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Nucleic Acids Research Jul 2016We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method...
We describe a simple, specific and sensitive microRNA (miRNA) detection method that utilizes Chlorella virus DNA ligase (SplintR(®) Ligase). This two-step method involves ligation of adjacent DNA oligonucleotides hybridized to a miRNA followed by real-time quantitative PCR (qPCR). SplintR Ligase is 100X faster than either T4 DNA Ligase or T4 RNA Ligase 2 for RNA splinted DNA ligation. Only a 4-6 bp overlap between a DNA probe and miRNA was required for efficient ligation by SplintR Ligase. This property allows more flexibility in designing miRNA-specific ligation probes than methods that use reverse transcriptase for cDNA synthesis of miRNA. The qPCR SplintR ligation assay is sensitive; it can detect a few thousand molecules of miR-122. For miR-122 detection the SplintR qPCR assay, using a FAM labeled double quenched DNA probe, was at least 40× more sensitive than the TaqMan assay. The SplintR method, when coupled with NextGen sequencing, allowed multiplex detection of miRNAs from brain, kidney, testis and liver. The SplintR qPCR assay is specific; individual let-7 miRNAs that differ by one nucleotide are detected. The rapid kinetics and ability to ligate DNA probes hybridized to RNA with short complementary sequences makes SplintR Ligase a useful enzyme for miRNA detection.
Topics: Animals; DNA Ligases; DNA Probes; Humans; Mice; MicroRNAs; Nucleic Acid Hybridization; Rats; Real-Time Polymerase Chain Reaction; Viral Proteins
PubMed: 27154271
DOI: 10.1093/nar/gkw399 -
Journal of Clinical Microbiology Nov 2002Group A streptococcus (GAS) antigen testing has become a routine point-of-care (POC) test in acute care settings. Concern about performance parameters (PP) of these...
Group A streptococcus (GAS) antigen testing has become a routine point-of-care (POC) test in acute care settings. Concern about performance parameters (PP) of these tests as well as inappropriate antibiotic use has resulted in various recommendations regarding diagnosis of GAS. There were two objectives in this study. The first was to evaluate the rapid GAS antigen test presently in use (Thermo BioStar, Boulder, Colo.) and the GAS Direct probe test (Gen-Probe, San Diego, Calif.) compared to culture. The second was to define the optimal use of these technologies in a large acute care pediatric clinic. A total of 520 consecutive pediatric patients presenting with symptoms of pharyngitis at any of three Lahey Clinic acute care facilities were evaluated. Pharyngeal specimens were collected using a double-swab collection device (Copan, Corona, Calif.). One swab was used for the antigen test, the second was used for the probe test, and the pledget was placed in the collection device for culture on 5% sheep blood agar, incubated for 48 h anaerobically, and subsequently placed in Todd-Hewitt broth. After discrepant analysis, sensitivity, specificity, and positive and negative predictive values were as follows: 94.8, 100, 100, and 96.9% for the probe test and 86.1, 97.1, 93.7, and 93.4% for the antigen test, respectively. Sensitivity using an enhanced culture technique was 99.4% (163 of 164). False-positive (FP) antigen results were often seen from patients previously diagnosed and/or treated for GAS. No FP results were seen with the probe test. Colony counts for the false-negative (FN) antigen tests were higher than those for the FN probe tests. Compared to culture and DNA probe, the rapid antigen test (RAT) offered a result at the time of the patient's visit, with acceptable PP when prevalence of disease is high. Follow-up testing with the RAT of GAS patients who previously tested as positive should be avoided due to increased FP results. The probe test was comparable to culture in performance. Results indicate the probe test can be used as the primary test or as a backup to negative antigen tests. The probe test offers the advantage over culture of same-day reporting of a final result but, in contrast to a POC test, necessitates follow-up communication to the patient. Preliminary data show the specificity of the probe test to be greater than that of the RAT for patients previously diagnosed with GAS.
Topics: Adolescent; Ambulatory Care; Antigens, Bacterial; Child; Child, Preschool; Culture Media; DNA Probes; DNA, Ribosomal; Humans; Immunoassay; Infant; Infant, Newborn; Luminescent Measurements; Pediatrics; Pharyngitis; Predictive Value of Tests; RNA, Ribosomal; Sensitivity and Specificity; Streptococcal Infections; Streptococcus pyogenes; Time Factors
PubMed: 12409399
DOI: 10.1128/JCM.40.11.4207-4210.2002 -
PloS One 2017Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short...
Core microRNA (miRNA) sequences exist as populations of variants called isomiRs made up of different lengths and nucleotide compositions. In particular, the short sequences of miRNA make single-base isomiR mismatches very difficult to be discriminated. Non-specific hybridizations often arise when DNA probe-miRNA target hybridization is the primary, or initial, mode of detection. These errors then become exacerbated through subsequent amplification steps. Here, we present the design of DNA probes modified with poly-guanine (PG) tracts that were induced to form G-quadruplexes (G4) for hi-fidelity discrimination of miRNA core target sequence from single-base mismatched isomiRs. We demonstrate that, when compared to unmodified probes, this G4 'gate-keeping' function within the G4-modified probes enables more stringent hybridization of complementary core miRNA target transcripts while limiting non-specific hybridizations. This increased discriminatory power of the G4-modified probes over unmodified probes is maintained even after further reverse transcriptase extension of probe-target hybrids. Enzymatic extension also enhanced the clarity and sensitivity of readouts and allows different isomiRs to be distinguished from one another via the relative positions of the mismatches.
Topics: DNA Probes; G-Quadruplexes; Isomerism; MicroRNAs
PubMed: 29145502
DOI: 10.1371/journal.pone.0188163 -
Journal of Clinical Microbiology Oct 1993The direct detection of gonococcal DNA in rectal and pharyngeal specimens was evaluated by using a DNA probe-based assay (Gen-Probe, Inc., San Diego, Calif.). Rectal...
The direct detection of gonococcal DNA in rectal and pharyngeal specimens was evaluated by using a DNA probe-based assay (Gen-Probe, Inc., San Diego, Calif.). Rectal (234) and pharyngeal (608) swab specimens were obtained from 249 men and 372 women attending sexually transmitted disease clinics in Las Vegas and Reno, Nevada. The prevalence of gonococcal infection by culture at the pharyngeal and rectal sites was 2.9% (16 of 548 specimens) in women and 2.7% (8 of 294 specimens) in men. No false-positive reactions were observed among the 234 rectal specimens tested. Two probe-positive, culture-negative specimens were detected among the 361 pharyngeal specimens obtained from women. Both of these samples were confirmed as Neisseria gonorrhoeae by a probe competition assay. The overall correlation of the DNA probe test with pharyngeal and rectal cultures was 99.4% (837 of 842 cultures), with a sensitivity of 87.5% (21 of 24 cultures) and specificity of 99.7% (816 of 818 cultures). The positive and negative predictive values of the DNA assay were 91.3 and 99.8%, respectively. The direct DNA probe assay provides an alternative to culture screening for rectal and/or pharyngeal gonococcal infections.
Topics: DNA Probes; DNA, Bacterial; Female; Gonorrhea; Humans; Male; Neisseria gonorrhoeae; Pharynx; Rectum
PubMed: 8253983
DOI: 10.1128/jcm.31.10.2783-2785.1993 -
Nucleic Acids Research Sep 2017Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells....
Quantitative measurement of mRNA levels in single cells is necessary to understand phenotypic variability within an otherwise isogenic population of cells. Single-molecule mRNA Fluorescence In Situ Hybridization (FISH) has been established as the standard method for this purpose, but current protocols require a long region of mRNA to be targeted by multiple DNA probes. Here, we introduce a new single-probe FISH protocol termed sFISH for budding yeast, Saccharomyces cerevisiae using a single DNA probe labeled with a single fluorophore. In sFISH, we markedly improved probe specificity and signal-to-background ratio by using methanol fixation and inclined laser illumination. We show that sFISH reports mRNA changes that correspond to protein levels and gene copy number. Using this new FISH protocol, we can detect >50% of the total target mRNA. We also demonstrate the versatility of sFISH using FRET detection and mRNA isoform profiling as examples. Our FISH protocol with single-fluorophore sensitivity significantly reduces cost and time compared to the conventional FISH protocols and opens up new opportunities to investigate small changes in RNA at the single cell level.
Topics: Carbocyanines; DNA Probes; Fluorescent Dyes; Gene Expression Regulation, Fungal; In Situ Hybridization, Fluorescence; RNA, Messenger; Saccharomyces cerevisiae; Sensitivity and Specificity; Single Molecule Imaging
PubMed: 28666354
DOI: 10.1093/nar/gkx568 -
Nature Communications Sep 2017Analysis of the spatial arrangement of molecular features enables the engineering of synthetic nanostructures and the understanding of natural ones. The ability to...
Analysis of the spatial arrangement of molecular features enables the engineering of synthetic nanostructures and the understanding of natural ones. The ability to acquire a comprehensive set of pairwise proximities between components would satisfy an increasing interest in investigating individual macromolecules and their interactions, but current biochemical techniques detect only a single proximity partner per probe. Here, we present a biochemical DNA nanoscopy method that records nanostructure features in situ and in detail for later readout. Based on a conceptually novel auto-cycling proximity recording (APR) mechanism, it continuously and repeatedly produces proximity records of any nearby pairs of DNA-barcoded probes, at physiological temperature, without altering the probes themselves. We demonstrate the production of dozens of records per probe, decode the spatial arrangements of 7 unique probes in a homogeneous sample, and repeatedly sample the same probes in different states.The spatial organisation of nanostructures is fundamental to their function. Here, the authors develop a non-destructive, proximity-based method to record extensive spatial organization information in DNA molecules for later readout.
Topics: Base Sequence; DNA; DNA Probes; Models, Molecular; Nanostructures; Nanotechnology; Nucleic Acid Conformation; Streptavidin; Thermodynamics
PubMed: 28947733
DOI: 10.1038/s41467-017-00542-3 -
Analytical Chemistry Jan 2021
Review
Topics: Biosensing Techniques; DNA; DNA Probes; Humans; Nucleic Acid Amplification Techniques
PubMed: 33147015
DOI: 10.1021/acs.analchem.0c04364 -
Journal of Clinical Microbiology Dec 1990The objective of this hospital-based study was to determine the relationship between colonizing and infecting strains of Candida species and Torulopsis glabrata....
The objective of this hospital-based study was to determine the relationship between colonizing and infecting strains of Candida species and Torulopsis glabrata. Surveillance cultures from high-risk patients were paired with subsequent bloodstream isolates. Organisms were typed by using restriction endonuclease digestion of chromosomal DNA with BstNI and EcoRI, followed by Southern hybridization with a DNA probe (pBD4) derived from Saccharomyces cerevisiae. Sixteen patients for whom documented colonization preceded documented bloodstream infection were identified. The mean time between obtainment of surveillance isolates and obtainment of bloodstream isolates was 8 days, with a range of 1 to 423 days. For 15 (94%) of 16 patients, the DNA fingerprint pattern (using BstNI) of the surveillance isolate was identical to that of the bloodstream isolate. Isolates from 13 (81%) of 16 patients were unique to those patients. Typing by Southern hybridization with the pBD4 probe was less discriminating. We conclude that for a well-defined subset of hospitalized patients who were colonized by Candida species before developing nosocomial candidemia, the colonizing and infecting strains were identical, suggesting endogenous acquisition of infection. Restriction endonuclease digestion of chromosomal DNA was shown to be a discriminating and reproducible typing method for Candida species and T. glabrata.
Topics: Blotting, Southern; Candida; Candidiasis; Cross Infection; DNA Probes; DNA Restriction Enzymes; DNA, Fungal; Humans
PubMed: 2177750
DOI: 10.1128/jcm.28.12.2733-2738.1990 -
Journal of Applied Microbiology Jun 1997A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using...
A 300 bp DNA fragment of Lactobacillus plantarum isolated by randomly amplified polymorphic DNA (RAPD) analysis was cloned and sequenced. This fragment was tested using a dot-blot DNA hybridization to technique for its ability to identify Lact. plantarum strains. This probe hybridized with all Lact. plantarum strains tested and with some strains of Lact. pentosus, albeit more weakly. Two internal primers of this probe were selected (LbP11 and LbP12) and polymerase chain reaction (PCR) was carried out. All Lact. plantarum strains tested amplified a 250 bp fragment contrary to the other LAB species tested. This specific PCR for Lact. plantarum was also performed from colonies grown on MRS medium with similar results. These methods enabled the rapid and specific detection and identification of Lact. plantarum.
Topics: Base Sequence; DNA Probes; DNA, Bacterial; Lactobacillus; Molecular Sequence Data; Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 9202445
DOI: 10.1046/j.1365-2672.1997.00157.x -
The Analyst Sep 2015We have developed a novel approach for DNA detection as well as genetic screening of mutations by uniquely combining DNA-responsive and optically diffracting materials....
We have developed a novel approach for DNA detection as well as genetic screening of mutations by uniquely combining DNA-responsive and optically diffracting materials. This approach entails the polymerization of a photonic crystal within a hydrogel network that alters the diffraction of light in response to a target DNA strand. The utility of this approach, which permits label-free sensing, was demonstrated via the detection of a target sequence from the DNA binding domain of the major tumor suppressor protein p53. Using a complementary capture probe strand, we were able to detect down to picomole concentrations of the target p53 sequence. Moreover, we demonstrated that this approach could readily detect a single base pair mutation in the target strand, which corresponds to the hotspot cancer mutation R175H in p53. The sensitivity of detection was increased by lowering the rate of annealing of the target strand and adjusting the solution ionic strength during optical characterization. Changes in ionic strength during characterization impact the melting temperature of the bound target DNA and the Donnan potential between the hydrogel and solution, which influence detection. We further showed that this approach is sensitive to epigenetic changes via the detection of a fully methylated form of the target p53 sequence. Ultimately, this approach represents a new paradigm for DNA detection and specifically genetic screening of p53 as well as other disease markers and nucleotide modifications that alter the properties of DNA (e.g., epigenetic alterations and adducts with chemical carcinogens).
Topics: Base Sequence; Biosensing Techniques; DNA; DNA Methylation; DNA Probes; Genes, p53; Hydrogels; Mutation, Missense; Nucleic Acid Hybridization; Optical Phenomena; Osmolar Concentration; Transition Temperature
PubMed: 26270146
DOI: 10.1039/c5an01191d