EurekaMag.com logo
+ Site Statistics
References:
53,517,315
Abstracts:
29,339,501
+ Search Articles
+ Subscribe to Site Feeds
EurekaMag Most Shared ContentMost Shared
EurekaMag PDF Full Text ContentPDF Full Text
+ PDF Full Text
Request PDF Full TextRequest PDF Full Text
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter
Follow on Google+Follow on Google+
Follow on LinkedInFollow on LinkedIn

+ Translate

Sequence-specific cleavage of small-subunit (SSU) rRNA with oligonucleotides and RNase H: a rapid and simple approach to SSU rRNA-based quantitative detection of microorganisms



Sequence-specific cleavage of small-subunit (SSU) rRNA with oligonucleotides and RNase H: a rapid and simple approach to SSU rRNA-based quantitative detection of microorganisms



Applied and Environmental Microbiology 70(6): 3650-3663



A rapid and simple approach to the small-subunit (SSU) rRNA-based quantitative detection of a specific group of microorganisms in complex ecosystems has been developed. The method employs sequence-specific cleavage of rRNA molecules with oligonucleotides and RNase H. Defined mixtures of SSU rRNAs were mixed with an oligonucleotide (referred to as a "scissor probe") that was specifically designed to hybridize with a particular site of targeted rRNA and were subsequently digested with RNase H to proceed to sequence-dependent rRNA scission at the hybridization site. Under appropriate reaction conditions, the targeted rRNAs were correctly cut into two fragments, whereas nontargeted rRNAs remained intact under the same conditions. The specificity of the cleavage could be properly adjusted by controlling the hybridization stringency between the rRNA and the oligonucleotides, i.e., by controlling either the temperature of the reaction or the formamide concentration in the hybridization-digestion buffer used for the reaction. This enabled the reliable discrimination of completely matched rRNA sequences from single-base mismatched sequences. For the detection of targeted rRNAs, the resulting RNA fragment patterns were analyzed by gel electrophoresis with nucleotide-staining fluorescent dyes in order to separate cleaved and intact rRNA molecules. The relative abundance of the targeted SSU rRNA fragments in the total SSU rRNA could easily be calculated without the use of an external standard by determining the signal intensity of individual SSU rRNA bands in the electropherogram. This approach provides a fast and easy means of identification, detection, and quantification of a particular group of microbes in clinical and environmental specimens based on rRNA.

(PDF same-day service: $19.90)

Accession: 012547216

Download citation: RISBibTeXText

PMID: 15184170

DOI: 10.1128/AEM.70.6.3650-3663.2004



Related references

Quantitative analysis of rRNA populations of bacterial groups in dairy cattle rumen samples by sequence-specific small-subunit rRNA cleavage method. 2005

Sequence-specific cleavage of 16S rRNA for rapid and quantitative detection of particular groups of anaerobes in bioreactors. Water Science and Technology 52(1-2): 107-113, 2005

Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis. Genome 48(3): 355-366, 2005

Intermolecular hybridization of 5S rRNA with 18S rRNA: identification of a 5'-terminally-located nucleotide sequence in mouse 5S rRNA which base-pairs with two specific complementary sequences in 18S rRNA. Biochimica et Biophysica Acta, Gene Structure and Expression 1088(1): 57-70, 1991

Quantitative detection of previously characterized syntrophic bacteria in anaerobic wastewater treatment systems by sequence-specific rRNA cleavage method. Water Research 46(7): 2167-2175, 2012

Characterization of the rDNA unit and sequence analysis of the small subunit rRNA and 5.8S rRNA genes from Tritrichomonas foetus. Molecular and Biochemical Parasitology 52(1): 75-83, 1992

Isolation of the 5S rRNA of Phaseolus aureus. Determination of the structure of the oligonucleotides of the pyrimidyl-RNase from a hydrolysate of 5S rRNA. Chemistry of natural compounds (pub 1986) 21(6): 813-815, 1986

Imp3p and Imp4p mediate formation of essential U3-precursor rRNA (pre-rRNA) duplexes, possibly to recruit the small subunit processome to the pre-rRNA. Proceedings of the National Academy of Sciences of the United States of America 101(43): 15301-6, 2004

Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis. Acta Parasitologica 57(4): 342-353, 2013

Characterization of babesia bigemina rrna genes and utilization of oligonucleotide probes complementary to the small subunit rrna for its detection in bovine blood. Journal of Cellular Biochemistry Supplement (16 PART A): 129, 1992

Inhibition of ribosomal subunit association and protein synthesis by oligonucleotides corresponding to defined regions of 18S rRNA and 5S rRNA. Biochemical and Biophysical Research Communications 248(1): 51-56, 1998

16S rRNA-based assays for quantitative detection of universal, human-, cow-, and dog-specific fecal Bacteroidales: a Bayesian approach. Water Research 41(16): 3701-3715, 2007

The development of novel rRNA based oligonucleotides for specific PCR detection of Salmonella. FEMS Congress of European Microbiologists Abstract Book (1): 34, 2003

Euglena gracilis chloroplast small subunit rRNA. Sequence and base pairing potential of the 3' terminus, cleavage by colicin E3. Journal of Biological Chemistry 257(17): 10430-9, 1982

Proposed sequence homology between the 5'-end regions of prokaryotic 23 S rRNA and eukaryotic 28 S rRNA. Relevance to the hypothesis that 5.8 S rRNA is homologous to the 5'-end region of 23 S rRNA. Febs Letters 126(2): 150-151, 1981