Introduction to the Workshop
URLs Provided by Attendees

Abstracts

Mapping

Informatics

Sequencing

Instrumentation

Ethical, Legal, and Social Issues

Infrastructure

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.

PROGRESS TOWARD AUTOMATION OF THE FRONT-END OF DNA SEQUENCING

Richard A. Guilfoyle, Jim Uzgiris, Huamin Ji, Qinghua Liu, Dan Chen, Michael Westphall, Robert Brumley, Brian Boville, Jessica Hayden, Zhen Guo, Andy Thiel, Arthur Johnson, Todd Francisco, Ricardo Gee, and Lloyd M. Smith
University of Wisconsin, Dept. of Chemistry, Madison, WI 53706

Results will be presented addressing recent progress made in our laboratory regarding the continued development of our front-end strategy for high-throughput M13-based shotgun cloning and sequencing. Our "front-end" will be discussed in terms of ongoing technology development and strategy designs as pertaining to new methods for: (1) purification and restriction mapping of cosmid inserts, (2) random fragmentation of purified cosmid inserts, (3) M13 library constructions, (4) isolation of M13 clones, (5) ordering of M13 shotgun clones and selection of the minimally overlapping inserts, (6) purification of M13 templates, (7) quantitation of M13 templates, and (8) 4-dye fluorescent dideoxy sequencing reactions. Also, an initiated plan for robotic integration of these technologies will be discussed in terms of achieving robust compatibility with the ABI373A and horizontal ultra-thin gel electrophoresis (HUGE).

Specifically, and as directly related to the above, ongoing and planned experimentation to be discussed will include: (1) a new vector facilitating triple-helix-affinity-capture (TAC) purification and restriction mapping of cosmid inserts, (2) CviJ1 digestion vs. nebulization for random fragmentation of target DNA, (3) M13 library constructions in the direct selection vector, M13-100, now equipped with universal primers and a TAC sequence for purification of ssDNA, (4) isolation of M13-100 clones by flow cytometric sorting vs. plaque-picking and limiting dilution of transfected cells, (5) SBH format 2-related ordering of M13 clones by hybridization to oligo arrays on glass supports, (6) purification of ssDNA M13 templates by TAC vs. glass-fiber bottom microtiter wells, (7) YOYO cyanine dye-based quantitation of ssDNA template concentrations in microtiter plates using the Molecular Dynamics Fluorimager 575, and (8) direct (eg. non-cycle sequencing) Bst DNA polymerase catalyzed sequencing reactions performed on the Gilson 215 robotic liquid handling station. ABI373A and HUGE base-calling data will also be presented which examine an automated solid-phase scheme (biotin-streptavidin/magnetic bead) for the purification of DNA sequencing reactions. Robotic integration will be discussed in terms of initial and long-term objectives utilizing the ROBOLAB 9600 System (Robocon, Austria).