PMID- 10919826
OWN - NLM
STAT- MEDLINE
DCOM- 20010104
LR  - 20210526
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 66
IP  - 8
DP  - 2000 Aug
TI  - Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA 
      of fluorescently labeled oligonucleotide probes.
PG  - 3603-7
AB  - Target site inaccessibility represents a significant problem for fluorescence in 
      situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, 
      unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site 
      were evaluated for their potential to increase weak probe hybridization signals 
      in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence 
      signal of all six probes examined at least fourfold. In one case, the signal of 
      probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. 
      In another case, four unlabeled helpers raised the FISH signal of a formerly weak 
      probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes 
      available for E. coli. The temperature of dissociation and the mismatch 
      discrimination of probes were not significantly influenced by the addition of 
      helpers. Therefore, using helpers should not cause labeling of additional 
      nontarget organisms at a defined stringency of hybridization. However, the helper 
      action is based on sequence-specific binding, and there is thus a potential for 
      narrowing the target group which must be considered when designing helpers. We 
      conclude that helpers can open inaccessible rRNA regions for FISH with 
      oligonucleotide probes and will thereby further improve the applicability of this 
      technique for in situ identification of microorganisms.
FAU - Fuchs, B M
AU  - Fuchs BM
AD  - Max-Planck-Institut fur Marine Mikrobiologie, D-28359 Bremen, Germany. 
      bfuchs@mpi-bremen.de
FAU - Glockner, F O
AU  - Glockner FO
FAU - Wulf, J
AU  - Wulf J
FAU - Amann, R
AU  - Amann R
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (Oligonucleotides)
RN  - 0 (RNA, Ribosomal, 16S)
SB  - IM
MH  - Azospirillum/genetics
MH  - Escherichia coli/genetics
MH  - Flow Cytometry
MH  - *Fluorescent Dyes
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - *Oligonucleotide Probes
MH  - *Oligonucleotides
MH  - RNA, Ribosomal, 16S/*genetics
PMC - PMC92190
EDAT- 2000/08/05 11:00
MHDA- 2001/02/28 10:01
PMCR- 2000/08/01
CRDT- 2000/08/05 11:00
PHST- 2000/08/05 11:00 [pubmed]
PHST- 2001/02/28 10:01 [medline]
PHST- 2000/08/05 11:00 [entrez]
PHST- 2000/08/01 00:00 [pmc-release]
AID - 0322 [pii]
AID - 10.1128/AEM.66.8.3603-3607.2000 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2000 Aug;66(8):3603-7. doi: 
      10.1128/AEM.66.8.3603-3607.2000.