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The Journal of Physical Chemistry. B May 2013In this work, DNA-Hg(II) interactions were investigated by monitoring the translocation of DNA hairpins in a protein ion channel in the absence and presence of metal...
In this work, DNA-Hg(II) interactions were investigated by monitoring the translocation of DNA hairpins in a protein ion channel in the absence and presence of metal ions. Our experiments demonstrate that target-specific hairpin structures could be stabilized much more significantly by mercuric ions than by the stem length and the loop size of the hairpin due to the formation of Thymine-Hg(II)-Thymine complexes. In addition, the designed DNA probe allows the development of a highly sensitive nanopore sensor for Hg(2+) with a detection limit of 25 nM. Further, the sensor is specific, and other tested metal ions including Pb(2+), Cu(2+), Cd(2+), and so on with concentrations of up to 2 orders of magnitude greater than that of Hg(2+) would not interfere with the mercury detection.
Topics: DNA Probes; Electrochemical Techniques; Hemolysin Proteins; Inverted Repeat Sequences; Ions; Lipid Bilayers; Mercury; Nanopores; Thymine
PubMed: 23565989
DOI: 10.1021/jp309541h -
Nature Nanotechnology May 2017Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result...
Cells interact with the extracellular environment through molecules expressed on the membrane. Disruption of these membrane-bound interactions (or encounters) can result in disease progression. Advances in super-resolution microscopy have allowed membrane encounters to be examined, however, these methods cannot image entire membranes and cannot provide information on the dynamic interactions between membrane-bound molecules. Here, we show a novel DNA probe that can transduce transient membrane encounter events into readable cumulative fluorescence signals. The probe, which translocates from one anchor site to another, mimicking motor proteins, is realized through a toehold-mediated DNA strand displacement reaction. Using this probe, we successfully monitored rapid encounter events of membrane lipid domains using flow cytometry and fluorescence microscopy. Our results show a preference for encounters within the same lipid domains.
Topics: Animals; Biological Transport, Active; Cattle; Cell Line; Cell Membrane; DNA Probes; Microscopy, Fluorescence; Molecular Imaging; Serum Albumin, Bovine
PubMed: 28319616
DOI: 10.1038/nnano.2017.23 -
Angewandte Chemie (International Ed. in... Aug 2016A sensing approach is applied to encode quantitative enzymatic activity information into DNA sequence populations. The method utilizes DNA-linked peptide substrates as...
A sensing approach is applied to encode quantitative enzymatic activity information into DNA sequence populations. The method utilizes DNA-linked peptide substrates as activity probes. Signal detection involves chemical manipulation of a probe population downstream of sample exposure and application of purifying, selective pressure for enzyme products. Selection-induced changes in DNA abundance indicate sample activity. The detection of protein kinase, protease, and farnesyltransferase activities is demonstrated. The assays were employed to measure enzyme inhibition by small molecules and activity in cell lysates using parallel DNA sequencing or quantitative PCR. This strategy will allow the extensive infrastructure for genetic analysis to be applied to proteomic assays, which has a number of advantages in throughput, sensitivity, and sample multiplexing.
Topics: Base Sequence; DNA; DNA Probes; Enzyme Activation; Farnesyltranstransferase; Humans; Peptide Hydrolases; Polymerase Chain Reaction; Protein Kinases
PubMed: 27355201
DOI: 10.1002/anie.201603387 -
Parasitology Research Jun 1998Repetitive DNA sequences present in the genome of Dicrocoelium dendriticum were identified by hybridization of genomic DNA that had been digested with different...
Repetitive DNA sequences present in the genome of Dicrocoelium dendriticum were identified by hybridization of genomic DNA that had been digested with different restriction enzymes with 32P-labeled genomic D. dendriticum DNA. DNA fragments containing repetitive sequences were isolated from PstI-digested D. dendriticum DNA and were subcloned into a plasmid vector. Plasmids containing repetitive sequences were identified by colony hybridization. One of these plasmids, designated Ddr-IV, was isolated and used as a probe in further studies. Ddr-IV is specific for D. dendriticum since it does not hybridize to DNA isolated from other trematodes. In addition, Ddr-IV was capable of detecting D. dendriticum metacercariae in ants (Formica cunicularia, F. rufibarbis, and Lasius sp.), which act as second intermediate hosts in the parasite's life cycle. Since metacercariae constitute the infectious stage of the parasite for grazing animals, Ddr-IV will provide a useful tool for epidemiology studies of dicrocoeliosis.
Topics: Animals; Ants; Base Sequence; Cloning, Molecular; DNA Probes; DNA, Helminth; Dicrocoelium; Molecular Sequence Data; Nucleic Acid Hybridization; Repetitive Sequences, Nucleic Acid
PubMed: 9660142
DOI: 10.1007/s004360050437 -
BioTechniques Nov 2018Described here is a novel, universal exogenous internal positive control (IPC), which is fully synthetic for unparalleled quality control. The IPC was rationally...
Described here is a novel, universal exogenous internal positive control (IPC), which is fully synthetic for unparalleled quality control. The IPC was rationally designed, is small and efficiently amplified, has been successfully utilized alone or in triplex qPCR reactions, and is not crossreactive to human DNA or to any of the numerous non-human DNA samples tested. It was sensitive to inhibition by at least four commonly encountered amplification inhibitors. The IPC was used in a hydrolysis probe qPCR assay; however, it may be adaptable for other applications such as intercalating dye qPCR, PCR, isothermal amplification and reverse transcription-PCR.
Topics: Animals; Base Sequence; DNA; DNA Probes; False Negative Reactions; Humans; Quality Control; Real-Time Polymerase Chain Reaction; Reference Standards
PubMed: 30394127
DOI: 10.2144/btn-2018-0034 -
Nucleic Acids Research Dec 2001The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we...
The hybridization of complementary strands of DNA is the underlying principle of all microarray-based techniques for the analysis of DNA variation. In this paper, we study how probe immobilization at surfaces, specifically probe density, influences the kinetics of target capture using surface plasmon resonance (SPR) spectroscopy, an in situ label-free optical method. Probe density is controlled by varying immobilization conditions, including solution ionic strength, interfacial electrostatic potential and whether duplex or single stranded oligonucleotides are used. Independent of which probe immobilization strategy is used, we find that DNA films of equal probe density exhibit reproducible efficiencies and reproducible kinetics for probe/target hybridization. However, hybridization depends strongly on probe density in both the efficiency of duplex formation and the kinetics of target capture. We propose that probe density effects may account for the observed variation in target-capture rates, which have previously been attributed to thermodynamic effects.
Topics: DNA; DNA Probes; DNA, Single-Stranded; Kinetics; Nucleic Acid Hybridization; Osmolar Concentration; Surface Plasmon Resonance
PubMed: 11812850
DOI: 10.1093/nar/29.24.5163 -
Memorias Do Instituto Oswaldo Cruz 1992The development of a repetitive DNA probe for Babesia bigemina was reviewed. The original plasmid (p(Bbi)16) contained an insert of B. bigemina DNA of approximately 6.3... (Comparative Study)
Comparative Study Review
The development of a repetitive DNA probe for Babesia bigemina was reviewed. The original plasmid (p(Bbi)16) contained an insert of B. bigemina DNA of approximately 6.3 kb. This probe has been evaluated for specificity and analytical sensitivity by dot blot hybridization with isolates from Mexico, the Caribbean region and Kenya. A partial restriction map has been constructed and insert fragments have been subcloned and utilized as specific DNA probes. A comparison of 32P labelled and non-radioactive DNA probes was presented. Non-radioactive detection systems that have been used include digoxigenin dUTP incorporation, and detection by colorimetric substrate methods. Derivatives from the original DNA probe have been utilized to detect B. bigemina infection in a) experimentally inoculated cattle, b) field exposed cattle, c) infected Boophilus microplus ticks, and d) the development of a PCR amplification system.
Topics: Animals; Babesia; Babesiosis; Caribbean Region; Cattle; Cattle Diseases; DNA Probes; DNA, Protozoan; Kenya; Mexico; Nucleic Acid Hybridization; Polymerase Chain Reaction; Sensitivity and Specificity; Ticks
PubMed: 1343692
DOI: 10.1590/s0074-02761992000700034 -
Journal of Clinical Microbiology Sep 1997Randomly amplified polymorphic DNA (RAPD) analysis, multilocus enzyme electrophoresis (MLEE), and Southern blot hybridization with moderately repetitive DNA probes have... (Comparative Study)
Comparative Study
Parity among the randomly amplified polymorphic DNA method, multilocus enzyme electrophoresis, and Southern blot hybridization with the moderately repetitive DNA probe Ca3 for fingerprinting Candida albicans.
Randomly amplified polymorphic DNA (RAPD) analysis, multilocus enzyme electrophoresis (MLEE), and Southern blot hybridization with moderately repetitive DNA probes have emerged as effective fingerprinting methods for the infectious fungus Candida albicans. The three methods have been compared for their capacities to identify identical or highly related isolates, to cluster weakly related isolates, to discriminate between unrelated isolates, and to assess microevolution within a strain. By computing similarity coefficients between 29 isolates from three cities within the continental United States, strong concordance of the results is demonstrated for RAPD analysis, MLEE, and Southern blot hybridization with the moderately repetitive probe Ca3, and weaker concordance of the results is demonstrated for these three fingerprinting methods and Southern blot hybridization with the moderately repetitive probe CARE2. All methods were also demonstrated to be able to resolve microevolution within a strain, with the Ca3 probe exhibiting the greatest resolving power. The strong correlations demonstrated between polymorphic markers assessed by the four independent fingerprinting methods and the nonrandom association between loci demonstrated by RAPD analysis and MLEE provide evidence for strong linkage disequilibrium and a clonal population structure for C. albicans. In addition, a synapomorphic allele, Pep-3A, was found to be present in all members of one of the three clusters discriminated by RAPD analysis, MLEE, and Ca3 fingerprinting, supporting the concordance of the clustering capacities of the three methods, the robustness of the clusters, and the clonal nature of the clusters.
Topics: Adaptor Proteins, Vesicular Transport; Alleles; Blotting, Southern; Candida albicans; DNA Fingerprinting; DNA Probes; DNA, Fungal; Electrophoresis, Starch Gel; Enzymes; Evolution, Molecular; Fungal Proteins; Genetic Linkage; Membrane Proteins; Multigene Family; Phylogeny; Random Amplified Polymorphic DNA Technique; Repetitive Sequences, Nucleic Acid; Saccharomyces cerevisiae Proteins
PubMed: 9276415
DOI: 10.1128/jcm.35.9.2348-2358.1997 -
Journal of Clinical Microbiology Jan 1995The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C.... (Comparative Study)
Comparative Study
The sensitivities of a PCR assay and a DNA probe assay were compared for the detection of Cowdria ruminantium in Amblyomma ticks that were fed on C. ruminantium-infected, clinically reacting, and recovered carrier animals. The PCR assay and DNA probe detected infection in 86.0 and 37.0%, respectively, of 100 ticks fed on a febrile animal. In 75 ticks fed on carrier animals, PCR and the DNA probe detected infection in 28.0 and 1.33% of ticks, respectively. This demonstrates that the DNA probe has poor sensitivity for the detection of low levels of infection in ticks and that PCR is necessary for this purpose. The PCR assay had a detection limit of between 1 and 10 C. ruminantium organisms and did not amplify DNA from Ehrlichia canis, which is phylogenetically closely related to C. ruminantium, Theileria parva, or uninfected Amblyomma hebraeum or A. variegatum. PCR detected infection in A. hebraeum and A. variegatum adult ticks infected with one of six geographically different C. ruminantium strains. Amplification was also possible from desiccated ticks and ticks fixed in 70% ethanol, 10% buffered formalin, or 2% glutaraldehyde. The PCR assay supersedes the DNA probe and older detection methods for the detection of C. ruminantium in ticks, particularly those fed on carrier animals, and is suitable for both prospective and retrospective studies which require accurate detection of C. ruminantium in individual ticks. Application of the PCR assay should significantly improve the understanding of heartwater epidemiology, particularly through the determination of field tick infection rates.
Topics: Animals; Archives; Base Sequence; Carrier State; Cattle; DNA Probes; DNA, Bacterial; Evaluation Studies as Topic; Female; Heartwater Disease; Male; Molecular Sequence Data; Nucleic Acid Hybridization; Polymerase Chain Reaction; Sensitivity and Specificity; Sheep; Ticks
PubMed: 7699036
DOI: 10.1128/jcm.33.1.166-172.1995 -
Nucleic Acids Research 2007Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5'-end and a non-fluorescent quencher...
Novel fluorogenic DNA probes are described. The probes (called Pleiades) have a minor groove binder (MGB) and a fluorophore at the 5'-end and a non-fluorescent quencher at the 3'-end of the DNA sequence. This configuration provides surprisingly low background and high hybridization-triggered fluorescence. Here, we comparatively study the performance of such probes, MGB-Eclipse probes, and molecular beacons. Unlike the other two probe formats, the Pleiades probes have low, temperature-independent background fluorescence and excellent signal-to-background ratios. The probes possess good mismatch discrimination ability and high rates of hybridization. Based on the analysis of fluorescence and absorption spectra we propose a mechanism of action for the Pleiades probes. First, hydrophobic interactions between the quencher and the MGB bring the ends of the probe and, therefore, the fluorophore and the quencher in close proximity. Second, the MGB interacts with the fluorophore and independent of the quencher is able to provide a modest (2-4-fold) quenching effect. Joint action of the MGB and the quencher is the basis for the unique quenching mechanism. The fluorescence is efficiently restored upon binding of the probe to target sequence due to a disruption in the MGB-quencher interaction and concealment of the MGB moiety inside the minor groove.
Topics: DNA Probes; Fluorescent Dyes; Kinetics; Nucleic Acid Hybridization; Temperature
PubMed: 17259212
DOI: 10.1093/nar/gkl1136