Present addresses. Department of Ecological Microbiology, University of Bayreuth, D-95440 Bayreuth, Germany; ‡Department of Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark.
Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries
Article first published online: 9 DEC 2002
Volume 4, Issue 11, pages 713–720, November 2002
How to Cite
Schramm, A., Fuchs, B. M., Nielsen, J. L., Tonolla, M. and Stahl, D. A. (2002), Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe validation and screening of clone libraries. Environmental Microbiology, 4: 713–720. doi: 10.1046/j.1462-2920.2002.00364.x
- Issue published online: 9 DEC 2002
- Article first published online: 9 DEC 2002
- Received 23 August, 2002; revised 3 October, 2002; accepted 23 October, 2002.
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 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 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.