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Applied and Environmental Microbiology, August 2000, p. 3603-3607, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Unlabeled Helper Oligonucleotides Increase the In Situ Accessibility to 16S rRNA of Fluorescently Labeled Oligonucleotide Probes

Bernhard M. Fuchs,* Frank Oliver Glöckner, Jörg Wulf, and Rudolf Amann

Max-Planck-Institut für Marine Mikrobiologie, D-28359 Bremen, Germany

Received 28 February 2000/Accepted 19 May 2000

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083T. The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.

* Corresponding author. Mailing address: Max-Planck-Institut für Marine Mikrobiologie, Celsiusstr. 1, D-28359 Bremen, Germany. Phone: 49 421 2028 938. Fax: 49 421 2028 790. E-mail: bfuchs{at}mpi-bremen.de.

Applied and Environmental Microbiology, August 2000, p. 3603-3607, Vol. 66, No. 8
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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