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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Automated Fluorescent Detection for Multiplex DNA Sequencing

Robert B. Weiss[1,3], F. Mark Ferguson[1], Leonard Di Sera[1], Alvin Kimball[1], Josh Cherry[1], Mark Stump[1], Andy Marks[1], Tony Schurtz[1], Diane Dunn[1,2] and Raymond F. Gesteland[1,2]
[1]Department of Human Genetics and the [2]Howard Hughes Medical Institute, University of Utah, Salt Lake City, UT 84112. [3]Corresponding author.

Instrumentation for automated hybridization and detection of DNA hybrids on nylon membranes is a focal point of our research. Recently, we have devised a method for amplifying fluorescent light output on nylon membranes by using an alkaline phosphatase-conjugated probe system combined with a fluorogenic alkaline phosphatase substrate [1]. The amplified signal allows sensitive detection of DNA hybrids in the sub-femtomole/band range.

Development of integrated instruments capable of multiple hybridization/detection cycles is underway. The hybridization apparatus contains a set of nested Plexiglas cylinders: a heated inner drum with nylon membranes fixed to its outer surface rotates through a fluid puddle formed by an eccentric outer drum enclosure. The inner drum has a viewable surface area of ~3000 cm2. Fluid delivery and drain are achieved using solenoid valve blocks, with segregated blocks for wash fluids, probe fluids, enzyme fluids and substrate fluids. A stepper motor supplies the drive system for drum rotation. This precision drive allows synchronization of the drum rotation with the charge transfer across one dimension of a two dimensional CCD camera during the detection process. The peltier-cooled 2048 x 96 pixel CCD camera collects a continuous image of the inner drum's surface by operating in the Time Delay and Integration mode.

Several hybridization/detection instruments have been constructed and are now undergoing testing with a variety of hybridization formats. Several probe-enzyme conjugates, both direct oligonucleotide-alkaline phosphatase conjugates and indirect biotin-streptavidin-AP or digoxigenin-anti-digoxigenin-AP, are being tested. Results will be shown from automated hybridization and detection of high-density colony grids of cosmid libraries using STS primers as probes. A major use of these instruments is in both multiplex mapping and sequencing of transposon inserts in large plasmid (~20 Kb) templates. Transposon inserts are mapped by probing Southern blots of restriction digests from multiplexed plasmids. Mapping membranes contain 96 lanes, and five of these membranes can be mounted on a single drum. A ten vector multiplex family allows mapping of 4,800 transposon inserts with a single drum load of the instrument, followed by 21 cycles of hybridization and detection. Multiplex sequence ladders in both 16 lane set and 32 lane set formats are being tested. Hybridization and detection of multiplex genotyping markers (PCR products of simple sequence repeats) is also under development. Continued refinement and development of these instruments will provide a key segment for the automation and integration of large-scale multiplex sequencing.

This work was funded by DOE grant DE-FG03-94ER-61817 (R.F.Gesteland, P.I.)

[1] Cherry, J.L., Young, H., Di Sera, L.J., Ferguson, F.M., Kimball, A.W., Dunn, D.M., Gesteland, R.F., and Weiss, R.B. (1994). Enzyme-linked fluorescent detection for automated multiplex DNA sequencing. Genomics 20, 68-74