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Rapid and high resolution detection of in situ hybridisation to polytene chromosomes using fluorochrome-labeled RNA

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Abstract

Fluorochrome-labeled RNA allows the rapid detection of in situ hybrids without the need for long exposure times as in the autoradiographical hybridisation methods. Resolution is high because of the high resolving power of fluorescence microscopy. The application of a previously reported method for the hybrido-cytochemical detection of DNA sequences to polytene chromosomes of Drosophilia is described. — The specificity and sensitivity of the method are demonstrated by the hybridisation with polytene chromosomes of 1) rhodamine-labeled 5S RNA, to the 5S rRNA sites of D. melanogaster (56F) and D. hydei (23 B), 2) rhodamine-labeled RNA complementary to a plasmid containing histone genes, to the 39DE region of D. melanogaster, 3) rhodamine-labeled D. melanogaster tRNA species (Gly-3 and Arg-2), to their respective loci in D. melanogaster, 4) rhodamine-labeled RNA complementary to the insert of plasmid 232.1 containing part of a D. melanogaster heat shock gene from locus 87 C, to D. hydei heat shock locus 2-32A. In the latter instance it was possible to demonstrate the labeling of a double band which escaped unambiguous detection by autoradiography in the radioactive cytochemical hybridisation procedure because of the low topological resolution of autoradiograms. — The sensitivity of the fluorochrome-labeled RNA method is compared with the radioactive methods which use 3H- or 125I-labeled RNAs. The factors governing the sensitivity and the number of bound fluorochrome molecules to be expected are discussed.

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Author notes

  1. J. G. J. Bauman

    Present address: Radiobiological Institute, TNO, Rijswijk, The Netherlands

  2. P. J. A. Sondermeijer

    Present address: , Transgene S.A., Strasbourg, France

Authors and Affiliations

  1. Laboratory for Histochemistry and Cytochemistry, State University Leiden, 2300, Leiden, RA, The Netherlands

    J. G. J. Bauman, J. Wiegant & P. Van Duijn

  2. Department of Genetics, University of Nijmegen, 6525, Nijmegen, ED, The Netherlands

    N. H. Lubsen, P. J. A. Sondermeijer & W. Hennig

  3. Zoologisches Institut der Universitat Zürich, Switzerland

    E. Kubli

Authors
  1. J. G. J. Bauman
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  2. J. Wiegant
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  3. P. Van Duijn
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  4. N. H. Lubsen
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  5. P. J. A. Sondermeijer
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  6. W. Hennig
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  7. E. Kubli
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Dedicated to Professor W. Beermann in honour of his 60th birthday

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Bauman, J.G.J., Wiegant, J., Van Duijn, P. et al. Rapid and high resolution detection of in situ hybridisation to polytene chromosomes using fluorochrome-labeled RNA. Chromosoma 84, 1–18 (1981). https://doi.org/10.1007/BF00293359

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  • DOI: https://doi.org/10.1007/BF00293359

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