A new method of in situ hybridization |
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| Authors: | Jerry E Manning N Davis Hershey Thomas R Broker Maria Pellegrini Herschel K Mitchell Norman Davidson |
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| Institution: | (1) Department of Chemistry, California Institute of Technology, 91125 Pasadena, California, USA;(2) Division of Biology, California Institute of Technology, 91125 Pasadena, California, USA;(3) Present address: Department of Molecular Biology and Biochemistry School of Biological Sciences, University of California, 92664 Irvine, Irvine, Calif, USA;(4) Present address: Cold Spring Harbor Laboratory, P.O. Box 100, 11724 Cold Spring Harbor, N.Y., USA |
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| Abstract: | A new method for gene mapping at the chromosome level using in situ hybridization and scanning electron microscopy is described and has been applied to mapping the rRNA genes of Drosophila melanogaster. Biotin is covalently attached to Drosophila rRNA via a cytochrome c bridge at a ratio of one cytochrome-biotin per 130 nucleotides by a chemical procedure. Polymethacrylate spheres with a diameter of ca. 60 nm are prepared by emulsion polymerization and are covalently attached to the protein avidin at a ratio of 5–20 avidins per sphere. The biotin-labeled rRNA is hybridized to denatured DNA in a chromosome squash. Upon incubation with a sphere solution, some of the biotin sites become labeled with spheres because of the strong non-covalent interaction between biotin and avidin. The chromosome squash is examined in the scanning electron microscope (SEM). Polymer spheres, which are visible in the SEM, are observed to label the nucleolus, where the rRNA genes are located.Contribution number 5121 from the Department of Chemistry. |
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