Title page for ETD etd-0221103-161521


Type of Document Dissertation
Author Xu, Yichuan
Author's Email Address yxu1@lsu.edu
URN etd-0221103-161521
Title 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
Degree Doctor of Philosophy (Ph.D.)
Department Chemistry
Advisory Committee
Advisor Name Title
Steve A. Soper Committee Chair
Kermit Murray Committee Member
Robin McCarley Committee Member
William Daly Committee Member
Keywords
  • DNA sequencing
  • microchip
  • fluorescence
  • capillary electrophoresis
Date of Defense 2002-12-17
Availability unrestricted
Abstract
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.

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