PMID- 12460279
OWN - NLM
STAT- MEDLINE
DCOM- 20030130
LR  - 20220321
IS  - 1462-2912 (Print)
IS  - 1462-2912 (Linking)
VI  - 4
IP  - 11
DP  - 2002 Nov
TI  - Fluorescence in situ hybridization of 16S rRNA gene clones (Clone-FISH) for probe 
      validation and screening of clone libraries.
PG  - 713-20
AB  - 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.
FAU - Schramm, Andreas
AU  - Schramm A
AD  - Department of Civil and Environmental Engineering, University of Washington, 
      Seattle, WA 98195-2700, USA.
FAU - Fuchs, Bernhard M
AU  - Fuchs BM
FAU - Nielsen, Jeppe L
AU  - Nielsen JL
FAU - Tonolla, Mauro
AU  - Tonolla M
FAU - Stahl, David A
AU  - Stahl DA
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - England
TA  - Environ Microbiol
JT  - Environmental microbiology
JID - 100883692
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Ribosomal, 16S)
SB  - IM
MH  - Cloning, Molecular
MH  - Gene Library
MH  - *In Situ Hybridization, Fluorescence
MH  - Oligonucleotide Probes/*genetics
MH  - RNA, Ribosomal, 16S/*genetics
OTO - NASA
OT  - Non-programmatic
EDAT- 2002/12/04 04:00
MHDA- 2003/01/31 04:00
CRDT- 2002/12/04 04:00
PHST- 2002/12/04 04:00 [pubmed]
PHST- 2003/01/31 04:00 [medline]
PHST- 2002/12/04 04:00 [entrez]
AID - 364 [pii]
AID - 10.1046/j.1462-2920.2002.00364.x [doi]
PST - ppublish
SO  - Environ Microbiol. 2002 Nov;4(11):713-20. doi: 10.1046/j.1462-2920.2002.00364.x.