Time-Resolved Laser-Induced Fluorescence Study on Dyes Used in DNA Sequencing

Abstract

Research on the time-resolved fluorescence of fluorescein isothiocyanate, NBD, tetramethylrhodamine isothiocyanate, and Texas Red—the dyes used for fluorescence-based DNA sequencing—is described. Mean fluorescence lifetimes in both aqueous buffer solution and 5.3%T, 4.8%C polyacrylamide gel were determined as a function of excitation wavelengths at 337, 470, and 550 nm and were found to be 3.5, 1.1, 2.5, and 4.3 ns; the detection limits are 10, 200, 200, and 200 amol for FITC, NBD, TEMR, and T. Red, respectively. Comparisons of fluorescence parameters between the conjugated dyes and the free dyes are also reported. Results on the optimization of the excitation source wavelengths to improve sensitivity and reduce background scattering in polyacrylamide gel are also reported. Time-resolved fluorescence was successfully applied to resolve spectral overlapping of emissions in both solution and in polyacrylamide gel.