The effect of nucleobase-specific fluorescence quenching on in situ hybridization with rRNA-targeted oligonucleotide probes.
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.
Behrens
Sebastian
S
Max Planck Institute of Marine Microbiology, Bremen, Germany.
Fuchs
Bernhard M
BM
Amann
Rudolf
R
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
D005452:Fluoresceins; D015345:Oligonucleotide Probes; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; C024098:6-carboxyfluorescein; D006147:Guanine