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In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test...

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Detalles Bibliográficos
Autores principales: Hutchison, NJ, Langer-Safer, PR, Ward, DC, Hamkalo, BA
Formato: Texto
Lenguaje:English
Publicado: The Rockefeller University Press 1982
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2112979/
https://www.ncbi.nlm.nih.gov/pubmed/6183277
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author Hutchison, NJ
Langer-Safer, PR
Ward, DC
Hamkalo, BA
author_facet Hutchison, NJ
Langer-Safer, PR
Ward, DC
Hamkalo, BA
author_sort Hutchison, NJ
collection PubMed
description In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.
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spelling pubmed-21129792008-05-01 In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold Hutchison, NJ Langer-Safer, PR Ward, DC Hamkalo, BA J Cell Biol Articles In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin. The Rockefeller University Press 1982-11-01 /pmc/articles/PMC2112979/ /pubmed/6183277 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).
spellingShingle Articles
Hutchison, NJ
Langer-Safer, PR
Ward, DC
Hamkalo, BA
In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold
title In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold
title_full In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold
title_fullStr In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold
title_full_unstemmed In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold
title_short In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold
title_sort in situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold
topic Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2112979/
https://www.ncbi.nlm.nih.gov/pubmed/6183277
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