The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes

Sebastian Behrens, Bernhard M. Fuchs, Rudolf Amann

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Oligonucleotide probes labeled with fluorescent dyes are used in a variety of in situ applications to detect specific DNA or RNA molecules. It has been described that probe fluorescence might be quenched upon hybridization in a sequence specific way. Here, a set of 17 oligonuleotides labeled with 6-carboxyfluorescein was used to examine the relevance of nucleotide specific quenching for fluorescence in situ hybridization (FISH) to whole fixed bacterial cells. Probes quenched upon hybridization to a guanine-rich region of purified RNA in solution were not quenched upon FISH. Among other factors the high protein concentration within cells may prevent quenching of probe fluorescence in situ.

Original languageEnglish (US)
Pages (from-to)565-572
Number of pages8
JournalSystematic and Applied Microbiology
Volume27
Issue number5
DOIs
StatePublished - Sep 2004
Externally publishedYes

Keywords

  • 6-carboxyfluorescein (6-FAM)
  • Fluorescence in situ hybridization (FISH)
  • Fluorescent dyes
  • Nucleobase-specific quenching
  • Oligonucleotide probes
  • Photoinduced electron transfer

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