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Tracking DNA and RNA Sequences at High Resolution

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Electron Microscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1117))

Abstract

In this chapter, the basic principles and protocols of the electron microscopical detections of specific DNA and RNA sequences are described. We focused primarily on a comparison of various methods of electron microscopy in situ hybridization (EM-ISH) with respect to their sensitivity and the structural preservation of the sample with the aim of helping the readers select the appropriate hybridization protocol. As the post-embedding EM-ISH most frequently represents the optimal choice, the protocol for the post-embedding EM-ISH approach is described in detail. Concurrently, the alternative methods based on the enzymatic synthesis of the labeled nucleic acids chains that can be used for the detection of DNA or RNA molecules in situ are mentioned. In this respect, the technique enabling the enzymatic detection of the polyadenylated RNA sequences is described in detail.

Dušan Cmarko and Anna Ligasová contributed equally to this work

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Acknowledgments

We thank Dr. J. Niedojadlo forkindly providing his micrograph. This work has been supported by the Grant Agency of the Academy of Sciences of the Czech Republic [KAN 200520801], by the Czech Science Foundation [P302/12/G157, P302/12/1885, 13-12317J], by the Technology Agency of the Czech Republic [TA03010598and TA03010719], by the Operational Programme Research and Development for Innovations [CZ.1.05/2.1.00/01.0030], by projects supported by Charles University in Prague [UNCE 204022 and Prvouk/1LF/1], and by BIOCEV - Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University (CZ.1.05/1.1.00/02.0109), from the European Regional Development Fund.

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Authors and Affiliations

  1. Institute of Cellular Biology and Pathology, 1st Faculty of Medicine, Charles University in Prague, Prague, Czech Republic

    Dušan Cmarko

  2. Institute of Molecular and Translational Medicine, Faculty of Medicine, Palacký University, Olomouc, Czech Republic

    Anna Ligasová & Karel Koberna

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  1. Dušan Cmarko
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  2. Anna Ligasová
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  3. Karel Koberna
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Editors and Affiliations

  1. University of Western Australia, Crawley, Australia

    John Kuo

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Cmarko, D., Ligasová, A., Koberna, K. (2014). Tracking DNA and RNA Sequences at High Resolution. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology, vol 1117. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-776-1_16

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  • DOI: https://doi.org/10.1007/978-1-62703-776-1_16

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-775-4

  • Online ISBN: 978-1-62703-776-1

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