| Multiplexed quantification of bacterial 16S rRNA by solution hybridization with oligonucleotide probes and affinity capture. | |
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MedLine Citation:
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PMID: 16132427 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Multiplexed and quantitative analysis of nucleic acid sequences in complex mixtures is essential in various applications of microbiological research. We have developed a method based on solution hybridization between biotinylated nucleic acid targets and multiple fluorophore-labeled oligonucleotide probes of distinct sizes. The biotin-nucleic acid-probe complexes are captured on magnetic streptavidin-coated microparticles and washed. The hybridized probes are eluted and their identity and quantity are determined by capillary electrophoresis. The signal intensities of the recorded probes correspond to the amount of target nucleic acid in the mixture, and the size indicates the target. Based on this principle and 16S rRNA-specific oligonucleotide probes, we set up an application for the relative quantification of different groups of clostridia and related organisms in a mixed bacterial population. The lower detection limit is 0.05 ng of total RNA and the linear range of measurement is 10(2). The method allowed accurate and highly repeatable quantification of the proportion of clostridia in human feces. Further, we discuss other applications of the method such as quantitative transcriptional analysis of eukaryotic microorganisms, which can be performed without conversion of mRNA to cDNA. |
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Authors:
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Reetta M Satokari; Kari Kataja; Hans Söderlund |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't Date: 2005-08-19 |
Journal Detail:
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Title: Microbial ecology Volume: 50 ISSN: 0095-3628 ISO Abbreviation: Microb. Ecol. Publication Date: 2005 Jul |
Date Detail:
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Created Date: 2005-09-30 Completed Date: 2005-11-14 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 7500663 Medline TA: Microb Ecol Country: United States |
Other Details:
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Languages: eng Pagination: 120-7 Citation Subset: IM |
Affiliation:
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VTT Biotechnology, Espoo, Finland. reetta.satokari@vtt.fi |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Bacteria
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genetics* Base Sequence DNA, Bacterial / analysis* In Situ Hybridization Oligonucleotide Array Sequence Analysis* Oligonucleotide Probes RNA, Ribosomal, 16S / analysis*, genetics* Reproducibility of Results Sensitivity and Specificity Sequence Analysis, DNA |
| Chemical | |
Reg. No./Substance:
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0/DNA, Bacterial; 0/Oligonucleotide Probes; 0/RNA, Ribosomal, 16S |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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