Visual detection of chromosomal encoded methyl coenzyme M reductase gene of a methanogen by two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with polydeoxyribonucleotide probes

DOI
  • KAWAKMI Shuji
    Department of Environmental Systems Engineering, Nagaoka University of Technology
  • KUBOTA Kengo
    Department of Environmental Systems Engineering, Nagaoka University of Technology
  • IMACHI Hiroyuki
    Department of Environmental Systems Engineering, Nagaoka University of Technology Japan Agency for Marine-Earth Science and Technology Research Scientist Extremobiosphere Research Center
  • HARADA Hideki
    Department of Environmental Systems Engineering, Nagaoka University of Technology Department of Civil Engineering, Graduate School of Engineering, Tohoku University
  • OHASHI Akiyoshi
    Department of Environmental Systems Engineering, Nagaoka University of Technology

Bibliographic Information

Other Title
  • Ｔｗｏ‐ｐａｓｓ　ＴＳＡ‐ＦＩＳＨ法によるメタン生成古細菌のｍｃｒ遺伝子の検出

Description

Applicability and reliability of two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with PCR generated polydeoxyribonucleotide probes, specific for chromosomal encoded gene, methyl coenzyme M reductase (mcr) in Methanococcus vannielli, was tested and evaluated. The probes were labeled with dinitrophenol (DNP) and the efficiency of probe-labeling was improved by optimizing the concentrations of DNP-labeled nucleotide and Mg2+ in PCR mixture. The target mcr gene was successfully detected, which was again verified by the disappearance of the signals after treating the target with DNase prior to hybridization or washing with high stringency buffer. However, a few nonspecific signals were observed when the method was applied to pure cultures of Methanoculleus bourgensis and Escherichia coli.

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Details 詳細情報について

  • CRID
    1390001204111490176
  • NII Article ID
    130003949508
  • DOI
    10.11532/proes1992.43.143
  • ISSN
    1884829X
    13415115
  • Text Lang
    ja
  • Article Type
    journal article
  • Data Source
    • JaLC
    • CiNii Articles
    • KAKEN
  • Abstract License Flag
    Disallowed

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