Micro-DNA sequence analysis using capillary electrophoresis and near-IR fluorescence detection

Our group is developing optical solid-state, near-IR (NIR) fluorescence detection systems for the analyses of DNA in restriction mapping and sequencing applications. Specifically, we are investigating a base-calling scheme using fluorescence lifetime discrimination in the NIR implementing intramolecular heavy-atom modified NIR fluorescence dyes which can be configured in a single lane, single fluor format. Results are presented concerning the ability to perform lifetime measurements on-line during capillary gel electrophoresis. Due to the high sensitivity associated with NIR fluorescence detection and small injection volumes in capillary electrophoresis, micro-reactor systems are also being developed which will potentially reduce the sample size for preparation of DNA sequencing ladders. The Sanger dideoxy-terminated fragments are prepared using solid-phase sequencing strategies in these micro-capillary reaction chambers. Our discussion focuses on the stability of the immobilized DNA template under typical sequencing conditions and the ability to sequence long templates using this strategy. Finally, we discuss our work on the preparation of nuclear staining dyes which show absorption and emission properties in the NIR for the low-level detection of restriction fragments. Fluorescence spectra of these dyes in the presence of dsDNAs is presented.

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