Introduction to the Workshop
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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.
Hybridization Technology Development at the Resource for Molecular Cytogenetics
H.-U. Weier[1,2], C. Thompson, M. Wang, K. Greulich, C. Collins, J. Gray[1,2], and D. Pinkel[1,2].
Resource for Molecular Cytogenetics, Life Sciences Division, Lawrence Berkeley Laboratory, Berkeley, CA and Department of Laboratory Medicine, University of California, San Francisco, CA.
The advancement of fluorescence in situ hybridization (FISH) techniques is a major goal of the Resource for Molecular Cytogenetics. We have focused on techniques to facilitate high resolution DNA probe mapping and ordering and to improve genetic analysis of human tissue samples.
Probe mapping and ordering: High resolution mapping and ordering of DNA molecules is required for tasks such as contig assembly. With current techniques it is often difficult or extremely laborious to establish the relative locations of subfragments of a large molecule, or to determine the amount of overlap of two elements in a contig. FISH has the ability to visualize hybridization to single extended DNA target molecules. Current techniques involve the use of genomic target DNA released from cell nuclei and spread on slides. We have employed techniques developed by collaborators (A. Bensimon, Institut Pasteur, Paris) that permit linear extension of individual cloned molecules on activated glass substrates by covalent attachment of one end. Hybridization of probes as small as 2kb can detected and the positions of hybridization sites determined to within 1kb.
FISH in thick tissue sections: FISH has the potential to provide genetic information from individual cells. Analysis of cells in context with surrounding cells in tissue samples provides information on the spatial distribution of genetic status, such as the evolution of genetic changes in tumors. Such studies require hybridization to tissue specimens that are thick enough to contain a large number of intact cells. We have developed protocols employing direct fluorochrome labeled probes that permit hybridization with centromeric probes throughout 20µm thick formalin-fixed/paraffin-embedded clinical specimens. Image analysis algorithms to obtain genetic information from such three dimensional specimens are under development (see Lockett et. al., this meeting).
This work was funded by the Office of Health and Environmental Research, Department of Energy, under contract DE-AC-03-76SF00098 and Imagenetics.