soper; William Daly; Kermit Murray; Robin McCarley
School:Louisiana State University in Shreveport
School Location:USA - Louisiana
Source Type:Master's Thesis
Date of Publication:02/25/2003