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Applied and Environmental Microbiology, March 2003, p. 1397-1407, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1397-1407.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes

Jörg Peplies, Frank Oliver Glöckner,* and Rudolf Amann

Department of Molecular Ecology, Max Planck Institute for Marine Microbiology, 28359 Bremen, Germany

Received 13 September 2002/ Accepted 10 December 2002

The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.

* Corresponding author. Mailing address: Max Planck Institute for Marine Microbiology, Celsiusstrasse 1, 28359 Bremen, Germany. Phone: 49 421 2028 938. Fax: 49 421 2028 580. E-mail: fog{at}mpi-bremen.de.

Applied and Environmental Microbiology, March 2003, p. 1397-1407, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1397-1407.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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