4,943 results on '"RNA Probes"'
Search Results
2. Cas12a/Guide RNA-Based Platform for Rapid and Accurate Identification of Major Mycobacterium Species.
- Author
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Xiao G, He X, Zhang S, Liu Y, Liang Z, Liu H, Zhang J, Ou M, Cai S, Lai W, Zhang T, Ren L, and Zhang G
- Subjects
- CRISPR-Cas Systems, Humans, Mycobacterium isolation & purification, Mycobacterium Infections, Nontuberculous microbiology, Mycobacterium avium Complex genetics, Mycobacterium avium Complex isolation & purification, Mycobacterium tuberculosis genetics, Mycobacterium tuberculosis isolation & purification, Nontuberculous Mycobacteria genetics, Nontuberculous Mycobacteria isolation & purification, RNA, Ribosomal, 16S genetics, Sensitivity and Specificity, Tuberculosis microbiology, Bacterial Proteins genetics, CRISPR-Associated Proteins genetics, Endodeoxyribonucleases genetics, Mycobacterium classification, RNA Probes, RNA, Guide, CRISPR-Cas Systems genetics, Tuberculosis diagnosis
- Abstract
Mycobacterium tuberculosis infection and nontuberculous mycobacteria (NTM) infections exhibit similar clinical symptoms; however, the therapies for these two types of infections are different. Therefore, the rapid and accurate identification of M. tuberculosis and NTM species is very important for the control of tuberculosis and NTM infections. In the present study, a Cas12a/guide RNA (gRNA)-based platform was developed to identify M. tuberculosis and most NTM species. By designing species-specific gRNA probes targeting the rpoB sequence, a Cas12a/gRNA-based platform successfully identified M. tuberculosis and six major NTM species ( Mycobacterium abscessus , Mycobacterium intracellulare , Mycobacterium avium , Mycobacterium kansasii , Mycobacterium gordonae , and Mycobacterium fortuitum ) without cross-reactivity. In a blind assessment, a total of 72 out of 73 clinical Mycobacterium isolates were correctly identified, which is consistent with previous rpoB sequencing results. These results suggest that the Cas12a/gRNA-based platform is a promising tool for the rapid, accurate, and cost-effective identification of both M. tuberculosis and NTM species., (Copyright © 2020 American Society for Microbiology.)
- Published
- 2020
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3. RNA imaging by chemical probes.
- Author
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Tomoike F and Abe H
- Subjects
- Fluorescent Dyes, Nucleic Acid Hybridization, RNA, RNA Probes
- Abstract
Sequence-specific detection of intracellular RNA is one of the most important approaches to understand life phenomena. However, it is difficult to detect RNA in living cells because of its variety and scarcity. In the last three decades, several chemical probes have been developed for RNA detection in living cells. These probes are composed of DNA or artificial nucleic acid and hybridize with the target RNA in a sequence-specific manner. This hybridization triggers a change of fluorescence or a chemical reaction. In this review, we classify the probes according to the associated fluorogenic mechanism, that is, interaction between fluorophore and quencher, environmental change of fluorophore, and template reaction with/without ligation. In addition, we introduce examples of RNA imaging in living cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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4. Hybridization Histochemistry of Neural Transcripts.
- Author
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Young WS, Song J, and Mezey É
- Subjects
- Animals, Humans, Digoxigenin, Histocytochemistry methods, In Situ Hybridization, RNA Probes, RNA, Messenger
- Abstract
This unit presents protocols to locate RNA transcripts in tissues. Numerous approaches are detailed, including those that use radiolabeled or colorimetric probes. Also, the probes may be modified oligodeoxynucleotides, singly or in pairs, as well as ribonucleic acids. High sensitivity and specificity are obtained, especially with sets of oligodeoxynucleotide pairs. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)
- Published
- 2018
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5. Profiling the transcriptome with RNA SPOTs.
- Author
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Eng CL, Shah S, Thomassie J, and Cai L
- Subjects
- Animals, Embryonic Stem Cells, Fibroblasts, High-Throughput Nucleotide Sequencing, Mice, NIH 3T3 Cells, Reproducibility of Results, Sensitivity and Specificity, Sequence Analysis, RNA, Gene Expression Profiling methods, In Situ Hybridization, Fluorescence methods, RNA Probes, RNA, Messenger genetics
- Abstract
Single-molecule FISH (smFISH) has been the gold standard for quantifying individual transcript abundances. Here, we scale up multiplexed smFISH to the transcriptome level and profile 10,212 different mRNAs from mouse fibroblast and embryonic stem cells. This method, called RNA sequential probing of targets (SPOTs), provides an accurate, flexible, and low-cost alternative to sequencing for profiling transcriptomes.
- Published
- 2017
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6. Characterizing restriction enzyme-associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom-designed RNA probes.
- Author
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Sánchez Barreiro F, Vieira FG, Martin MD, Haile J, Gilbert MT, and Wales N
- Subjects
- Polymorphism, Single Nucleotide, Sequence Analysis, DNA, Ambrosia classification, Ambrosia genetics, DNA, Ancient isolation & purification, DNA, Plant genetics, DNA, Plant isolation & purification, RNA Probes
- Abstract
Population genetic studies of nonmodel organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom-designed RNA probes could enrich for RRL loci (Restriction Enzyme-Associated Loci baits, or REALbaits). Starting with genotyping-by-sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20 000 RNA probes to target well-characterized genomic loci in herbarium voucher specimens dating from 1835 to 1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19- to 151-fold. Using our GBS capture pipeline on a data set of 38 herbarium samples, we discovered 22 813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7m reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL data sets., (© 2016 John Wiley & Sons Ltd.)
- Published
- 2017
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7. Flow-Cytometry-Based Method to Detect Escherichia coli and Shigella Spp. Using 16S rRNA-Based Probe.
- Author
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Xue Y, Wilkes JG, Moskal TJ, Williams AJ, Cooper WM, and Buzatu DA
- Subjects
- Colony Count, Microbial, Culture Media, Escherichia coli genetics, Food Microbiology, Limit of Detection, Shigella genetics, Escherichia coli isolation & purification, Flow Cytometry methods, RNA Probes, RNA, Ribosomal, 16S genetics, Shigella isolation & purification
- Abstract
Detection of microbial contamination in foods before they go on to the market can help prevent the occurrence of foodborne illness outbreaks. Current methods for the detection of Escherichia coli are limited by time-consuming procedures, which include multiple culture incubation steps, and require several days to get results. This unit describes the development of an improved rapid flow-cytometry-based detection method that has greater sensitivity and specificity. This method requires less time-to-results (TTR) and can detect a small number of E. coli in the presence of large numbers of other bacteria. Clear step-by-step protocols for cell concentration determination, sample preparation, and flow cytometric analysis are provided. © 2017 by John Wiley & Sons, Inc., (Copyright © 2017 John Wiley & Sons, Inc.)
- Published
- 2017
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8. Whole-Genome Enrichment Using RNA Probes and Sequencing of Chlamydia trachomatis Directly from Clinical Samples.
- Author
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Brown AC and Christiansen MT
- Subjects
- Chlamydia Infections microbiology, DNA, Bacterial isolation & purification, Female, Genome, Bacterial, High-Throughput Nucleotide Sequencing methods, Humans, Phylogeny, Sensitivity and Specificity, Chlamydia trachomatis genetics, RNA Probes, Whole Genome Sequencing methods
- Abstract
Whole-genome sequencing is a powerful, high-resolution tool that can be used to generate accurate data on bacterial population structure, phylogeography, and mutations associated with antimicrobial resistance. The ability to sequence pathogen genomes directly from clinical specimens, without the requirement for in vitro culturing, is attractive in terms of time- and labor-saving, especially in the case of slow-growing, or obligate intracellular pathogens, such as Chlamydia trachomatis. However clinical samples typically contain too low levels of pathogen nucleic acid, plus relatively high levels of human and natural microbiota DNA/RNA, to make this a viable option. Using a combination of whole-genome enrichment and deep sequencing, which has been proven to be a non-mutagenic approach, we can capture all known variations found within C. trachomatis genomes. The method is a consistent and sensitive tool that enables rapid whole-genome sequencing of C. trachomatis directly from clinical samples and has the potential to be adapted to other pathogens with a similar clonal nature.
- Published
- 2017
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9. High-Throughput Genotyping with TaqMan Allelic Discrimination and Allele-Specific Genotyping Assays.
- Author
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Heissl A, Arbeithuber B, and Tiemann-Boege I
- Subjects
- DNA, Complementary genetics, Humans, Microsatellite Repeats, Polymorphism, Single Nucleotide, Alleles, Genotype, RNA genetics, RNA Probes, Real-Time Polymerase Chain Reaction methods
- Abstract
Real-time PCR-based genotyping methods, such as TaqMan allelic discrimination assays and allele-specific genotyping, are particularly useful when screening a handful of single nucleotide polymorphisms in hundreds of samples; either derived from different individuals, tissues, or pre-amplified DNA. Although real-time PCR-based methods such as TaqMan are well-established, alternative methods, like allele-specific genotyping, are powerful alternatives, especially for genotyping short tandem repeat (STR) length polymorphisms. Here, we describe all relevant aspects when developing an assay for a new SNP or STR using either TaqMan or allele-specific genotyping, respectively, such as primer and probe design, optimization of reaction conditions, the experimental procedure for typing hundreds of samples, and finally the data evaluation. Our goal is to provide a guideline for developing genotyping assays using these two approaches that render reliable and reproducible genotype calls involving minimal optimization.
- Published
- 2017
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10. Spatiotemporal analysis with a genetically encoded fluorescent RNA probe reveals TERRA function around telomeres.
- Author
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Yamada T, Yoshimura H, Shimada R, Hattori M, Eguchi M, Fujiwara TK, Kusumi A, and Ozawa T
- Subjects
- Cell Line, Fluorescent Dyes, Humans, Spatio-Temporal Analysis, Heterogeneous Nuclear Ribonucleoprotein A1 metabolism, Optical Imaging methods, RNA Probes, RNA, Long Noncoding metabolism, Telomere metabolism, Telomere-Binding Proteins metabolism
- Abstract
Telomeric repeat-containing RNA (TERRA) controls the structure and length of telomeres through interactions with numerous telomere-binding proteins. However, little is known about the mechanism by which TERRA regulates the accessibility of the proteins to telomeres, mainly because of the lack of spatiotemporal information of TERRA and its-interacting proteins. We developed a fluorescent probe to visualize endogenous TERRA to investigate its dynamics in living cells. Single-particle fluorescence imaging revealed that TERRA accumulated in a telomere-neighboring region and trapped diffusive heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), thereby inhibiting hnRNPA1 localization to the telomere. These results suggest that TERRA regulates binding of hnRNPA1 to the telomere in a region surrounding the telomere, leading to a deeper understanding of the mechanism of TERRA function.
- Published
- 2016
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11. Hg(2+) detection using a phosphorothioate RNA probe adsorbed on graphene oxide and a comparison with thymine-rich DNA.
- Author
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Huang PJ, van Ballegooie C, and Liu J
- Subjects
- DNA chemistry, Thymine, Biosensing Techniques, Graphite, Mercury analysis, Oxides, RNA Probes
- Abstract
Mercury is a highly toxic heavy metal and many DNA-based biosensors have been recently developed for Hg(2+) detection in water. Among them, thymine-rich DNA is the most commonly used for designing Hg(2+) sensors. However, the thymine-Hg(2+) interaction is strongly affected by the buffer conditions. We recently reported a molecular beacon containing phosphorothioate (PS)-modified RNA linkages that can be cleaved by Hg(2+). In this work, the fluorescence quenching and DNA adsorption properties of nano-sized graphene oxide (NGO) were used to develop a new sensor using the PS-RNA chemistry. Three DNA probes, containing one, three and five PS-RNA linkages, respectively, were tested. Finally, a fluorophore-labeled poly-A DNA with five PS-RNA linkages was selected and adsorbed by NGO. In the presence of Hg(2+), the fluorophore was released from NGO due to the cleavage reaction, resulting in a fluorescence enhancement. This sensor is highly selective for Hg(2+) with a detection limit of 8.5 nM Hg(2+). For comparison, a fluorophore-labeled poly-T DNA was also tested, which responded to Hg(2+) more slowly and was inhibited by high NaCl concentrations, while the PS-RNA probe was more tolerant to different buffer conditions. This work indicates a new method for interfacing DNA with NGO for Hg(2+) detection.
- Published
- 2016
- Full Text
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12. Ultrasensitive genotyping with target-specifically generated circular DNA templates and RNA FRET probes.
- Author
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Zhou H, Wang H, Liu C, Wang H, Duan X, and Li Z
- Subjects
- DNA chemistry, DNA genetics, Fluorescence Resonance Energy Transfer, Genotype, Humans, Nucleic Acid Amplification Techniques, Ribonuclease H chemistry, Tumor Suppressor Protein p53 genetics, RNA Probes
- Abstract
A novel RNA FRET probe that can produce target-dependent signal amplification with the catalysis of RNase H has been developed for detection of rolling circle amplification (RCA) products with greatly improved sensitivity.
- Published
- 2015
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13. Combining single RNA sensitive probes with subdiffraction-limited and live-cell imaging enables the characterization of virus dynamics in cells.
- Author
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Alonas E, Lifland AW, Gudheti M, Vanover D, Jung J, Zurla C, Kirschman J, Fiore VF, Douglas A, Barker TH, Yi H, Wright ER, Crowe JE Jr, and Santangelo PJ
- Subjects
- Cell Line, Green Fluorescent Proteins genetics, Humans, Real-Time Polymerase Chain Reaction, Respiratory Syncytial Viruses genetics, Virus Assembly, RNA Probes, Respiratory Syncytial Viruses physiology
- Abstract
The creation of fluorescently labeled viruses is currently limited by the length of imaging observation time (e.g., labeling an envelope protein) and the rescue of viral infectivity (e.g., encoding a GFP protein). Using single molecule sensitive RNA hybridization probes delivered to the cytoplasm of infected cells, we were able to isolate individual, infectious, fluorescently labeled human respiratory syncytial virus virions. This was achieved without affecting viral mRNA expression, viral protein expression, or infectivity. Measurements included the characterization of viral proteins and genomic RNA in a single virion using dSTORM, the development of a GFP fusion assay, and the development of a pulse-chase assay for viral RNA production that allowed for the detection of both initial viral RNA and nascent RNA production at designated times postinfection. Live-cell measurements included imaging and characterization of filamentous virion fusion and the quantification of virus replication within the same cell over an eight-hour period. Using probe-labeled viruses, individual viral particles can be characterized at subdiffraction-limited resolution, and viral infections can be quantified in single cells over an entire cycle of replication. The implication of this development is that MTRIP labeling of viral RNA during virus assembly has the potential to become a general methodology for the labeling and study of many important RNA viruses.
- Published
- 2014
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14. The author file: Samie Jaffrey.
- Author
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Marx V
- Subjects
- Green Fluorescent Proteins, History, 21st Century, United States, Fluorescent Dyes, RNA Probes
- Published
- 2013
- Full Text
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15. Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.
- Author
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Kukutla P, Steritz M, and Xu J
- Subjects
- Animals, Digestive System chemistry, Digestive System microbiology, Metagenome, Nucleic Acid Hybridization methods, RNA, Messenger isolation & purification, Sequence Analysis, RNA, Anopheles genetics, Anopheles microbiology, RNA Probes, RNA, Messenger analysis, RNA, Messenger genetics, RNA, Ribosomal genetics
- Abstract
The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.
- Published
- 2013
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16. [Preparation of RNA probe for cd99l2 gene of zebrafish labeled with digoxingenin-UTP].
- Author
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Wen ZH, Zhang Y, Wu ZQ, Zhou XH, Han XQ, Zhang WQ, and Zhao T
- Subjects
- Animals, Central Nervous System embryology, Cloning, Molecular, Digoxigenin chemistry, Gene Expression Regulation, Developmental, Oligonucleotide Probes, Uridine Triphosphate chemistry, RNA Probes, Zebrafish embryology, Zebrafish genetics, Zebrafish Proteins genetics
- Abstract
Objective: To study the expression pattern of cd99l2 gene during zebrafish development, the RNA probes for whole-mount in situ hybridization were prepared in this study., Methods: The cd99l2 fragment obtained by RT-PCR was cloned into pGM-T Easy, then the plasmids were linearized with the restriction enzymes SacII or SalI. Using Sp6 or T(7) RNA polymerase, the digoxingenin-labeled antisense and sense probes were synthesized and confirmed by whole-mount in situ hybridization., Results: The plasmid cd99l2/pGM-T was constructed. cd99l2 gene expression pattern during embryogenesis of zebrafish was examined using the antisense probe, and intense expression was detected in the central nervous system during zebrafish development., Conclusion: The antisense probe can be used for study of the spatial and temporal distribution of cd99l2 during zebrafish development using the sense probe as control.
- Published
- 2010
17. Pyrrolo-C as a molecular probe for monitoring conformations of the tRNA 3' end.
- Author
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Zhang CM, Liu C, Christian T, Gamper H, Rozenski J, Pan D, Randolph JB, Wickstrom E, Cooperman BS, and Hou YM
- Subjects
- Aminoacylation, Cytidine analogs & derivatives, Cytosine chemistry, Escherichia coli genetics, RNA Nucleotidyltransferases chemistry, RNA, Bacterial chemistry, RNA, Transfer, Cys chemistry, Ribosomes metabolism, Cytosine analogs & derivatives, Fluorescent Dyes chemistry, Nucleic Acid Conformation, Pyrroles chemistry, RNA Probes, RNA, Transfer chemistry
- Abstract
All mature tRNA molecules have the conserved CCA sequence at the 3' end with a range of dynamic conformations that are important for tRNA functions. We present here the details of a general approach to fluorescent labeling of the CCA sequence with the fluorescent base analog pyrrolo-C (PyC) at position 75 as a molecular probe for monitoring the dynamics of the tRNA 3' end. Using Escherichia coli tRNA(Cys) as an example, we achieve such labeling by first synthesizing the tRNA as a transcript up to C74 and then employing the tRNA CCA-adding enzyme to incorporate PyC75 and A76, using pyrrolo-CTP (PyCTP) and ATP as the respective substrates. PyC-labeled full-length tRNA(Cys), separated from the unlabeled precursor tRNA by reverse phase high-pressure liquid chromatography, is an efficient substrate for aminoacylation by E. coli cysteinyl-tRNA synthetase (CysRS). Fluorescence binding measurement of the PyC-labeled tRNA(Cys) with E. coli CysRS reveals an equilibrium K(d) closely similar to the value determined from the fluorescence of intrinsic enzyme tryptophans. Kinetic measurements of translocation of the PyC-labeled tRNA from the ribosomal A to P sites identify a kinetic intermediate with a rate of formation and decay similar to the values reported for tRNAs labeled with the fluorescent proflavin at the tertiary core. These results highlight the potential of PyC to probe the dynamics of the tRNA CCA end in reactions ranging from aminoacylation to those on the ribosome.
- Published
- 2008
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18. SNP analysis using CataCleave probes.
- Author
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Harvey JJ, Brant SR, Knutson JR, and Han MK
- Subjects
- Humans, Insulin-Like Growth Factor II genetics, Nod2 Signaling Adaptor Protein genetics, Oligonucleotides chemistry, Oligonucleotides genetics, Ribonuclease H metabolism, Sequence Analysis, DNA, DNA Probes, Genotype, Polymorphism, Single Nucleotide genetics, RNA Probes
- Abstract
CataCleave probes are catalytically cleavable fluorescence probes having a chimeric deoxyribonucleic acid (DNA)-ribonucleic acid (RNA)-DNA structure that can be used for real-time detection of single nucleotide polymorphisms (SNPs), insertions, and deletions. Fluorescent donor emission is normally quenched by Förster resonance energy transfer (FRET). Upon binding to the target, if the RNA/DNA hybrid is correctly base-paired, ribonuclease (RNase) H will cleave the RNA moiety and the probe fragments will dissociate. FRET is lost and the donor fluorescence signal is recovered. A single-base mismatch within the hybrid region causes probe cleavage to be significantly reduced. We designed CataCleave probes to detect SNPs located in the insulin-like growth factor 2 (IGF-2) gene and at position 702 within the NOD2/CARD15 gene. Probes were also designed to detect a six-basepair deletion in the amelogenin gene and a partially methylated target DNA. Discrimination between wild-type and SNP is demonstrated for both genes in homogeneous reactions under isothermal and temperature cycling conditions. These probes were also able to identify a multibase deletion and methylated DNA. Cleavage rates were proportional to target concentration. Probe length and position of fluorescent labels may also be modified for use in multiplexing high-throughput SNP assays. This represents a novel method for the detection of SNPs., ((c) 2008 Wiley-Liss, Inc.)
- Published
- 2008
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19. Dinuclear ruthenium(II) complexes as potential probes for RNA bulge sites.
- Author
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Spillane CB, Smith JA, Buck DP, Collins JG, and Keene FR
- Subjects
- Magnetic Resonance Spectroscopy, Models, Molecular, Molecular Structure, RNA chemistry, RNA Probes, Ruthenium Compounds chemistry
- Abstract
1H NMR spectroscopy and molecular modelling have been used to investigate the binding of the DeltaDelta-and LambdaLambda-enantiomers of the dinuclear ruthenium(II) complex [[Ru(Me2bpy)2]2(mu-bpm)]4+ [Me2bpy = 4,4'-dimethyl-2,2'-bipyridine; bpm = 2,2'-bipyrimidine] to an RNA tridecanucleotide duplex containing a single-base bulge [r(CCGAGAAUUCCGG)2]], and the corresponding control dodecanucleotide [r(CCGGAAUUCCGG)2]. Both enantiomers bound the control RNA sequence weakly. From upfield shifts of the metal complex H3 and H3' protons throughout the titration of the control dodecanucleotide with DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+, a binding constant of 1 x 10(3) M(-1) was determined. In NOESY spectra of the control sequence with added DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+, NOEs were only observed to protons from the terminal base-pair residues. No significant changes in chemical shift were observed for either the metal complex or RNA protons upon addition of the LambdaLambda-enantiomer to the control dodecanucleotide. The DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ complex bound the bulge-containing RNA with a significantly greater affinity (6 x 10(4) M(-1)) than the non-bulge control RNA duplex. Competition binding experiments indicated that the LambdaLambda-isomer bound the tridecanucleotide with similar affinity to the DeltaDelta-enantiomer. Addition of DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ to the bulge-containing tridecanucleotide induced selective changes in chemical shift for the base H8 and sugar H1' resonances from the adenine bulge residue, and resonances from nucleotide residues adjacent to the bulge site. Intermolecular NOEs observed in NOESY spectra of the tridecanucleotide with added DeltaDelta-[[Ru(Me2bpy)2]2(mu-bpm)]4+ confirmed the selective binding of the ruthenium complex at the bulge site. Preliminary binding models, consistent with the NMR data, showed that the ruthenium complex could effectively associate in the RNA minor groove at the bulge site.
- Published
- 2007
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20. Human papillomavirus assay in genital warts--correlation with symptoms.
- Author
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Hadjivassiliou M, Stefanaki C, Nicolaidou E, Bethimoutis G, Anyfantakis V, Caroni C, and Katsambas A
- Subjects
- Adolescent, Adult, Aged, Alphapapillomavirus classification, Alphapapillomavirus pathogenicity, Anal Canal virology, Condylomata Acuminata complications, Humans, Male, Middle Aged, Perineum virology, Polymerase Chain Reaction, Risk Factors, Sensitivity and Specificity, Sexual Partners, Urethra virology, Alphapapillomavirus isolation & purification, Condylomata Acuminata virology, RNA Probes
- Abstract
Our purpose was to investigate the human papillomavirus (HPV) type distribution using the Hybrid Capture 2 (HC2) Microplate assay in males. We tested a urethral swab from 550 HIV-negative males with genital warts and 64 HIV-negative males clinically free of genital warts, partners of HPV-infected females, using the HC2 Microplate assay. A perianal swab was also obtained from patients with perianal warts. In the first group, HPV DNA of any type was detected in 280 (50.9%) patients. Relatively few patients with urethral or perianal warts demonstrated a negative test (both P < 0.0001). Low-risk types were commoner, accounting for 60.0% of the HPV cases, high/intermediate-risk types accounted for 23.6%, while 46 men (16.4%) were infected with both types. Of 13 subjects (20.3%) of the second group who tested positive for HPV DNA, 61.5% were infected by low-risk types, 23.1% by high/intermediate-risk types and 15.4% had a dual infection. In conclusion, male partners of infected females and males with genital warts are predominantly infected by low-risk HPV types, but a substantial proportion is also or only affected by high-risk types.
- Published
- 2007
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21. In situ protocol for butterfly pupal wings using riboprobes.
- Author
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Ramos D and Monteiro A
- Subjects
- Animals, Butterflies genetics, Gene Expression, Pupa metabolism, Butterflies growth & development, Butterflies metabolism, In Situ Hybridization methods, RNA Probes, Wings, Animal growth & development, Wings, Animal metabolism
- Abstract
Here we present, in video format, a protocol for in situ hybridizations in pupal wings of the butterfly Bicyclus anynana using riboprobes. In situ hybridizations, a mainstay of developmental biology, are useful to study the spatial and temporal patterns of gene expression in developing tissues at the level of transcription. If antibodies that target the protein products of gene transcription have not yet been developed, and/or there are multiple gene copies of a particular protein in the genome that cannot be differentiated using available antibodies, in situs can be used instead. While an in situ technique for larval wing discs has been available to the butterfly community for several years, the current protocol has been optimized for the larger and more fragile pupal wings.
- Published
- 2007
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22. Construction of a multiprobe for the simultaneous detection of viroids infecting citrus trees.
- Author
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Cohen O, Batuman O, Stanbekova G, Sano T, Mawassi M, and Bar-Joseph M
- Subjects
- Blotting, Northern, Nucleic Acid Hybridization, Viroids genetics, Viroids isolation & purification, Citrus virology, Plant Viruses isolation & purification, RNA Probes, Viroids classification
- Abstract
Infections with different viroid species are common among cultivated fruit trees and grapevines, and many old-clone citrus varieties contain up to five citrus viroids (CVds) within a single tree. This paper describes the construction of a CVd-Multiprobe consisting of full-length clones of Hop stunt viroid, Citrus exocortis viroid, Citrus bent leaf viroid and CVd-III. The CVd-Multiprobe was tested against RNA transcripts of the four viroids and RNA extracts from plants singly infected with CEVd or HSVd or multiply infected with different CVds. The viroids were effectively diagnosed with the DIG labeled CVd-Multiprobe when tested by Northern hybridization or dot blot analyses. The CVd-Multiprobe does not provide information on the specific viroid resulting in a positive signal. However, this should not be considered as a problem, since most citrus certification programs will discard budwood source trees infected with any of the known CVds.
- Published
- 2006
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23. Combinatorial fluorescence energy transfer molecular beacons for probing nucleic acid sequences.
- Author
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Li X, Li Z, Martí AA, Jockusch S, Stevens N, Akins DL, Turro NJ, and Ju J
- Subjects
- Fluorescence Polarization, Nucleic Acid Hybridization, Spectrophotometry, Ultraviolet, Base Sequence, DNA Probes, Fluorescence Resonance Energy Transfer instrumentation, RNA Probes
- Abstract
We report the design, synthesis, and characterization of molecular beacons (MB) consisting of three distinct fluorophores, 6-carboxyfluorescein (Fam), N,N,N',N'-tetramethyl-6-carboxyrhodamine (Tam), and Cyanine-5 (Cy5). The primary light absorber/energy donor (Fam) is located on one terminus of the MB, whereas the primary energy acceptor/secondary donor (Tam) and secondary acceptor (Cy5) are located at the other terminus of the MB. In the absence of target DNA or RNA, the MB exists in the stem-closed form. Excitation of Fam initiates an energy transfer cascade from Fam to Tam and further to Cy5 generating unique fluorescence signatures defined as the ratio of the emission from each of the three fluorophores. This energy transfer cascade was investigated in detail by steady-state and time-resolved fluorescence spectroscopy, as well as fluorescence depolarization studies. In the presence of the complementary target DNA, the MB opened efficiently and hybridized with the target separating Fam and Tam by a large distance, so that energy transfer from Fam to Tam was blocked in the stem-open form. This opening of the MB generates a "bar code" fluorescence signature, which is different from the signature of the stem-closed MB. The fluorescence signature of this combinatorial fluorescence energy transfer MB can be tuned by variation of the spacer length between the individual fluorophores.
- Published
- 2006
- Full Text
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24. Discrimination of four soybean dwarf virus strains by dot-blot hybridization with specific probes.
- Author
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Yamagishi N, Terauchi H, Honda K, Kanematsu S, and Hidaka S
- Subjects
- Luteovirus classification, Luteovirus genetics, Nucleic Acid Hybridization methods, RNA Probes, RNA, Viral genetics, Glycine max virology
- Abstract
Soybean dwarf virus (SbDV) is divided into four strains (YS, YP, DS, and DP) on the basis of host symptoms in infected soybean plants and on aphid vector specificity. To detect and discriminate each strain of SbDV by dot-blot hybridization, probes Y, D, S, and P were prepared. Probes Y and D, covering most of the 3'-noncoding region of the viral genome containing the sequence of small subgenomic RNA, could discriminate strains in accord with the host symptoms. Probes S and P were derived from the 5'-half of open reading frame 5 encoding the N-terminal half of the readthrough domain which is closely related to the aphid vector specificity of each strain. Thus, the four SbDV strains could be discriminated by the combination of these probes. This method, based on a procedure specific to the SbDV sequence, is a good alternative for routine examination of infected plants in soybean breeding programs for evaluation of resistance to SbDV and for assessment of the distribution of each strain in epidemiological studies.
- Published
- 2006
- Full Text
- View/download PDF
25. In situ hybridization using riboprobes on free-floating brain sections.
- Author
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Owens NC, Hess FM, and Badoer E
- Subjects
- Animals, Digoxigenin metabolism, Histological Techniques, Microtomy, Neurons cytology, Neurons metabolism, RNA, Messenger analysis, Rats, Tissue Fixation, Brain cytology, Brain metabolism, In Situ Hybridization methods, RNA Probes
- Abstract
A method is described for in situ hybridization of riboprobes to free-floating brain sections. Brain sections are hybridized and processed free-floating in buffer, i.e., without attachment to a support such as a slide. To withstand the extra wear compared with sections processed on-slide, the brain tissue must be well fixed (4% paraformaldehyde) and sections cut at thickness of typically 40 microm. Sections were exposed to a prehybridization treatment before a riboprobe is added to form the hybridization solution. Riboprobes were prepared from cDNA via an in vitro transcription reaction and are labeled with digoxigenin. The sections are subsequently processed to remove nonspecific binding and the digoxigenin label detected via an antibody conjugated to alkaline phosphatase. This method may be readily combined with neuronal tracing and is ideal for further processing by immunohistochemistry to detect specific proteins.
- Published
- 2006
- Full Text
- View/download PDF
26. Localization of cannabinoid CB1 receptor mRNA using ribonucleotide probes: methods for double- and single-label in situ hybridization.
- Author
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Hohmann AG
- Subjects
- Animals, Autoradiography, Digoxigenin chemistry, RNA, Messenger genetics, Rats, In Situ Hybridization methods, RNA Probes, RNA, Messenger analysis, Receptor, Cannabinoid, CB1 genetics
- Abstract
This chapter presents a reliable, detailed method for performing double-label in situ hybridization (ISH) that has been validated for use in studies identifying the co-localization of cannabinoid CB1 receptor mRNA with other distinct species of mRNAs. This method permits simultaneous detection of two different species of mRNA within the same tissue section. Double-label ISH may be accomplished by hybridizing tissue sections with a combination of radiolabeled and digoxigenin-labeled RNA probes that are complementary to their target mRNAs. Single-label ISH may be accomplished by following the procedures described for use with radioisotopic probes (here [35S]-labeled) only. Silver grains derived from conventional emulsion autoradiography are used to detect the radiolabeled cRNA probe. An alkaline phosphatase-dependent chromogen reaction product is used to detect the nonisotopic (here, digoxigenin-labeled) cRNA probe. Necessary controls that are required to document the specificity of the labeling of the digoxigenin and radiolabeled probes are described. The methods detailed herein may be employed to detect even low levels of a target mRNA. These methods may be utilized to study co-localization and coregulation of expression of a particular gene within identified neurons in multiple systems.
- Published
- 2006
27. Antisense and sense RNA probe hybridization to immobilized crude cellular lysates: a tool to screen growth hormone antagonists.
- Author
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Rosengren L, Simko H, Aryan L, Axelsson-Lendin P, Chmielewska J, Mode A, and Parrow V
- Subjects
- Animals, Aryl Hydrocarbon Hydroxylases genetics, Down-Regulation, Gene Expression drug effects, Growth Hormone pharmacology, Hepatocytes drug effects, Hepatocytes metabolism, Insulin-Like Growth Factor I biosynthesis, Mice, Nucleic Acid Hybridization methods, RNA, Messenger metabolism, Steroid Hydroxylases genetics, Drug Evaluation, Preclinical methods, Growth Hormone antagonists & inhibitors, Insulin-Like Growth Factor I genetics, Liver Extracts chemistry, RNA Probes, RNA, Antisense, RNA, Messenger analysis
- Abstract
The growth-promoting effect of growth hormone (GH) is primarily mediated by insulin-like growth factor-1 (IGF-1). The liver is the main source of circulating IGF-I. The authors have used rodent primary hepatocytes for studies on pharmacological intervention of IGF-I mRNA expression. A 96-well nonradioactive IGF-1 mRNA quantification assay was developed, based on the hybridization of sense and antisense RNA probes, to replicate membranes with crude hepatocyte lysates. The sense hybridization was used as an internal standard. The antagonistic properties of a set of GH-receptor binding compounds were evaluated. Two compounds were found to down-regulate IGF-I mRNA. Effects due to metabolic inhibition or toxicity were excluded using a cell proliferation assay. To investigate potential unspecific transcriptional effects, the mRNA levels of the housekeeping genes, beta-actin and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were determined. Two other GH-regulated genes, cytochrome P450 2C12 (CYP2C12) and a rat homologue to the human alpha1B-glycoprotein (A1BG), were quantified by RNase protection assays and found to be down-regulated, confirming the antagonistic property of 1 compound. In conclusion, a direct filter hybridization assay of hepatocyte lysates using nonradioactive sense and antisense probes can be used for quantitative mRNA measurements and could constitute a valuable tool in screening for pharmacologically active compounds.
- Published
- 2005
- Full Text
- View/download PDF
28. Ribosomal RNA probes and microarrays: their potential use in assessing microbial biodiversity.
- Author
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Metfies K and Medlin L
- Subjects
- Base Sequence, Gene Expression Profiling methods, Genetic Variation, Genotype, Molecular Probe Techniques, Nucleic Acid Hybridization, Polymerase Chain Reaction, RNA, Ribosomal isolation & purification, Microbiological Techniques, Oligonucleotide Array Sequence Analysis methods, RNA Probes, RNA, Ribosomal genetics
- Abstract
The awareness that global biological diversity is affected by numerous, mostly human-made threats has made biodiversity assessment an important scientific issue for decades. Biodiversity includes different levels of complexity, such as community diversity, habitat diversity, genetic diversity, and species diversity. The application of molecular methods to answer ecological questions permits issues of biodiversity to be addressed at all levels. Microorganisms dominate global biological diversity in terms of their species numbers. However, their small size and limited morphological features make it challenging to obtain a comprehensive view of their biodiversity. The application of ribosomal RNA (rRNA) probes contributes significantly to the assessment of biodiversity at the molecular level. DNA microarrays offer a great potential to facilitate the application of molecular probes and other DNA analytical methods to answer ecological and biodiversity questions. We provide an introduction into the application of rRNA probes and DNA microarrays for the assessment of microbial biodiversity, as well as protocols for the implementation of DNA microarrays.
- Published
- 2005
- Full Text
- View/download PDF
29. Detection of hepatitis C virus RNA in formalin-fixed paraffin-embedded sections with digoxigenin-labeled cRNA probes.
- Author
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Qian X, Guerrero RB, Plummer TB, Alves VF, and Lloyd RV
- Subjects
- Biopsy, Digoxigenin chemistry, Fixatives, Formaldehyde chemistry, Hepacivirus chemistry, Hepacivirus genetics, Hepatitis Antibodies chemistry, Humans, Immunochemistry, Liver pathology, Paraffin Embedding, Prolactin chemistry, RNA, Viral genetics, Retrospective Studies, Reverse Transcriptase Polymerase Chain Reaction, Viral Proteins analysis, Viral Proteins genetics, Hepacivirus isolation & purification, Hepatitis C diagnosis, In Situ Hybridization methods, Liver virology, RNA Probes, RNA, Viral analysis
- Abstract
Although recent studies have analyzed Hepatitis C (HCV) infections in liver tissue by in situ hybridization (ISH), many of these studies have been of limited diagnostic utility because of the low copy numbers of HCV in formalin-fixed paraffin-embedded (FFPE) tissue and failure to correlate the ISH analysis with other methods of detecting HCV. Thirty six cases of liver biopsies from patients with known HCV antibody status including 20 cases of serum HCV positive and 16 cases of serum HCV negative were analyzed. All cases showed histologic features suggestion of HCV infection. Analyses of all 36 cases were done by RT-PCR combined with Southern hybridization (RT-PCR-SH) and in situ hybridization (ISH). A prolactin riboprobe was used as a negative control. Immunohistochemistry (IHC) with an antibody against HCV (Rb 246) was also used to analyze HCV viral protein in the tissues. Of the 20 serum antibody-positive cases, RT-PCR-SH detected 18 positive cases, while ISH and IHC detected 19 and 16 positive cases, respectively. Of the 16 serum antibody-negative cases, RT-PCR-SH detected 8 positive cases while ISH and IHC detected 8 and 11 positive cases, respectively. A positive ISH signal for HCV was also detected in some lymphocytes and bile ducts in the liver. These results show that ISH with a highly specific riboprobe is comparable to RT-PCR-SH for detection of HCV infection in liver tissue.
- Published
- 2004
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- View/download PDF
30. Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe.
- Author
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Smith JJ, Gunasekera TS, Barardi CR, Veal D, and Vesey G
- Subjects
- Animals, Fluorescent Antibody Technique methods, Half-Life, Microscopy, Fluorescence methods, Permeability, RNA, Protozoan analysis, RNA, Ribosomal analysis, Ribonucleases antagonists & inhibitors, Ribonucleases genetics, Ribonucleosides metabolism, Species Specificity, Vanadates metabolism, Cryptosporidium parvum physiology, In Situ Hybridization, Fluorescence methods, Oocysts physiology, RNA Probes
- Abstract
Aims: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum., Methods and Results: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h., Conclusions: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity., Significance and Impact of the Study: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.
- Published
- 2004
- Full Text
- View/download PDF
31. Probe selection for high-density oligonucleotide arrays.
- Author
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Mei R, Hubbell E, Bekiranov S, Mittmann M, Christians FC, Shen MM, Lu G, Fang J, Liu WM, Ryder T, Kaplan P, Kulp D, and Webster TA
- Subjects
- Cell Line, Humans, Models, Molecular, Open Reading Frames, Oligonucleotide Array Sequence Analysis, RNA Probes
- Abstract
High-density oligonucleotide microarrays enable simultaneous monitoring of expression levels of tens of thousands of transcripts. For accurate detection and quantitation of transcripts in the presence of cellular mRNA, it is essential to design microarrays whose oligonucleotide probes produce hybridization intensities that accurately reflect the concentration of original mRNA. We present a model-based approach that predicts optimal probes by using sequence and empirical information. We constructed a thermodynamic model for hybridization behavior and determined the influence of empirical factors on the effective fitting parameters. We designed Affymetrix GeneChip probe arrays that contained all 25-mer probes for hundreds of human and yeast transcripts and collected data over a 4,000-fold concentration range. Multiple linear regression models were built to predict hybridization intensities of each probe at given target concentrations, and each intensity profile is summarized by a probe response metric. We selected probe sets to represent each transcript that were optimized with respect to responsiveness, independence (degree to which probe sequences are nonoverlapping), and uniqueness (lack of similarity to sequences in the expressed genomic background). We show that this approach is capable of selecting probes with high sensitivity and specificity for high-density oligonucleotide arrays.
- Published
- 2003
- Full Text
- View/download PDF
32. Probing the importance of tRNA anticodon: human immunodeficiency virus type 1 (HIV-1) RNA genome complementarity with an HIV-1 that selects tRNA(Glu) for replication.
- Author
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Dupuy LC, Kelly NJ, Elgavish TE, Harvey SC, and Morrow CD
- Subjects
- Base Sequence, Molecular Sequence Data, Nucleic Acid Conformation, RNA, Viral chemistry, Sequence Homology, Nucleic Acid, Anticodon, Genome, Viral, HIV-1 genetics, RNA Probes, RNA, Transfer genetics, RNA, Viral genetics
- Abstract
The initiation of human immunodeficiency virus type 1 (HIV-1) reverse transcription occurs at the primer binding site (PBS) that is complementary to the 3'-terminal nucleotides of tRNA(3)(Lys). Why all known strains of HIV-1 select tRNA(3)(Lys) for replication is unknown. Previous studies on the effect of altering the PBS of HIV-1 on replication identified an HIV-1 with a PBS complementary to tRNA(Glu). Since the virus was not initially designed to use tRNA(Glu), the virus had selected tRNA(Glu) from the intracellular pool of tRNA for use in replication. Further characterization of HIV-1 that uses tRNA(Glu) may provide new insights into the preference for tRNA(3)(Lys). HIV-1 constructed with the PBS complementary to tRNA(Glu) was more stable than HIV-1 with the PBS complementary to tRNA(Met) or tRNA(His); however, all of these viruses eventually reverted back to using tRNA(3)(Lys) following growth in SupT1 cells or peripheral blood mononuclear cells (PBMCs). New HIV-1 mutants with nucleotides in U5 complementary to the anticodon of tRNA(Glu) remained stable when grown in SupT1 cells or PBMCs, although the mutants grew more slowly than the wild-type virus. Sequence analysis of the U5 region and the PBS revealed additional mutations predicted to further promote tRNA-viral genome interaction. The results support the importance of the tRNA anticodon-genome interaction in the selection of the tRNA primer and highlight the fact that unique features of tRNA(3)(Lys) are exploited by HIV-1 for selection as the reverse transcription primer.
- Published
- 2003
- Full Text
- View/download PDF
33. Double-color fluorescence in situ hybridization with RNA probes.
- Author
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Xi X, Roane DS, Zhou J, Ryan DH, and Martin RJ
- Subjects
- ATP-Binding Cassette Transporters, Animals, Dopamine Plasma Membrane Transport Proteins, In Situ Hybridization, Fluorescence instrumentation, In Vitro Techniques, Potassium Channels, Inwardly Rectifying, RNA, Messenger metabolism, Rats, Rats, Sprague-Dawley, Receptors, Drug, Sulfonylurea Receptors, Tissue Distribution, DNA-Binding Proteins metabolism, Gene Expression Profiling methods, In Situ Hybridization, Fluorescence methods, Membrane Glycoproteins, Membrane Transport Proteins, Nerve Tissue Proteins, Potassium Channels metabolism, RNA Probes, Saccharomyces cerevisiae Proteins, Substantia Nigra cytology, Substantia Nigra metabolism
- Published
- 2003
- Full Text
- View/download PDF
34. Fluorescence based rRNA sensor systems for detection of whole cells of Saccharomonospora spp. and Thermoactinomyces spp.
- Author
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Neef A, Schäfer R, Beimfohr C, and Kämpfer P
- Subjects
- Air Microbiology, Air Pollutants analysis, Air Pollutants classification, Colony Count, Microbial instrumentation, Colony Count, Microbial methods, In Situ Hybridization, Fluorescence instrumentation, Micromonosporaceae classification, Micromonosporaceae isolation & purification, Reproducibility of Results, Sensitivity and Specificity, Species Specificity, Actinomycetales classification, Actinomycetales isolation & purification, Biosensing Techniques, In Situ Hybridization, Fluorescence methods, RNA Probes, RNA, Ribosomal, 16S
- Abstract
Airborne thermophilic actinomycetes (TPAs) are a growing hygienic challenge in different occupational situations e.g. large scale composting. This study describes first results of a new approach for highly specific and rapid detection of organisms of this group using fluorescently labelled oligonucleotide probes as sensors for whole cells. Three genus-specific 16S rRNA-targeted probes, two for Saccharomonospora spp. and one for Thermoactinomyces spp. were developed and evaluated in a fluorescence in situ hybridisation (FISH) format with agar-grown whole cells. For optimal sensitivity and specificity of FISH, conditions for cell wall permeabilisation and hybridisation stringency were evaluated independently for both genera. Performing specified pretreatment protocols, all three probes yielded strong fluorescence signals. However, the relative fraction of detectable cells or spores clearly depended on the single bacterial species. The probes can serve as cell sensors for direct detection of TPAs in natural samples.
- Published
- 2003
- Full Text
- View/download PDF
35. Enumeration of Bacteroides species in human faeces by fluorescent in situ hybridisation combined with flow cytometry using 16S rRNA probes.
- Author
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Rigottier-Gois L, Rochet V, Garrec N, Suau A, and Doré J
- Subjects
- Adult, Bacteroides classification, Bacteroides genetics, Humans, RNA, Ribosomal, 16S genetics, Reference Standards, Species Specificity, Staining and Labeling, Bacteroides isolation & purification, Feces microbiology, Flow Cytometry, In Situ Hybridization, Fluorescence methods, RNA Probes, RNA, Ribosomal, 16S analysis
- Abstract
Bacteroides is a predominant group of the faecal microbiota in healthy adults. To investigate the species composition of Bacteroides by fluorescent in situ hybridisation (FISH) combined with flow cytometry, we developed five species-specific probes targeting the 16S rRNA. Probes were designed to identify cells belonging to Bacteroides distasonis, B. fragilis, B. ovatus, B. vulgatus and B. putredinis. The species-specificity of the probes was assessed against a collection of reference strains from the Cytophaga-Flavobacterium-Bacteroides group. The results of the FISH experiments showed that the probes were specific as they only detected strains of the target species. Determining the fluorescence intensity of each probe relative to that of the EUB 338 probe (domain bacteria) further showed that each species probe easily accessed the targeted site. The probes were applied to quantify the Bacteroides species in faeces collected from 20 healthy adults. All five species were detected in the faecal samples. Cells hybridised with Bfra 998 were the most frequent as they were observed in 90% of individuals (18/20 samples, mean proportion of 3.9 +/- 2.2%). The cells hybridised with Bvulg 1017 were observed in 85% of individuals (17/20 samples) and represented with a mean proportion of 4.2 +/- 6.1%, the most abundant Bacteroides species in human faeces. Cells hybridising with probes for B. ovatus, B. distasonis and B. putredinis were less frequently detected. The large distribution of B. vulgatus and B. fragilis in human faeces is in accordance with previous reports based on culture or molecular studies. This work showed that fluorescent in situ hybridisation is a tool appropriate for a high-resolution analysis of the species composition of complex ecosystems and especially of the Bacteroides group within the faecal microbiota.
- Published
- 2003
- Full Text
- View/download PDF
36. Development of a fluorescence in situ hybridization method for cheese using a 16S rRNA probe.
- Author
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Ercolini D, Hill PJ, and Dodd CE
- Subjects
- Dairy Products, RNA, Ribosomal, 16S genetics, Cheese microbiology, In Situ Hybridization, Fluorescence methods, RNA Probes, RNA, Bacterial analysis, RNA, Ribosomal, 16S analysis
- Abstract
A 16S rRNA fluorescence in situ hybridization (FISH) method for cheese was developed to allow detection in situ of microorganisms within the dairy matrix. An embedding procedure using a plastic resin was applied to Stilton cheese, providing intact embedded cheese sections withstanding the hybridization reaction. The use of a fluorescein-labelled 16S rRNA Domain Bacteria probe allowed observation of large colonies of microbial cells homogeneously distributed in the cheese matrix. FISH experiments performed on cheese suspensions provided images of the different microbial morphotypes occurring. The technique has great potential to study the spatial distribution of microbial populations in situ in foods, especially where the matrix is too fragile to allow manipulation of cryosections., (Copyright 2003 Elsevier Science B.V.)
- Published
- 2003
- Full Text
- View/download PDF
37. Chicken alpha-globin gene cluster is preceded by the nonerythroid-specific gene, the beginning of which is colocated with the replication origin.
- Author
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Sjakste N, Iarovaia OV, Sjakste T, Razin SV, and Ioudinkova ES
- Subjects
- Animals, Base Sequence, Cells, Cultured, Chickens, Erythroblasts metabolism, Gene Expression Profiling methods, Humans, Molecular Sequence Data, Replication Origin genetics, Sequence Homology, Species Specificity, Gene Expression Regulation genetics, Globins genetics, RNA Probes, Sequence Alignment methods, Sequence Analysis, DNA methods
- Published
- 2003
- Full Text
- View/download PDF
38. Increased concentrations of radioisotopically-labeled complementary ribonucleic acid probe, dextran sulfate, and dithiothreitol in the hybridization buffer can improve results of in situ hybridization histochemistry.
- Author
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Hrabovszky E and Petersen SL
- Subjects
- Animals, Autoradiography, Brain metabolism, Buffers, Female, Galanin genetics, Galanin metabolism, Image Processing, Computer-Assisted, Indicators and Reagents, RNA, Messenger analysis, Rats, Rats, Sprague-Dawley, Receptors, Androgen genetics, Receptors, Androgen metabolism, Receptors, GABA-A genetics, Receptors, GABA-A metabolism, Receptors, Progesterone genetics, Receptors, Progesterone metabolism, Sulfur Radioisotopes, Dextran Sulfate, Dithiothreitol, In Situ Hybridization methods, RNA Probes, RNA, Complementary
- Abstract
The goal of the present studies was to optimize mRNA detection with radioisotopic in situ hybridization histochemistry (ISHH). Test experiments performed on sections of rat brain tissue used computer-assisted image analysis to compare autoradiographic signals resulting when varying concentrations of (35)S-labeled cRNA probes, dextran sulfate (DS), and dithiothreitol (DTT) were used for ISHH. We found that greatly enhanced corrected signal density (total density of signal area minus background density) was obtained using concentrations of probe and/or DS that were several-fold higher than those widely recommended in published ISHH procedures (probe concentration >4 x 10(4) cpm/microl; DS concentration >10%). Extended hybridization reaction (>16 hr) also significantly augmented the corrected signal density. Finally, nonspecific probe binding was greatly reduced and corrected signal density enhanced by including 750-1000 mM, rather than the widely used 10-200 mM DTT, in the hybridization buffer. These observations indicate that the low efficiency of hybridization and the formation of high background may largely compromise the sensitivity of routine ISHH procedures. We suggest that the new method using increased concentrations of (35)S-labeled cRNA probe, DS, and DTT will be especially important for the cellular localization of rare mRNA species.
- Published
- 2002
- Full Text
- View/download PDF
39. [Development of the method of RNA labeling in vitro using the terminal transferase with the purpose to obtain hybridization probes].
- Author
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Syrchin SA and Mendzhul MI
- Subjects
- Isotope Labeling, Phosphorus Radioisotopes, Transferases, Nucleic Acid Hybridization methods, RNA Probes
- Abstract
A possibility to use terminal transferase to obtain hybridized RNA-probes has been investigated. Optimal conditions of the reaction proceeding were elaborated on the model objects--RNA of the phage MS2: 200 mM of potassium cacodylate, pH 7.2, 1 mM CoCl2, 1 mM DTT, under the considerable surplus of the enzyme. The elaborated method was used to determine the area of early genes of cyanophage LPP-3. With this purpose total RNA preparations were isolated from cyanobacterium Plectonema boryanum CALU 465. The above preparations were labeled by means of terminal transferase and [alpha-32P] ATP and used as the probes in hybridization with the DNA restriction fragments of LPP-3. Proceeding from the results of Southern-blots, a conclusion was made, that the KpnI-fragment of genome of cyanophage LPP3 10 kb in size includes the early genes. A possibility of practical use of the developed method is discussed.
- Published
- 2002
40. Quantitation of in situ hybridization using image analysis of radioactively labeled RNA probes.
- Author
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Bisucci T, Hewitson TD, and Darby IA
- Subjects
- Animals, Collagen genetics, Mice, Software, Image Processing, Computer-Assisted methods, In Situ Hybridization methods, Isotope Labeling methods, RNA Probes, RNA, Messenger isolation & purification
- Published
- 2002
- Full Text
- View/download PDF
41. Labeling fluorescence in situ hybridization probes for genomic targets.
- Author
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Morrison LE, Ramakrishnan R, Ruffalo TM, and Wilber KA
- Subjects
- Fluorescent Dyes chemistry, Molecular Structure, Polymerase Chain Reaction, DNA Probes, In Situ Hybridization, Fluorescence methods, RNA Probes
- Published
- 2002
- Full Text
- View/download PDF
42. Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function.
- Author
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Manefield M, Whiteley AS, Ostle N, Ineson P, and Bailey MJ
- Subjects
- Biomarkers analysis, Carbon Isotopes metabolism, Mass Spectrometry, Phenol metabolism, Pseudomonas putida classification, Pseudomonas putida metabolism, Reverse Transcriptase Polymerase Chain Reaction, Pseudomonas putida genetics, RNA Probes, RNA, Bacterial analysis, Soil Microbiology
- Abstract
An ongoing challenge within microbial ecology is the development of methodologies that attribute microbial community functions to microbial diversity. One approach, involving the incorporation of stable isotopes from labelled tracer compounds into biological signature molecules (biomarkers), may overcome this current limitation. To examine the potential of RNA as the biomarker in stable isotope probing we have generated a series of atom % (13)C-enriched RNA samples through exploitation of the anabolic abilities of a phenol-degrading environmental isolate. Isotope ratio mass spectrometry was used to determine the atom % (13)C of each RNA sample (ca. 1-100%). The corresponding buoyant density (1.755-1.795 g mL(-1)) was determined by equilibrium density gradient centrifugation and agarose gel electrophoresis. This empirically defined relationship between the atom % (13)C of RNA and its buoyant density suggests ribonucleic acids with atom % (13)C enrichments greater than 10% can be isolated by equilibrium density centrifugation. The processing and analysis of isolated RNA by reverse transcription polymerase chain reaction, denaturing gradient gel electrophoresis, cloning and sequencing are discussed. The RNA-based stable isotope probing protocol presented here will find particular utility in assessing the roles of microbial community members in the biodegradation of natural and anthropogenic xenobiotic compounds., (Copyright 2002 John Wiley & Sons, Ltd.)
- Published
- 2002
- Full Text
- View/download PDF
43. Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes.
- Author
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Simard C, Lemieux R, and Côté S
- Subjects
- Animals, Blotting, Northern methods, Cell Line, Mice, Nucleic Acid Denaturation, Tumor Cells, Cultured, DNA analysis, Formamides, Nucleic Acid Hybridization methods, RNA analysis, RNA Probes, Urea
- Abstract
Since their introduction some three decades ago, methods for hybridization analysis of nucleic acids immobilized on solid supports have evolved to improve the sensitivity, speed, and convenience of their application. However, in many cases these methods still require the use of solutions containing formamide, a recognized hazardous solvent with potential toxicity. Here, we have compared the efficiency of urea to that of formamide as denaturing agent in nucleic acid hybridization with RNA probes. We show that urea at concentrations of 2-4 molar in solution performs as good as 50% formamide to reduce heterologous background hybridization in Northern blotting experiments realized at 68 degrees C. Presence of urea at higher concentrations resulted in reduced hybridization sensitivity, possibly due to increased viscosity. When tested in Southern blot analysis of genomic DNA, our results revealed that the use of urea in hybridization solution is also suitable to carry out single-copy gene detection. Together, these findings show that urea can efficiently and safely replace formamide in solutions.
- Published
- 2001
- Full Text
- View/download PDF
44. Methods for visualizing RNA processing and transport pathways in living cells.
- Author
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Dirks RW, Molenaar C, and Tanke HJ
- Subjects
- Animals, Biological Transport, Humans, In Situ Hybridization, Fluorescence methods, RNA Probes, RNA Processing, Post-Transcriptional genetics
- Abstract
Recent advances in fluorescence microscopy, imaging, and probe technology provided possibilities to study the spatial and temporal distribution of RNA species in living cells. While some methods have been developed to localize all nascent or poly (A) containing transcripts others have been developed to study the in vivo distribution of specific RNA species. Irrespective of the method that has been used, the results of these studies provided important information concerning the localization and the cellular transport pathways of RNAs. Also, the picture emerges that RNA molecules travel through the nucleus at much faster speed, equaling that of free diffusion, than previously anticipated. Still, a major challenge proves to be the development of a microscopic detection technique that allows specific, in vivo, detection of low levels of RNA species by fluorescence in situ hybridization, without interfering fluorescent background signals derived from non-hybridized probe sequences and autofluorescent cell components. By applying photoactivatable caged fluorochrome-, molecular beacon-, or fluorescence resonance energy transfer (FRET)-based detection methods an important step in the future of living cell analysis has already been made.
- Published
- 2001
- Full Text
- View/download PDF
45. Developmental patterns of mST3GalV mRNA expression in the mouse: in situ hybridization using DIG-labeled RNA probes.
- Author
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Ji MY, Lee YC, Do S 2nd, Nam SY, Jung KY, Kim HM, Park LK, and Choo YK
- Subjects
- Animals, Female, Mice, Pregnancy, G(M3) Ganglioside biosynthesis, Gene Expression Regulation, Developmental, Gene Expression Regulation, Enzymologic, In Situ Hybridization, RNA Probes, RNA, Messenger analysis, Sialyltransferases genetics
- Abstract
mST3GalV synthesizes ganglioside GM3, the precursor for simple and complex a- and b- series gangliosides, and the expression and regulation of mST3GalV (CMP-NeuAc: lactosylceramide alpha2,3-sialyltransferase) activity is central to the production of almost all gangliosides, a class of glycosphingolipids implicated in variety of cellular processes such as transmembrane signaling, synaptic transmission, specialized membrane domain formation and cell-cell interactions. To understand the developmental expression of mST3GalV in mice, we investigated the spatial and temporal expression of mST3GalV mRNA during the mouse embryogenesis [embryonic (E) days; E9, E11, E13, E15] by in situ hybridization with digoxigenin-labeled RNA probes. All tissues from E9 and E11 were positive for mST3GalV mRNA. On E13, mST3GalV mRNA was expressed in various neural and non-neural tissues. In contrast to these, on E15, the telencephalon and liver produced a strong expression of mST3Gal V which was a quite similar to that of E13. In this stage, mST3GalV mRNA was also expressed in some non-neural tissues. These data indicate that mST3GalV is differently expressed at developmental stages of embryo, and this may be importantly related with regulation of organogenesis in mice.
- Published
- 2000
- Full Text
- View/download PDF
46. Ultrastructural cytochemical, immunocytochemical and in situ hybridization methods with polyuridine probes detect mRNA in human mast cell granules.
- Author
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Dvorak AM and Morgan ES
- Subjects
- Biotin, Cell Nucleus metabolism, Cell Nucleus ultrastructure, Cytoplasm metabolism, Cytoplasm ultrastructure, Cytoplasmic Granules ultrastructure, Gold, Humans, Hydrogen-Ion Concentration, Immunohistochemistry, In Situ Hybridization, Mast Cells ultrastructure, Microscopy, Electron, Cytoplasmic Granules metabolism, Mast Cells metabolism, Poly U, RNA Probes, RNA, Messenger analysis
- Abstract
Mature human mast cells are classical secretory cells that are filled with secretory-storage granules but are poorly endowed with visible free or membrane-bound cytoplasmic ribosomes. We recently reported close associations of ribosomes and various components essential to RNA metabolism in and close to human mast cell granules using multiple ultrastructural imaging methods. In view of these findings and an increased awareness of RNA sorting and localization to specific subcellular sites and organelles, we used human mast cells purified from non-tumour portions of lung samples resected at surgery for carcinoma and ultrastructural methods to investigate this further. Poly(U) probes were used to detect direct en grid binding, and radiolabelled as well as non-radiolabelled poly(U) probes were used in in situ hybridization protocols to detect poly(A)-positive pre-mRNA and mRNA in nuclear, cytoplasmic and granular compartments of mature human mast cells. Negative controls verified specificity of label; expected nuclear and cytoplasmic locations of poly(A)-positive RNA served as positive controls for each sample. These findings lend support to the hypothesis that site-specific synthesis in secretory-storage granules may occur in secretory cells.
- Published
- 2000
- Full Text
- View/download PDF
47. Direct detection of bacteria in cellular blood products using bacterial ribosomal RNA-directed probes coupled to electrochemiluminescence.
- Author
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Chaney R, Rider J, and Pamphilon D
- Subjects
- Biotinylation, Blood Platelets microbiology, Electrodes, Erythrocyte Transfusion, Erythrocytes microbiology, Humans, Magnetics, Nucleic Acid Hybridization, Platelet Transfusion, Pseudomonas fluorescens isolation & purification, Ruthenium, Staphylococcus aureus isolation & purification, Yersinia enterocolitica isolation & purification, Bacteria genetics, Bacteria isolation & purification, Blood microbiology, Luminescent Measurements, RNA Probes, RNA, Bacterial blood, RNA, Ribosomal blood
- Abstract
Various techniques for detecting bacteria in blood products exist; however, none has been widely accepted. Our method permits direct bacterial detection in both platelet concentrates (PC) and packed red blood cells (RBC). This novel procedure targets bacterial ribosomal RNA (rRNA) but does not utilize culture or nucleic acid amplification. The assay comprises five steps: (1) release of bacterial rRNA by cell lysis with a combination of detergents and high heat; (2) hybridization of bacterial rRNA using a biotin- and a ruthenium (ORIGEN)-labelled oligonucleotide probe pair; (3) capture of labelled rRNA with streptavidin-coated magnetic beads; (4) concentration of labelled rRNA/bead complexes out of solution and onto an electrode surface with a magnet; (5) detection of ruthenium-labelled bacterial rRNA by application of voltage and consequent generation of the electrochemiluminescent (ECL) signal. Results using PC and RBC samples, spiked with clinically relevant gram-negative and -positive bacterial species, consistently demonstrated a linear relationship between ECL signal (equates to rRNA level) and colony forming units (CFU) mL(-1). Signals were generated in the range of 1400-80000 and 3500-500000 ECL units for unwashed and washed samples, respectively. This is equivalent to 10(5)-10(8)CFU mL(-1). These data demonstrate that therapeutic blood products significantly contaminated with bacteria may be identified prior to issue.
- Published
- 1999
- Full Text
- View/download PDF
48. Detection of vancomycin resistant genes vanA and vanB by cycling probe technology.
- Author
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Modrusan Z, Marlowe C, Wheeler D, Pirseyedi M, and Bryan RN
- Subjects
- Drug Resistance genetics, Genes, Bacterial, Humans, Bacterial Proteins genetics, Carbon-Oxygen Ligases genetics, DNA Probes, RNA Probes, Vancomycin
- Abstract
Cycling Probe Technology (CPT) has been used to develop gene-based assays for detection of vancomycin resistance genes vanA and vanB in enterococci (VRE). Cycling Probe Technology utilizes a chimeric DNA-RNA-DNA probe that is cleaved by the enzyme RNase H when hybridized to its complementary DNA target. Conversion of full-length probe into the cleaved probe fragments is the basis for detection and quantification of the CPT reaction. Two gene-specific probes, each one unique to either the vanA or vanB gene, were utilized for development of vanA and vanB CPT assays, respectively. Both vanA and vanB CPT assays were used to determine the presence or absence of the corresponding gene in 440 clinical enterococcal isolates. The presence of vanA and vanB gene sequences was detected in 154 and 131 isolates, respectively. Phenotypic characterization of all isolates was determined through interpretation of conventional susceptibility data obtained with the disk diffusion method. Comparison between disk diffusion characterization and CPT assays revealed 11 discrepant isolates. The identity of these isolates was resolved by polymerase chain reaction (PCR) which confirmed the vanA and vanB CPT assay data. Therefore, compared to conventional phenotyping, both the vanA and vanB CPT assays appeared superior for accurate identification of VanA and VanB isolates., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
49. Probe generation by PCR coupled with ligation.
- Author
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Ilyin SE and Plata-Salamán CR
- Subjects
- Base Sequence, DNA Primers, Promoter Regions, Genetic, Templates, Genetic, Polymerase Chain Reaction methods, RNA Probes
- Published
- 1999
- Full Text
- View/download PDF
50. Rapid detection of mecA in methicillin resistant Staphylococcus aureus using cycling probe technology.
- Author
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Cloney L, Marlowe C, Wong A, Chow R, and Bryan R
- Subjects
- Humans, Microbial Sensitivity Tests, Penicillin-Binding Proteins, Polymerase Chain Reaction methods, Time Factors, Bacterial Proteins genetics, Carrier Proteins genetics, DNA Probes, Hexosyltransferases, Methicillin Resistance genetics, Muramoylpentapeptide Carboxypeptidase genetics, Peptidyl Transferases, RNA Probes, Staphylococcus aureus genetics
- Abstract
A novel method has been developed for the detection of the mecA gene, that confers the principle mechanism of methicillin resistance in staphylococci. Cycling Probe Technology (CPT) is a rapid, simple, isothermal method for the detection of specific target sequences. CPT utilizes a unique chimeric DNA-RNA-DNA probe sequence that provides an RNase H sensitive scissile link when hybridized to a complementary target DNA sequence. In the presence of target DNA, the cycling reaction converts full-length chimeric probe into cleaved probe fragments, which accumulate and are quantified. A cycling probe designed for detection of a specific sequence within the mecA gene was used to develop a culture confirmation assay for methicillin resistant Staphylococcus aureus. The CPT assay was used to screen 238 S. aureus isolates and the results were in complete agreement with detection of the mecA gene by polymerase chain reaction (PCR). Detection of mecA should be considered the gold standard for determining methicillin resistance in S. aureus. This study demonstrates the feasibility of using CPT to meet this need., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF
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