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Applied and Environmental Microbiology, August 2006, p. 5311-5317, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.03039-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Improved In Situ Hybridization Efficiency with Locked-Nucleic-Acid-Incorporated DNA Probes

Kengo Kubota,1 Akiyoshi Ohashi,1* Hiroyuki Imachi,1,2 and Hideki Harada1

Department of Environmental Systems Engineering, Nagaoka University of Technology, Nagaoka, Niigata 940-2188, Japan,1 Subground Animalcule Retrieval Program, Extremobiosphere Research Center, Japan Agency for Marine-Earth Science & Technology (JAMSTEC), Yokosuka, Kanagawa 237-0061, Japan2

Received 25 December 2005/ Accepted 13 June 2006

Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.

* Corresponding author. Mailing address: Department of Environmental Systems Engineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan. Phone: 81-258-47-9637. Fax: 81-258-47-9637. E-mail: ecohashi{at}vos.nagaokaut.ac.jp.

Applied and Environmental Microbiology, August 2006, p. 5311-5317, Vol. 72, No. 8
0099-2240/06/$08.00+0     doi:10.1128/AEM.03039-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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