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.

Generation and Characterization of a Large Insert Chromosome 2 PAC Library

Denise Boehrer and Jeffrey C. Gingrich
Human Genome Center, L-452, Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Livermore, California, 94550.

As one part of the National Laboratory Gene Library Project we are generating large insert, ~50 kb to ~250 kb, bacterial clone libraries using the pCYPAC cloning vector developed at Livermore [1]. For a chromosome 2-specific PAC library, PAC clones are being generated from the hybrid cell line GM10826. DNA partially digested with MboI is size fractionated using PFGE and the DNA cloned into the BamHI cloning site of the pCYPAC-2 vector. As with the bacteriophage P1 cloning system, sucrose is being used to select for recombinants. Ligation mixes containing clones over 80 kb average size are being plated and the chromosome 2 clones identified by colony hybridization using both human and hamster probes. To date, approximately 2,500 chromosome 2 clones, ~1X chromosome 2 coverage, has been arrayed into microliter plates and we are continuing to array additional clones. The chromosome 2 representation of the library is being tested by PCR screening using STS primers distributed across the chromosome. Combining the chromosome 2 specific PAC library with the cosmid and fosmid library being generated here by the Gene Library Project (see abstract by Garnes et al.) in conjunction with STSs being developed here (see abstract by Gingrich et al.), and elsewhere, will allow for the construction of a high quality, high resolution physical map of chromosome 2.

This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory, contract No. W-7405-ENG-48.

[1] Ioannou et al. (1994) Nature Genetics, 6, 84-89.