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Protocol for In Situ Detection of Functional Genes of Microorganisms by Two-Pass TSA-FISH

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Hydrocarbon and Lipid Microbiology Protocols

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Abstract

An approach is presented for the detection of functional genes on chromosomal DNA in prokaryotes by two-pass tyramide signal amplification–fluorescence in situ hybridization (two-pass TSA-FISH). Functional genes are hybridized with 2,4-dinitrophenol (DNP)-labeled polynucleotide probes or digoxigenin-labeled oligonucleotide probes. Horseradish peroxidase (HRP)-labeled antibody is then immunologically bounded, and a first round of TSA with DNP-labeled tyramide is carried out. After the second immunological reaction with HRP-labeled anti-DNP antibody, cells hybridized with the probes are detected upon a second round of TSA with fluorescent-labeled tyramide. As a case study, we describe the use of two-pass TSA-FISH to detect the methanogenesis marker gene mcrA, which encodes the alpha subunit of methyl coenzyme M reductase in methanogenic archaea. Practical suggestions for using the two-pass TSA-FISH method are presented as well.

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Acknowledgment

The authors thank Prof. Hideki Harada of Tohoku University, Prof. Akiyoshi Ohashi of Hiroshima University, and Dr. Hiroyuki Imachi of the Japan Agency for Marine-Earth Science and Technology (JAMSTEC) for their helpful advice regarding protocol development.

Author information

Authors and Affiliations

  1. Department of Civil and Environmental Engineering, Tohoku University, 6-6-06 Aza-Aoba, Aramaki, Aoba-ku, Sendai, Miyagi, 980-8579, Japan

    Kengo Kubota

  2. Department of Construction Systems Engineering, Anan National College of Technology, 265 Aoki Minobayashi, Anan, Tokushima, 774-0017, Japan

    Shuji Kawakami

Authors
  1. Kengo Kubota
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  2. Shuji Kawakami
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Corresponding author

Correspondence to Kengo Kubota .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Essex, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

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© 2015 Springer-Verlag Berlin Heidelberg

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Kubota, K., Kawakami, S. (2015). Protocol for In Situ Detection of Functional Genes of Microorganisms by Two-Pass TSA-FISH. In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_71

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  • DOI: https://doi.org/10.1007/8623_2015_71

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  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-49132-4

  • Online ISBN: 978-3-662-49134-8

  • eBook Packages: Springer Protocols

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