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      Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) improves signal intensity and increases rRNA accessibility.

      Applied and Environmental Microbiology
      Conserved Sequence, genetics, Escherichia coli, Evolution, Molecular, Flow Cytometry, Fluorescence, Fluorescent Dyes, chemistry, In Situ Hybridization, Fluorescence, methods, Nucleic Acid Conformation, Oligonucleotide Probes, Phylogeny, RNA, Bacterial, analysis, RNA, Ribosomal, RNA, Ribosomal, 16S, RNA, Ribosomal, 18S, Sequence Alignment, Species Specificity, Thermodynamics

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          Abstract

          Fluorescence in situ hybridization (FISH) with singly labeled rRNA-targeted oligonucleotide probes is widely applied for direct identification of microbes in the environment or in clinical specimens. Here we show that a replacement of singly labeled oligonucleotide probes with 5'-, 3'-doubly labeled probes at least doubles FISH signal intensity without causing specificity problems. Furthermore, Cy3-doubly labeled probes strongly increase in situ accessibility of rRNA target sites and thus provide more flexibility for probe design.

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