Analysis of near-infrared dye -labeled Sanger sequencing fragments with gel electrophoresis using time-resolved fluorescence lifetime identification methods

The research presented in this dissertation involves the identification of sequencing fragments with time-resolved methods. For this application, near-infrared heavy-atom tricarbocyanine dyes were developed in our laboratory, which can be excited with a single laser and emission collected using a single detection channel. The dyes have four spectroscopically unique, but relatively short lifetimes that can be altered by the intramolecular heavy-atom they contain. The work described here involves the optimization of dye-primer chemistry for preparing Sanger sequencing reactions for longer reads and the optimization of the separation matrix for capillary gel electrophoresis that produces favorable statistical analysis of the aforementioned dyes' lifetimes.;The performance of a two-lifetime experiment in which we modified an automated DNA sequencer to allow implementation of lifetime identification of DNA fragments labeled with near-IR fluorochromes and fractionation via slab-gel electrophoresis was investigated. A two-dye/two-lane sequencing experiment was carried out, in which two terminal bases, labeled with near-infrared dyes, were run in one lane and the other two bases in an adjacent lane. A lifetime evaluation of the resulting electropherogram on a pixel-by-pixel basis allowed the identification of the terminal nucleotide comprising a DNA band. The read accuracy was found to be better than a one-dye/four-lane approach using the software of the commercial instrument in spite of the fact that a spectroscopic call was implemented.;An automated peak recognition and base calling algorithm was also implemented and evaluated on two-tract dye-primer and dye-terminator capillary electrophoresis runs. The base calling accuracy was greater than 97% for both.