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.

Agency
National Institute of Health (NIH)
Institute
National Human Genome Research Institute (NHGRI)
Type
Research Project (R01)
Project #
5R01HG001161-06
Application #
6181619
Study Section
Genome Study Section (GNM)
Program Officer
Schloss, Jeffery
Project Start
1994-09-30
Project End
2003-03-31
Budget Start
2000-04-01
Budget End
2003-03-31
Support Year
6
Fiscal Year
2000
Total Cost
$184,695
Indirect Cost
Name
Duke University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
071723621
City
Durham
State
NC
Country
United States
Zip Code
27705
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Li, L; McGown, L B (2000) Improving signal to background ratio for on-the-fly fluorescence lifetime detection in capillary electrophoresis. Electrophoresis 21:1300-4
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Li, L C; He, H; Nunnally, B K et al. (1997) On-the-fly fluorescence lifetime detection of labeled DNA primers. J Chromatogr B Biomed Sci Appl 695:85-92
Nunnally, B K; He, H; Li, L C et al. (1997) Characterization of visible dyes for four-decay fluorescence detection in DNA sequencing. Anal Chem 69:2392-7

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