KAWAKMI Shuji
Visual detection of single copy genes by two-pass TSA-FISH
OHASHI Akiyoshi
Since catalyzed reporter deposition - fluorescence in situ hybridization (TSA-FISH) was first
introduced for microbial community analyses in 1997, it has been developed for detection of rRNA
and mRNA. However, it has not yet applied for detection of genes on prokaryotic chromosomes due
to its insufficient sensitivity. Recently, two-pass TSA-FISH was reported, which yields intensified
signals rather than TSA-FISH. In this study, we focus on two-pass TSA-FISH and attempted to
detect genes on prokaryotic chromosomes by this technique. 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.
Methanococcus maripaludis strain S2 was employed as a model organism and the alpha subunit
of methyl-coenzyme M reductase (mcrA), which is single copy gene on the archaeal chromosome,
was selected for a target. Probes labeled with hapten were hybridized after denaturation of DNA and
probes simultaneously. Subsequently, HRP-conjugated antibody was applied and first tyramide
signal amplification (TSA) was carried out with hapten-labeled tyramide. Afterward, HRP-
conjugated antibody was again applied and then second TSA was performed with
fluorphore-labeled tyramide for visualization. Detection of mcrA gene was successfully
carried out by two-pass TSA-FISH, whereas detection by TSA-FISH resulted in no signals.
However, the staining was very heterogeneous, with certain cells showing no signals. In addition,
strong unspecific background was obtained from glass slide due to, perhaps, unspecific binding of
HRP-labeled antibody to the slide. Despite the many attempts it is still difficult to overcome these
problems. Our results suggest that two-pass TSA-FISH has a potential for identification of
microorganisms based on functional genes.
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