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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.

Strand-Specific In Situ Hybridization: Application of the Method

Julianne Meyne, Edwin H. Goodwin, Susan M. Bailey, Loanne R. Smith, Denise I. Quigley and Robert K. Moyzis
Life Sciences Division, Los Alamos National Laboratory, Los Alamos, NM 87545

Strand-specific fluorescence in situ hybridization (FISH) is a modification of the standard FISH method that allows hybridization of a single-stranded deoxyoligomer probe to only one chromatid of a metaphase chromosome. This is accomplished by removing one strand of the DNA helix from each chromatid. Because the method allows determination of the relative chromosomal orientation of highly repetitive, tandemly organized DNA sequences it is also referred to as CO-FISH (Chromosome Orientation FISH) [1,2]. We have hybridized a variety of repetitive DNA sequences localized within the pericentromeric regions of mammalian chromosomes using the CO-FISH method. All of the tandemly repeated satellite sequences studied to date have been oriented in the head-to-tail fashion except the heterochromatic C-band positive region of the long arm of human chromosome 9. The satellite 2 and 3 sequences in this region are arranged in mixed orientation.

CO-FISH has been a very reliable method to detect compound lateral asymmetry in the heterochromatic C-band positive region of the long arm of human chromosome 1. This polymorphic region exhibits a variety of simple and compound lateral asymmetry combinations, as revealed by studies of chromosomes from both cultured fibroblasts and lymphocytes. Similar patterns are observed using repetitive probes present in the pericentromeric regions of other mammalian species.

When slides prepared using the CO-FISH method are hybridized with both a repetitive DNA probe and a probe for the telomere, one can also determine the direction of the DNA sequence and its complement. It is known that the G-rich strand of the telomere overhangs the 3' end of the DNA duplex within each chromatid. Therefore, by selecting the appropriate single-stranded telomere probe one can label either the 3' or 5' end of each chromatid and determine the direction of that strand. Centromeric twists and sister chromatid exchanges can be discerned by analyzing the relative frequencies of the observed hybridization patterns.

[1] Goodwin, E.H. and Meyne, J. (1993) Strand-specific FISH reveals orientation of chromosome 18 alphoid DNA. Cytogenet Cell Genet 63: 126-127.
[2] Meyne, J., Goodwin, E.H. and Moyzis, R.K. Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA. Chromosoma 103: 99-103

This work was supported by grants from the U.S. Department of Energy.