Near-infrared, laser-induced fluorescence detection for DNA sequencing applications

Laser-induced fluorescence detection has become the detection strategy of choice in many large-scale DNA sequencing applications due to its ease of Implementation, sensitivity and the ability to identify the constituent bases of DNA in a single separation lane when the probes used have a distinct spectral characteristic. While the common strategy is to use fluorescent dyes which show absorption and emission properties in the visible region (400-600 nm) of the electromagnetic spectrum, our efforts have been directed toward developing near-IR (700-1000 nm) fluorescence as a viable detection strategy for DNA sequencing. In this paper, we discuss our results concerning the use of near-IR fluorescence detection for DNA sequencing carried out in a capillary gel column, where the capillary column has an internal diameter of 75 μm, and the loading level of DNA onto this column is in the nL regime, requiring ultra-sensitive detection. In addition, we discuss our efforts toward the development of a highly efficient, single lane, single fluor, base-calling strategy using lifetime discrimination of heavy-atom modified near-IR dyes. The dyes developed for this application contain an intramolecular heavy atom (halogen) on a remote section of the chromophore, resulting in a perturbation in the fluorescence lifetime without altering the absorption or emission maximum of the base chromophore. This will allow the dye series to be excited with a single laser with the fluorescence processed on a single detector and the identity of the terminal base accomplished via lifetime discrimination. In order to effectively carry out lifetime measurements during capillary electrophoretic separation of the oligonucleotides, a simple solid-state time-correlated single photon counting instrument was constructed