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Fluorescence in situ hybridization of 16S rRNA gene clones (Clone‐FISH) for probe validation and screening of clone libraries
Author(s) -
Schramm Andreas,
Fuchs Bernhard M.,
Nielsen Jeppe L.,
Tonolla Mauro,
Stahl David A.
Publication year - 2002
Publication title -
environmental microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.954
H-Index - 188
eISSN - 1462-2920
pISSN - 1462-2912
DOI - 10.1046/j.1462-2920.2002.00364.x
Subject(s) - biology , clone (java method) , library , fluorescence in situ hybridization , microbiology and biotechnology , cloning (programming) , ribosomal rna , 16s ribosomal rna , gene , plasmid , bacterial artificial chromosome , genomic library , polymerase chain reaction , t7 rna polymerase , genetics , escherichia coli , genome , bacteriophage , chromosome , peptide sequence , computer science , programming language
Summary 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.