William F. Kolbe, Jocelyn C. Schultz and Jian Jin
Ernest Orlando Lawrence Berkeley National Laboratory, University of California, Engineering Science Department, Human Genome Group, 1 Cyclotron Road, Berkeley, CA 94720, Phone: (510) 486-7199, Fax: (510) 486-5857, wfkolbe@lbl.gov
In order to address the need for increased sequencing capability, we have undertaken a program of gel-based fluorescent sequencer development. Currently available slab-gel fluorescent sequencers are limited in the number of sample lanes they can accommodate due to limitations in the spatial resolution of their detection systems. We have developed a fiber optic based detection system permitting at least 100 lanes to be used in a 25 cm width. This detection system has been applied to two different gel platforms: a conventional sized (30 cm x 25 cm x 350 µm) and an ultrathin gel apparatus (15 cm x 25 cm x 100 µm).
The detection system uses a laser beam passing transversely through the gel to excite all the DNA lanes simultaneously and a fiber optic array to collect the fluorescence produced. A gradient index lens array is employed to image the fluorescence on to the ends of the fibers. The output end of the fiber array is formed into a compact rectangular shape compatible with a cooled charge coupled device camera used to detect the light. Both single color and four color operation have been investigated. To provide four color detection capability, a motorized translation stage containing interference filters is placed between the camera and the fiber optic array.
Because of the high spatial resolution of the optical system, alignment of the laser beam and detector array is critical. In addition, the laser beam is found to interact with the gel material eventually producing a damage zone which causes a deflection and subsequent broadening of the beam. To eliminate these problems, the alignment of the laser beam is stabilized by means of a feedback system employing a motorized steering mirror and beam translation stage controlled by photo diode detectors positioned at each end of the fiber optic array. To eliminate gel damage by the laser beam, the detector array assembly together with the stabilized laser beam is slowly scanned over the course of a sequencing run. The scanning rate employed (0.5 mm per hour) is sufficiently slow that no observable effect on the quality of the sequencing data is produced.
The two systems were characterized using sequence data generated with M13mpl8 templates. The performance was evaluated on the basis of resolution, speed and sequence accuracy using in-house base calling software.
This work was supported by the Director, Office of Energy Research, Office of Health and Environmental Research, Human Genome Program, of the U.S. Department of Energy under Contract No. DE-AC03-76SF00098.