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.

Construction And Characterization of Region-Specific Microdissection Libraries for Human Chromosome 2

Fa-Ten Kao and Jingwei Yu
Eleanor Roosevelt Institute for Cancer Research, 1899 Gaylord St., Denver, CO 80206, and Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, CO.

Using microdissection and microcloning techniques developed in this laboratory [1], we have constructed 4 region-specific libraries for the entire short arm of human chromosome 2: 2p23-p25 (designated 2P1 library), 2p21-p23 (2P2), 2p14-p16 (2P3), and 2p11-p13 (2P4). These libraries are large, comprising 300,000 - 1,000,000 recombinant microclones with a mean insert size of 250 bp (ranging between 50-800 bp). 40-60% of the microclones contain unique sequences. Southern blot hybridization showed that 54-86% of the single-copy microclones were derived from the respective dissected region. A subset of single-copy microclones from each library was characterized and the hybridizing HindIII fragments in the human genome determined, including 26 microclones for the 2P1 library, 60 clones for 2P2, 66 clones for 2P3, and 30 clones for 2P4. Details of these libraries have been described [2-5]. In addition, 14 of the 26 microclones in the 2P1 library were further mapped to the 2p23.3-p25.1 region by dosage analysis using a patient with an interstitial deletion of this region [2], and 10 of the 60 microclones from the 2P2 library were mapped to 2p21 using an interstitial deletion in 2p21 [4]. The 2P3 library and the single-copy microclones have been used in a team effort which led to the cloning of the hereditary nonpolyposis colorectal cancer (HNPCC) gene [6].

Six region-specific libraries for the entire long arm of chromosome 2, plus a library for the centromere region, are under various stages of construction and characterization. One library for the distal long arm, region 2q35-q37(2Ql), has already been constructed and described [7]. 26 single-copy microclones from the library were characterized and 4 of these clones were further mapped to the 2q37 region using a cell hybrid containing only this region. This library has been deposited in the American Type Culture collection (ATCC No. 77419). Other libraries will be similarly deposited for general distribution to the genome community.

Region-specific libraries and the single-copy microclones from the libraries are useful resources for genome studies [8]. For example, the libraries can be used to screen for highly polymorphic microsatellite markers for high resolution linkage analysis and to narrow down the distance between a probe and a disease locus under study [6]. In addition, single copy microclones with short inserts can be conveniently sequenced to prepare STSs, and it is relatively simple to isolate many single-copy microclones to provide sufficient STSs (e.g. 1 STS per 50-100 kb or less) for constructing high density physical maps and to prepare sequence-ready reagents for each dissected region of 10-20 mb.

[1] PNAS 88, 1844-1848, 1991.
[2] Hum. Genet. 93, 557-562, 1994.
[3] Somat. Cell Mol. Genet. 20, 133-136, 1994.
[4] Cytogenet. Cell Genet. in press.
[5] Somat. Cell Mol. Genet. in press.
[6] Cell 75, 1215-1225, 1993.
[7] Genomics 14, 769-774, 1992.
[8] BioEssays 15, 141-146, 1993.