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Schramm, Andreas ; Fuchs, Bernhard M. ; Nielsen, Jeppe L. ; Tonolla, Mauro ; Stahl, David A.:
Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries.
In: Environmental Microbiology.
Bd. 4
(2002)
Heft 11
.
- S. 713-720.
ISSN 1462-2920
DOI: https://doi.org/10.1046/j.1462-2920.2002.00364.x
Abstract
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 behavior and calculated Td 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 sulfate-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.
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| Publikationsform: | Artikel in einer Zeitschrift |
|---|---|
| Begutachteter Beitrag: | Ja |
| Zusätzliche Informationen: | BAYCEER13372 |
| Institutionen der Universität: | Forschungseinrichtungen > Forschungszentren > Bayreuther Zentrum für Ökologie und Umweltforschung - BayCEER Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Biologie > Lehrstuhl Ökologische Mikrobiologie Fakultäten Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Biologie Forschungseinrichtungen Forschungseinrichtungen > Forschungszentren |
| Titel an der UBT entstanden: | Ja |
| Themengebiete aus DDC: | 500 Naturwissenschaften und Mathematik |
| Eingestellt am: | 24 Sep 2015 09:38 |
| Letzte Änderung: | 24 Sep 2015 09:38 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/19612 |