Mark A. Quesada

Laser‐excited confocal‐fluorescence gel scanner

A high‐sensitivity, laser‐excited, confocal‐fluorescence scanner has been developed for the detection of fluorescently labeled nucleic acids separated on slab gels. The gel is placed on a motor‐driven, two‐dimensional scan stage and raster scanned past the optical detection system. The 488‐nm argon ion laser beam is introduced into the confocal optical system at a long‐pass dichroic beam splitter and focused within the gel to an ∼2 μm diameter spot by a high‐numerical aperture microscope objective. The resulting fluorescence is gathered by the objective, passed back through the first long‐pass beam splitter, and relayed to a second dichroic beam splitter that separates the red and green emissions. The fluorescence is then focused on confocal spatial filters to reduce stray and scattered light, passed through spectral filters, and detected with photomultipliers. The resulting signals are amplified, filtered, and digitized for display on a computer. This system can detect as little as 5×10−12 M fluorescein,...

Capillary array electrophoresis: an approach to high-speed high-throughput DNA sequencing

A laser-excited, confocal-fluorescence scanner is used for high-sensitivity, on-column detection of electrophoresis performed using an array of capillaries. The capabilities of this method and apparatus are illustrated by application to high-speed, high-throughput DNA sequencing. Sanger DNA sequencing fragments are separated on an array of capillaries and then distinguished by using a binary coding scheme that employs only two different fluorescently labeled dye primers to identify four sets of fragments. DNA sequencing results are presented using a 25-capillary array. This apparatus has the capability of sequencing DNA at a rate of approximately 25,000 bases/hour.