Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

Xi, Chuanwu; Balberg, Michal; Boppart, Stephen A.; Raskin, Lutgarde

doi:10.1128/aem.69.9.5673-5678.2003

Cited by 65 publications

(75 citation statements)

References 35 publications

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“…The relative quantification of DNA using fluorogenic primers is comparable in sensitivity and dynamic range to other published methods of quantification, such as TaqMan probes (Best et al, 2005), molecular beacons (Xi et al, 2003) and DNA-binding dyes (SYBR Green) (De Medici et al, 2003). However, direct comparisons among methods for quantification are difficult as these methods are functionally different and may be affected differently by various factors.…”

mentioning

confidence: 75%

Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers

Balcázar

Vendrell

Blas

et al. 2007

Journal of Medical Microbiology

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In this study a real-time PCR assay using self-quenched primers labelled with a single fluorophore for the detection of Aeromonas salmonicida was developed. Probe specificity was confirmed by amplification of 16 A. salmonicida strain templates and by the lack of a PCR product with 26 non-A. salmonicida strains. With a pure culture of A. salmonicida, the assay was linear over a range of 0.5 pg to 50 ng and was able to detect 16 c.f.u. per reaction. A similar sensitivity was observed in DNA extracted from a mixture of A. salmonicida and fish tissue. Results using artificially inoculated tissues and diseased fish from outbreaks indicated that the assay can provide sensitive species-specific detection and quantification of A. salmonicida in fish tissue.

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confidence: 75%

Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers

Balcázar

Vendrell

Blas

et al. 2007

Journal of Medical Microbiology

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“…This is accomplished through a PNA coupled to a cell-penetrating peptide (CPP) as well as a photoactivatable compound. PNAs are nucleic acid analogs in which the sugar-phosphate backbone is replaced by a polyamide skeleton without altering the position of nucleobases (10,11). The PNA binds RNA with high specificity and selectivity, and the PNA-RNA hybrids are more stable and form faster (11), with increased specificity (12), than the corresponding nucleic acid hybrids.…”

Section: Rna-binding Proteinsmentioning

confidence: 99%

“…PNAs are nucleic acid analogs in which the sugar-phosphate backbone is replaced by a polyamide skeleton without altering the position of nucleobases (10,11). The PNA binds RNA with high specificity and selectivity, and the PNA-RNA hybrids are more stable and form faster (11), with increased specificity (12), than the corresponding nucleic acid hybrids. These characteristics, in combination with their resistance to proteases and nucleases (13), have permitted PNAs to be used in various molecular biological (11,(14)(15)(16) as well as therapeutic contexts (17)(18)(19)(20).…”

Section: Rna-binding Proteinsmentioning

confidence: 99%

In vivo identification of ribonucleoprotein-RNA interactions

Zielinski

Kilk

Peritz

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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To understand the role of RNA-binding proteins (RBPs) in the regulation of gene expression, methods are needed for the in vivo identification of RNA-protein interactions. We have developed the peptide nucleic acid (PNA)-assisted identification of RBP technology to enable the identification of proteins that complex with a target RNA in vivo. Specific regions of the 3 and 5 UTRs of ankylosis mRNA were targeted by antisense PNAs transported into cortical neurons by the cell-penetrating peptide transportan 10. An array of proteins was isolated in complex with or near the targeted regions of the ankylosis mRNA through UV-induced crosslinking of the annealed PNA-RNA-RBP complex. The first evidence for pharmacological modulation of these specific protein-RNA associations was observed. These data show that the PNA-assisted identification of the RBP technique is a reliable method to rapidly identify proteins interacting in vivo with the target RNA.

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“…Hybridizations with DNA MBs exhibit several other advantages, such as a low background signal (28), high specificity (5), and high sensitivity (51). Because of these characteristics, DNA MB probes have been shown to be advantageous in applications in real-time PCR (35,51), visualization of mRNA in living cells (6), and detection of nucleic acids in array techniques (4, 57) but have been less successful in quantifying rRNA in solution-based hybridizations (55).…”

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confidence: 99%

“…Apart from PNA-conferred improvements in probe composition, novel probe chemistries have also been devised for both DNA-and PNA-based probes. These include molecular beacons (MBs) (50,55), adjacent fluorescence resonance energy transfer probes (9,49), light-up probes (38,46), and quenched autoligation probes (40,41). The first DNA MB probes were developed by Tyagi and Kramer (50).…”

mentioning

confidence: 99%

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

Morgenroth

Raskin

2008

Appl Environ Microbiol

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The potential of a solution-based hybridization assay using peptide nucleic acid (PNA) molecular beacon (MB) probes to quantify 16S rRNA of specific populations in RNA extracts of environmental samples was evaluated by designing PNA MB probes for the genera Dechloromonas and Dechlorosoma. In a kinetic study with 16S rRNA from pure cultures, the hybridization of PNA MB to target 16S rRNA exhibited a higher final hybridization signal and a lower apparent rate constant than the hybridizations to nontarget 16S rRNAs. A concentration of 10 mM NaCl in the hybridization buffer was found to be optimal for maximizing the difference between final hybridization signals from target and nontarget 16S rRNAs. Hybridization temperatures and formamide concentrations in hybridization buffers were optimized to minimize signals from hybridizations of PNA MB to nontarget 16S rRNAs. The detection limit of the PNA MB hybridization assay was determined to be 1.6 nM of 16S rRNA. To establish proof for the application of PNA MB hybridization assays in complex systems, target 16S rRNA from Dechlorosoma suillum was spiked at different levels to RNA isolated from an environmental (bioreactor) sample, and the PNA MB assay enabled effective quantification of the D. suillum RNA in this complex mixture. For another environmental sample, the quantitative results from the PNA MB hybridization assay were compared with those from clone libraries.Accurate quantification of specific populations in complex microbial communities is important in many areas of microbiology. Today, real-time PCR-based techniques are often used for this purpose (45). Other PCR-based techniques, such as denaturing gradient gel electrophoresis (30) and terminal restriction fragment length polymorphism (29), have also been used to study microbial population dynamics, but they are less suitable as quantitative techniques (52). Oligonucleotide probe-based microbial quantification methods that do not rely on PCR usually target the small subunit rRNA and include quantitative membrane hybridization, quantitative fluorescence in situ hybridization (FISH), and phylogenetic microarrays. While membrane hybridization has been used successfully to study microbial population dynamics in a variety of complex microbial systems (39, 58), this technique is time consuming and labor intensive. Quantitative FISH also has been used effectively to quantify microbial populations (34, 59). However, in some applications, traditional FISH methods do not provide satisfactory quantitative results because of poor cell permeability for oligonucleotide probes (10), low accessibility of target sites in rRNA (18), or poor sensitivity when the cellular rRNA content is low (20). Microarray techniques have the potential to quantify rRNA extracted from environmental samples (16, 23), but reproducibility and specificity issues have not been addressed satisfactorily (37,43).The peptide nucleic acid (PNA) oligomer probe was studied for its hybridization properties for the first time about 15 years ago (15). PNA di...

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Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells

Cited by 65 publications

References 35 publications

Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers

Quantitative detection of Aeromonas salmonicida in fish tissue by real-time PCR using self-quenched, fluorogenic primers

In vivo identification of ribonucleoprotein-RNA interactions

Quantitative rRNA-Targeted Solution-Based Hybridization Assay Using Peptide Nucleic Acid Molecular Beacons

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