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

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.

FIG. 1.

(A) Effect of double labeling of the EUB338 probe on the FISH signal intensity of four reference organisms. For each organism, the signal intensity conferred by a doubly labeled EUB338 probe was normalized to the signal intensity obtained with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5- and FLUOS-labeled probe EUB338, respectively. (B) Effect of double labeling of the probe Gam42a (in the presence of the unlabeled competitor probe Bet42a, specific for most members of the Betaproteobacteria [14]) on the FISH signal intensities of four reference organisms. For each organism, the signal intensity conferred by the doubly labeled probe Gam42a was normalized to the signal intensity obtained for E. coli with the same probe as a singly labeled derivative. Hatched, light-gray, and dark-gray bars depict results with the Cy3-, Cy5-, and FLUOS-labeled probe, respectively. The weak unspecific signals observed with some DOPE-FISH probes for B. subtilis are also detectable at comparable intensities with singly labeled probes (data not shown). (C) Cy3-doubly labeled but not FLUOS-doubly labeled probes improve in situ accessibility of E. coli 16S rRNA target sites. E. coli was hybridized with five probes representing brightness classes V and VI (3). FISH signals were recorded for Cy3-singly and -doubly labeled probes and normalized to the FISH signal obtained for E. coli with the singly labeled probe EUB338. Light-gray and dark-gray bars depict results with Cy3-singly and doubly labeled probes, respectively. FLUOS-singly and -doubly labeled probes showed no signal. For all panels, all experiments were performed in triplicate. Error bars indicate the standard deviation. ND, not detectable.

FIG. 2.

Comparison of probe dissociation profiles of singly and doubly labeled probes. For each profile, the microscopic settings were adjusted for the lowest formamide concentration and subsequently kept constant. Dashed and solid lines represent sigmoid fittings for singly and doubly labeled probes, respectively. (A) Dissociation profiles of the singly and doubly labeled probe Gam42a with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. (B) Dissociation profiles of the singly and doubly labeled probe Gam42a with B. cepacia as a nontarget organism having a single mismatch to probe GAM42a. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe GAM42a, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe GAM42a, respectively. The melting curves for FLUOS-singly labeled and Cy5-doubly labeled probes are almost identical and thus overlap. In the presence of the unlabeled probe Bet42a as a competitor, no probe-conferred signal was recordable for both singly and doubly labeled GAM42a probes. (C) Dissociation profiles of the singly and doubly labeled probe EUB338 with E. coli as the target organism. Empty circles, squares, and triangles represent data obtained with the Cy3-, Cy5-, and FLUOS-singly labeled probe EUB338, respectively. Filled circles, squares, and triangles depict the data measured for the Cy3-, Cy5-, and FLUOS-doubly labeled probe EUB338, respectively. For all panels, error bars are not shown since they were always smaller than the symbols.