ON-THE-FLY FLUORESCENCE LIFETIME DETECTION OF DYE-LABELED DNA PRIMERSFOR MULTIPLEX ANALYSIS

Mixtures of dye-labeled, M13-forward DNA primers were separated by cap illary 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-la beled primer was identified on the basis of the lifetime of the conjug ated dye using nonlinear least squares or the maximum entropy method t o analyze the lifetime data. Overlapping electrophoretic peaks were ge nerated by making multiple injections of mixtures of the dye-labeled p rimers. The overlapping peaks were resolved by fitting the data to two -, three- or four-component lifetime models used in nonlinear least-sq uares analysis in which each lifetime component was fixed to the prede termined lifetime of the corresponding dye-labeled primer. In two of t he dye systems, the lifetimes of the four dye-labeled primers were suf ficiently different to allow peak resolution. In the other dye system, addition of 10% DMSO to the run buffer changed the lifetime of one dy e-labeled primer, allowing it to be resolved from another dye-labeled primer with similar lifetime.