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          Institute: MPI für marine Mikrobiologie     Collection: Abteilung Molekulare Ökologie     Display Documents



ID: 13914.0, MPI für marine Mikrobiologie / Abteilung Molekulare Ökologie
Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries
Authors:Schramm, A.; Fuchs, B. M.; Nielsen, J. L.; Tonolla, M.; Stahl, D. A.
Language:English
Date of Publication (YYYY-MM-DD):2002-11
Title of Journal:Environmental Microbiology
Journal Abbrev.:Environ. Microbiol.
Volume:4
Issue / Number:11
Start Page:713
End Page:720
Review Status:Peer-review
Audience:Not Specified
Abstract / Description:A method is presented for fluorescence in situ hybridization (FISH) of 16S rRNA gene clones targeting in vivo transcribed plasmid inserts (Clone-FISH). Several different cloning approaches and treatments to generate target-rRNA in the clones were compared. Highest signal intensities of Clone-FISH were obtained using plasmids with a T7 RNA polymerase promoter and host cells with an IPTG-inducible T7 RNA polymerase. Combined IPTG-induction and chloramphenicol treatment of those clones resulted in FISH signals up to 2.8-fold higher than signals of FISH with probe EUB338 to cells of Escherichia coli. Probe dissociation curves for three oligonucleotide probes were compared for reference cells containing native (FISH) or cloned (Clone-FISH) target sequences. Melting behaviour and calculated T-d values were virtually identical for clones and cells, providing a format to use 16S rRNA gene clones instead of pure cultures for probe validation and optimization of hybridization conditions. The optimized Clone-FISH protocol was also used to screen an environmental clone library for insert sequences of interest. In this application format, 13 out of 82 clones examined were identified to contain sulphate-reducing bacterial rRNA genes. In summary, Clone-FISH is a simple and fast technique, compatible with a wide variety of cloning vectors and hosts, that should have general utility for probe validation and screening of clone libraries.
Comment of the Author/Creator:Date: 2002, NOV
External Publication Status:published
Document Type:Article
Communicated by:N. N.
Affiliations:MPI für marine Mikrobiologie
External Affiliations:Univ Washington, Dept Civil & Environm Engn, Seattle, WA 98195; USA; Univ Washington, Dept Civil & Environm Engn, Seattle, WA 98195 USA; Max Planck Inst Marine Microbiol, D-28359 Bremen, Germany; Univ Geneva, Cantonal Inst Bacteriol Microbial Ecol, CH-6904 Lugano, Switzerland
Identifiers:ISI:000179540100011 [ID No:1]
ISSN:1462-2912 [ID No:2]
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