High-resolution in situ hybridization using DNA halo preparations

To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string,...

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Bibliographic Details
Published in:Human molecular genetics 1992-11, Vol.1 (8), p.587-591
Main Authors: Wiegant, J., Kalle, W., Mullenders, L., Brookes, S., Hoovers, J.M.N., Dauwerse, J.G., van Ommen, G.J.B., Raap, A.K.
Format: Article
Language:English
Subjects:
Biological and medical sciences
Cells, Cultured
Cosmids
Cytogenetics
DNA
DNA - analysis
DNA - chemistry
fluorescence in situ hybridization
Fundamental and applied biological sciences. Psychology
Genetics of eukaryotes. Biological and molecular evolution
Humans
In Situ Hybridization, Fluorescence - methods
methodology
Methods and miscellaneous
Nucleic Acid Conformation
preparations
resolution
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Summary:To improve DNA resolution of fluorescence in situ hybridization we have adapted a nuclear extraction technique, resulting in highly extended DNA loops arranged around the nuclear matrix in a halo-like structure. In situ hybridization signals from alphoid and cosmid DNAs appear as beads-on-a-string, which, according to preliminary experiments, results from the association of individual probe fragments. By multicolor hybridizations we have been able to determine relative map position and to easily detect 10 kb overlap between individual cosmid clones, each of which shows linear beaded signals of ca. 10 μm, suggesting that the DNA is essentially linearized in our protocol. The map configuration can be typically derived from analysis of 5–10 cells only. The resolution range of the technique is at least 10–200 kb, and probably as little as a few kb, thus greatly extending the abilities of the existing FISH methodologies. This novel technique is much more efficient and practicable than pronuclei hybridizations, another method for high resolution FISH, and readily produces results with probes of a variety of genomic origin. In conclusion the DNA halo technique should be able to contribute significantly to the assessment of cosmid and YAC overlaps as well as to the sizing of gaps between adjacent contigs generated in genome projects.
ISSN:0964-6906
1460-2083
DOI:10.1093/hmg/1.8.587