PMID- 8890483
OWN - NLM
STAT- MEDLINE
DCOM- 19961209
LR  - 20190720
IS  - 0008-4166 (Print)
IS  - 0008-4166 (Linking)
VI  - 42
IP  - 10
DP  - 1996 Oct
TI  - Differential sensitivity of 16S rRNA targeted oligonucleotide probes used for 
      fluorescence in situ hybridization is a result of ribosomal higher order 
      structure.
PG  - 1061-71
AB  - The use of 16S rRNA targeted gene probes for the direct analysis of microbial 
      communities has revolutionized the field of microbial ecology, yet a 
      comprehensive approach for the design of such probes does not exist. The 
      development of 16S rRNA targeted oligonucleotide probes for use with fluorescence 
      in situ hybridization (FISH) procedures has been especially difficult as a result 
      of the complex nature of the rRNA target molecule. In this study a systematic 
      comparison of 16S rRNA targeted oligonucleotide gene probes was conducted to 
      determine if target location influences the hybridization efficiency of 
      oligonucleotide probes when used with in situ hybridization protocols for the 
      detection of whole microbial cells. Five unique universal 12-mer oligonucleotide 
      sequences, located at different regions of the 16S rRNA molecule, were identified 
      by a computer-aided sequence analysis of over 1000 partial and complete 16S rRNA 
      sequences. The complements of these oligomeric sequences were chemically 
      synthesized for use as probes and end labeled with either [gamma-32P]ATP or the 
      fluorescent molecule tetramethylrhodamine-5/-6. Hybridization sensitivity for 
      each of the probes was determined by hybridization to heat-denatured RNA 
      immobilized on blots or to formaldehyde fixed whole cells. All of the probes 
      hybridized with equal efficiency to denatured RNA. However, the probes exhibited 
      a wide range of sensitivity (from none to very strong) when hybridized with whole 
      cells using a previously developed FISH procedure. Differential hybridization 
      efficiencies against whole cells could not be attributed to cell wall type, since 
      the relative probe efficiency was preserved when either Gram-negative or 
      -positive cells were used. These studies represent one of the first attempts to 
      systematically define criteria for 16S rRNA targeted probe design for use against 
      whole cells and establish target site location as a critical parameter in probe 
      design.
FAU - Frischer, M E
AU  - Frischer ME
AD  - Department of Biology, MRC 306 Rensselaer Polytechnic Institute, Troy, NY 
      12180-3590, USA.
FAU - Floriani, P J
AU  - Floriani PJ
FAU - Nierzwicki-Bauer, S A
AU  - Nierzwicki-Bauer SA
LA  - eng
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - Canada
TA  - Can J Microbiol
JT  - Canadian journal of microbiology
JID - 0372707
RN  - 0 (Fluorescent Dyes)
RN  - 0 (Oligonucleotide Probes)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 63231-63-0 (RNA)
SB  - IM
MH  - Cell Wall/chemistry
MH  - Electronic Data Processing
MH  - Fluorescent Dyes/analysis
MH  - Gram-Negative Bacteria/cytology
MH  - Gram-Positive Bacteria/cytology
MH  - In Situ Hybridization, Fluorescence/*methods
MH  - Molecular Structure
MH  - Oligonucleotide Probes/*analysis
MH  - RNA/isolation & purification
MH  - RNA, Ribosomal, 16S/*chemistry/*ultrastructure
MH  - Sensitivity and Specificity
EDAT- 1996/10/01 00:00
MHDA- 1996/10/01 00:01
CRDT- 1996/10/01 00:00
PHST- 1996/10/01 00:00 [pubmed]
PHST- 1996/10/01 00:01 [medline]
PHST- 1996/10/01 00:00 [entrez]
AID - 10.1139/m96-136 [doi]
PST - ppublish
SO  - Can J Microbiol. 1996 Oct;42(10):1061-71. doi: 10.1139/m96-136.