Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments.
The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.
Neumann
M
M
Physikalisch-Chemisches-Institut, Universität Heidelberg, Germany. v58@ix.urz.uni-heidelberg.de
Herten
D P
DP
Dietrich
A
A
Wolfrum
J
J
Sauer
M
M
D004247:DNA / Q000737:chemistry*; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D004247:DNA