title: The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes.
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.
authors.affiliation: Max Planck Institute of Marine Microbiology, Bremen, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Behrens
authors.forename: Bernhard M
authors.initials: BM
authors.lastname: Fuchs
authors.forename: Rudolf
authors.initials: R
authors.lastname: Amann
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D006147:Guanine / Q000737:chemistry; D017404:In Situ Hybridization, Fluorescence; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis*; D012680:Sensitivity and Specificity
chemical_list: D005452:Fluoresceins; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; C024098:6-carboxyfluorescein; D006147:Guanine