![]() The combined confocal laser-focusing and emission-collection optics are mounted on a moving stage the confocal approach reduces the number of optical elements required, and consequently the number of optical adjustments is also kept to a minimum. The laser beam is focused to a spot size of 80 µm at the electrophoretic gel and is scanned in a horizontal line across the face of the gel at a rate of 150 mm/s. These wavelengths excite all four fluorophores and are largely outside of the emission spectra of the dyes. The laser is a 40-mW multiline air-cooled device emitting primarily at 488 and 514.5 nm. The optical configuration of the model 377 sequencer includes an argon-ion laser, focusing and collection lenses, holographic grating spectrograph, and CCD camera (see Fig. Unlike the CCD camera, however, a PMT does not discriminate wavelengths so an instrument based on a PMT detector required four independent laser scans and a rotating wheel of wavelength-selective optical filters in order to determine the identity of each fluorophore (see Fig. Earlier Perkin-Elmer instruments used a photomultiplier tube (PMT) for detection of the four fluorophore colors. The result is an increase in overall system throughput by a factor of four. The newest Perkin-Elmer sequencer (model 377) incorporates a holographic grating spectrograph and charge-coupled-device (CCD) camera-based optical detector for simultaneous detection of four or more emission colors from the laser-excited fluorophores used to label DNA fragments. Among many approaches to increasing the throughput of DNA sequencers are recent advances in the optical design of a commercial system produced by Perkin-Elmer. 16), which has as its goal the complete sequencing of all 3 ¥ 109 base pairs that make up the human genome. Very-large-scale automated sequencing has assumed growing importance due, primarily, to the Human Genome Project (see Laser Focus World, June 1994, p. 141), but only Perkin-Elmer systems are able to simultaneously detect, from a single lane on the electrophoretic gel, emissions from all four of the fluorescent dyes (fluorophores) used to label the four bases that make up DNA. ![]() Each manufacturer has adopted a different approach to detection of the laser-stimulated fluorescence from labeled DNA molecules (see "Other approaches to automated DNA sequencing," p. Several firms manufacture laser-based sequencers including Pharmacia Biotech (Piscataway, NJ), LI-COR (Lincoln, NE), Molecular Dynamics (Sunnyvale, CA), and Perkin-Elmer`s Applied Biosystems Division (Foster City, CA). New optical design with a CCD detector quadruples throughput of commercial DNA sequencers.ĭuring the past eight years, laser-based automated deoxyribonucleic acid (DNA) sequencing instruments have revolutionized molecular biology by enabling researchers to automatically determine the sequence of nucleotide bases in an already formed strand of DNA (see Laser Focus World, May 1994, p. Sequencers benefit from solid-state detectors
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