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 co nstituent bases of DNA in a single separation lane when the probes use d have a distinct spectral characteristic, While the common strategy i s to use fluorescent dyes which show absorption and emission propertie s in the visible region (400-600 mm) 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, The p rinciple advantages of near-IR fluorescence include, the lower backgro und levels observed in this spectral region, improving the signal-to-n oise ratio (SNR) and the ability to use simple diode lasers and avalan che photodiodes to construct an all-solid-state laser fluorescence det ector, In this paper, we will discuss our results concerning the use o f near-IR fluorescence detection for DNA sequencing carried out in a c apillary gel column, where the capillary column has an internal diamet er of 75 mu m, and the loading level of DNA onto this column is in the nL regime, requiring ultra-sensitive detection, In addition, we will discuss our efforts toward the development of a highly efficient, sing le lane, single fluor, base-calling strategy using lifetime discrimina tion 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 fluore scence lifetime without altering the absorption or emission maximum of the base chromophore. This will allow the dye series to be excited wi th a single laser with the fluorescence processed on a single detector and the identity of the terminal base accomplished via lifetime discr imination, In order to effectively carry out lifetime measurements dur ing capillary electrophoretic separation of the oligonucleotides, a si mple solid-state time-correlated single photon counting instrument was constructed.