Dyes of several classes were investigated as candidates for use in a multiplex, four-decay fluorescence detection scheme for DNA sequencing. The dyes include nitrobenzofuran dyes, rhodamine dyes, fluorescein dyes, cyanine dyes, Nile Red, and BODIPY dyes. Based on the results of fluorescence spectral and lifetime studies, an initial set of four dyes was selected for further study: NBD-aminohexanoic acid (NBD-HA, r = 1.1 ns), tetramethyl-rhodamine, methyl ester (r = 2.2 ns), rhodamine green (r = 4.3 ns), and BODIPY 505/515 (r = 5.9 ns). Limits of lifetime detection of the four dyes were investigated, and lifetime resolution was demonstrated for mixtures of the free dyes in batch solution. Lifetime of dye-labeled DNA primers also were determined in batch solution and detected on-the-fly in capillary electrophoresis (CE). Conjugation of the dyes to DNA improved the resolution of their individual lifetimes in mixtures in batch measurements. When attached to the primer, tetramethyl-rhodamine exhibited biexponential decay with a dominant lifetime of 3.8 ns, making it unsuitable for four-decay sequencing. Contact with the CE gel lengthened the lifetime of NBD-HA-labeled primer from 1.3 to 2.1 ns but did not affect the lifetimes of the other dyes. Lifetime detectability of labeled primers at individual points along an electrophoretic peak in the attomole range.

On-the-fly fluorescence lifetime detection in capillary electrophoresis (CE) is demonstrated. Virtually continuous detection is achieved by interfacing a commercial CE instrument with a commercial, multiharmonic Fourier transform lifetime fluorometer (MHF). The CE capillary cartridge was modified to allow the capillary to pass through a specially constructed capillary column mount capable of micropositioning in the MHF sample chamber. Optimization of the CE/MHF interface was achieved through the incorporation of a focusing lens, appropriate alignment of the laser beam on the capillary, and use of appropriate optical filters in the emission beam. Both fluorescence intensity and lifetime are recovered from the dynamic MHF data, which is analyzed using either conventional nonlinear least-squares or the maximum entropy method, which allows for lifetime recovery without a priori knowledge of the system. Continuous, on-the-fly fluorescence lifetime detection using the MHF technology adds the dimension of fluorescence lifetime without sacrificing the resolution and speed of CE. Its application to the CE separation of a mixture of the fluorescent dyes NBD-hexanoic acid and fluorescein is demonstrated.

Mixtures of dye-labeled, M13-forward DNA primers were separated by capillary gel electrophoresis and detected on-the-fly, using fluorescence lifetime measurements, to evaluate four-decay detection for multiplex DNA sequencing. Three different four-dye systems were used, two that were excited at 488 nm and one that was excited at 514 nm. Each dye-labeled primer was identified on the basis of the lifetime of the conjugated dye using nonlinear least squares or the maximum entropy method to analyze the lifetime data. Overlapping electrophoretic peaks were generated by making multiple injections of mixtures of the dye-labeled primers. The overlapping peaks were resolved by fitting the data to two-, three- or four-component lifetime models used in nonlinear least-squares analysis in which each lifetime component was fixed to the predetermined lifetime of the corresponding dye-labeled primer. In two of the dye systems, the lifetimes of the four dye-labeled primers were sufficiently different to allow peak resolution. In the other dye system, addition of 10% DMSO to the run buffer changed the lifetime of one dye-labeled primer, allowing it to be resolved from another dye-labeled primer with similar lifetime.

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