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

  • Kengo KubotaEmail author
  • Shuji Kawakami
Part of the Springer Protocols Handbooks book series (SPH)


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.


Catalyzed reporter deposition Fluorescence in situ hybridization Functional genes Methyl coenzyme M reductase (mcr) gene Prokaryotic cells Tyramide signal amplification 



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.


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© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Department of Civil and Environmental EngineeringTohoku UniversitySendaiJapan
  2. 2.Department of Construction Systems EngineeringAnan National College of TechnologyAnanJapan

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