A new scheme for detection of electrophoretically separated, fluorescent-labeled DNA fragments for genome sequencing is under investigation. In this multiplex scheme, fluorescence lifetime replaces spectra selectivity for distinguishing between the four base-specific labels. Thus, the scheme is based on detection of four fluorescence decays instead of four colors in a single-lane separation of the DNA fragments. Fluorescence lifetime is a concentration-independent characteristic that provides """"""""digital"""""""" detection, offering important advantages over analog, intensity-dependent measurements. Since the fluorescence lifetimes of the four dyes are pre-determined, data analysis is simple, straightforward even, when there is incomplete electrophoretic resolution. Continuous, on-the-fly fluorescence lifetime detection in four-decay sequencing is performed in the frequency-domain using a state-of-the-art, commercial instrument that incorporates multiharmonic Fourier transform (MHF) technology to allow measurements of phase and modulation simultaneously at many modulation frequencies. The MHF instrument is interfaced to a commercial capillary electrophoresis (CE) system. Fluorescence lifetime and intensity are simultaneously derived from the same measurement to produce a lifetime-intensity electropherogram in no more time than is required for a simple intensity or color electropherogram. In fact, the improved accuracy and resolution that is expected from lifetime detection should enable faster sequencing since the demands on the electrophoretic resolution will be eased. The goal for the next project period is the implementation of four-decay detection in DNA sequencing. Concurrently, studies will be directed towards optimization of lifetime detection and improved detection limits, which will be achieved through (1) further investigations of visible dyes and new studies of energy-transfer strategies and near-infrared dyes, (2) design and implementation of a confocal optical detection system, (3) optimization of the CE/MHF interface design, and (4) investigations of different CE packing materials and buffers to enhance on-capillary fluorescence lifetime detection.
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