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Journal of Virological Methods 1991
Review
Topics: DNA Probes; DNA Replication; Humans; Ligases; Nucleic Acid Amplification Techniques; Polymerase Chain Reaction; Q beta Replicase
PubMed: 1816250
DOI: 10.1016/0166-0934(91)90127-l -
Bioorganic & Medicinal Chemistry Letters Nov 2021Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly...
Micro RNAs (miRNAs) are involved in a variety of biological functions and are attracting attention as diagnostic and prognostic markers for various diseases. Highly sensitive RNA detection methods are required to determine miRNA expression levels and intracellular localization. In this study, we designed new double-stranded peptide nucleic acid (PNA)/DNA probes consisting of a fluorophore-PNA-quencher (fPq) and a quencher-DNA (qD) for miR-221 detection. We optimized the fPq structure, PNA-DNA hybrid length, and hybrid position. The resultant fPq-2/qD-6b probe was a 6-bp hybrid probe with a 10-base fPq and a 6-base qD. The signal-to-background ratios of the probes showed that fPq-2/qD-6b had a higher target sensitivity than fPq (PNA beacon)-type and fP/qD-type probes. The results of the detection limit and target specificity indicate that the fPq/qD probe is promising for RNA detection in both cells and cell extracts as well as for miRNA diagnosis.
Topics: DNA Probes; Fluorescent Dyes; Humans; MicroRNAs; Peptide Nucleic Acids
PubMed: 34534675
DOI: 10.1016/j.bmcl.2021.128359 -
Current Protocols in Cell Biology Sep 2004Fluorescence in situ Hybridization (FISH) involves the preparation of two main components: the DNA probe and the target DNA to which the probe will be hybridized. The... (Review)
Review
Fluorescence in situ Hybridization (FISH) involves the preparation of two main components: the DNA probe and the target DNA to which the probe will be hybridized. The DNA probe typically comes from cloned sources such as plasmids, cosmids, PACs, YACs, or BACs; where the insert may contain a specific gene or originate from a specific chromosomal locus. Whole-chromosome paints may also be used but are usually applicable to metaphase preparations. The purified DNA can then be labeled and detected indirectly using haptens, or labeled directly using fluorochrome or dye-conjugated nucleotides. Labeling strategies are also variable, employing standard nick translation or PCR labeling methods. The target DNA can take the form of chromosomes spreads or interphase nuclei. The sources of interphase targets may come from cytogenetic preparations or from paraffin-embedded tissues. Both the labeled DNA probe and DNA target are denatured to a single-stranded state and permitted to hybridize to each other. Post-hybridization washes and fluorescently-labeled antibody incubations follow the 24-hour hybridization, and the specimen is ready for visualization by fluorescent microscopy. Successful interpretation of FISH experiments is dependent on the quality of the starting materials, hybridization efficiencies, and stringency of post-hybridization washes and antibody detections.
Topics: DNA Probes; Electronic Data Processing; Humans; In Situ Hybridization, Fluorescence; Models, Biological; Paraffin Embedding; Research Design; Staining and Labeling
PubMed: 18228455
DOI: 10.1002/0471143030.cb2204s23 -
Chemistry (Weinheim An Der Bergstrasse,... Aug 2023Accurate cancer diagnosis especially early diagnosis is of great importance for prompt therapy and elevated survival rate. mRNAs are widely used as biomarkers for cancer...
Accurate cancer diagnosis especially early diagnosis is of great importance for prompt therapy and elevated survival rate. mRNAs are widely used as biomarkers for cancer identification and treatment. mRNA expression levels are highly associated with cancer stage and malignant progression. Nevertheless, single type mRNA detection is insufficient and unreliable. Herein, we developed a DNA nano-windmill probe for in situ multiplexed mRNAs detection and imaging in this paper. The probe is designed to simultaneously target four types of mRNA through wind blades. Importantly, recognition of targets is independent from each other, which further facilitate cell type discrimination. The probe can specifically distinguish cancer cell lines from normal cells. In addition, it can identify changes in mRNA expression levels in living cells. The current strategy enriches the toolbox for improving the accuracy of cancer diagnosis and therapeutic solutions.
Topics: RNA, Messenger; DNA Probes; Cell Line, Tumor; DNA
PubMed: 37314386
DOI: 10.1002/chem.202301300 -
Analytical Sciences : the International... Feb 2022A novel and "turn-on" DNA-silver nanocluster probe (DNA/AgNCs) was developed that can detect the level of breast tumor-related mRNA markers: cyclin D1 mRNA. The...
A novel and "turn-on" DNA-silver nanocluster probe (DNA/AgNCs) was developed that can detect the level of breast tumor-related mRNA markers: cyclin D1 mRNA. The fluorescence of DNA/AgNCs probe rendered a "turn-on" response based on the secondary structure changes induced by the target strand. In the presence of target chain, the fluorescence enhances in a concentration-dependent manner, which enables the quantitative detection of analytes. By this method, a detection limit of 2.1 nM and a linear range of 15-125 nM were achieved. In addition, the probe displays excellent discrimination between a perfectly complementary target and single-base mismatch targets, providing an alternative approach for quick and specific recognition and quantification of mRNA or DNA.
Topics: Biosensing Techniques; DNA; DNA Probes; Humans; Limit of Detection; Metal Nanoparticles; Neoplasms; RNA, Messenger; Silver; Spectrometry, Fluorescence
PubMed: 35286639
DOI: 10.1007/s44211-022-00063-0 -
Bioelectrochemistry (Amsterdam,... Oct 2020Applications of molecular techniques to elucidate identity or function using biomarkers still remain highly empirical and biosensors are no exception. In the present...
Applications of molecular techniques to elucidate identity or function using biomarkers still remain highly empirical and biosensors are no exception. In the present study, target-specific oligonucleotide probes for E. coli K12 were designed thermodynamically and applied in an electrochemical DNA biosensor setup. Biosensor was prepared by immobilization of a stem-loop structured probe, modified with a thiol functional group at its 5' end and a biotin molecule at its 3' end, on a gold electrode through self-assembly. Mercaptopropionic acid (MPA) was used to optimize the surface probe density of the electrode. Hybridization between the immobilized probe and the target DNA was detected via the electrochemical response of streptavidin-horseradish peroxidase in the presence of the substrate. The amperometric response showed a linear relationship with the target DNA concentration, ranging from 10 and 400 nM, with a correlation coefficient of 0.989. High selectivity and good repeatability of the biosensor showed that the thermodynamic approach to oligonucleotide probe design can be used in development of electrochemical DNA biosensors.
Topics: Biosensing Techniques; Biotin; DNA Probes; Electrochemical Techniques; Horseradish Peroxidase; Limit of Detection; Propionates; Reproducibility of Results; Streptavidin; Thermodynamics
PubMed: 32442773
DOI: 10.1016/j.bioelechem.2020.107553 -
Angewandte Chemie (International Ed. in... Sep 2021Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has...
Threading intercalators bind DNA with high affinities. Here, we describe single-molecule studies on a cell-permeant luminescent dinuclear ruthenium(II) complex that has been previously shown to thread only into short, unstable duplex structures. Using optical tweezers and confocal microscopy, we show that this complex threads and locks into force-extended duplex DNA in a two-step mechanism. Detailed kinetic studies reveal that an individual stereoisomer of the complex exhibits the highest binding affinity reported for such a mono-intercalator. This stereoisomer better preserves the biophysical properties of DNA than the widely used SYTOX Orange. Interestingly, threading into torsionally constrained DNA decreases dramatically, but is rescued on negatively supercoiled DNA. Given the "light-switch" properties of this complex on binding DNA, it can be readily used as a long-lived luminescent label for duplex or negatively supercoiled DNA through a unique "load-and-lock" protocol.
Topics: Coordination Complexes; DNA; DNA Probes; Molecular Structure; Ruthenium
PubMed: 34378843
DOI: 10.1002/anie.202108077 -
Biosensors & Bioelectronics Jan 2017Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of...
Surface plasmon resonance (SPR) immunosensor using 4-mercaptobenzoic acid (4-MBA) modified gold (4-MBA/Au) SPR chip was developed first time for the detection of Brucella melitensis (B. melitensis) based on the screening of its complementary DNA target by using two different newly designed DNA probes of IS711 gene. Herein, interaction between DNA probes and target molecule are also investigated and result revealed that the interaction is spontaneous. The kinetics and thermodynamic results derived from the experimental data showed that the interaction between complementary DNA targets and probe 1 is more effective than that of probe 2. Equilibrium dissociation constant (K) and maximum binding capacity of analyte (B) values for the interaction of complementary DNA target with the immobilized DNA probes were calculated by using kinetic evaluation software, and found to be 15.3 pM (K) and 81.02m° (B) with probe 1 and 54.9pM and 55.29m° (B), respectively. Moreover, real serum samples analysis were also carried out using immobilized probe 1 and probe 2 with SPR which showed the applicability of this methodology and provides an alternative way for the detection of B. melitensis in less than 10min. This remarkable sensing response of present methodology offer a real time and label free detection of biological warfare agent and provide an opportunity to make miniaturized sensor, indicating considerable promise for diverse environmental, bio-defence, clinical diagnostics, food safety, water and security applications.
Topics: Benzoates; Brucella melitensis; Brucellosis; DNA Probes; DNA, Bacterial; Genes, Bacterial; Gold; Humans; Immobilized Nucleic Acids; Sulfhydryl Compounds; Surface Plasmon Resonance; Thermodynamics
PubMed: 27665519
DOI: 10.1016/j.bios.2016.09.063 -
Analytical Chemistry Feb 2017We have used temperature gradient surface plasmon resonance (SPR) measurements to quantitatively evaluate how the stability of different types of hybrids formed with DNA...
We have used temperature gradient surface plasmon resonance (SPR) measurements to quantitatively evaluate how the stability of different types of hybrids formed with DNA probes on surfaces is affected by probe spacing. SPR sensors with different average surface densities of probes were prepared by coadsorbing probes with lateral spacers strands comprised of phosphorothioated adenine nucleotides (A15*). Increasing the fraction of A15* spacers in the immobilization solution results in larger distances between probes on the sensor, determined here using a combination of SPR and X-ray photoelectron spectroscopy (XPS) measurements. The hybridization activities of probes were simultaneously measured over a temperature range that spanned the denaturation temperature (T) of hybrids by applying a spatial temperature gradient across the sensor surface. The resulting temperature profiles of hybridization activity show how the stability of hybrids increases as either the distance between probes or the ionic strength of the hybridization buffer increase. Additionally, hybridization activity profiles sharpen as the spacing between probes increases, indicating more homogeneous hybridization behavior of probes. The results provide quantitative experimental data for testing theoretical models of stability, supporting models that account for both repulsive interactions between DNA strands and local variability in probe surface density.
Topics: Base Pair Mismatch; DNA; DNA Probes; Nucleic Acid Hybridization; Photoelectron Spectroscopy; Surface Plasmon Resonance; Transition Temperature
PubMed: 28208255
DOI: 10.1021/acs.analchem.6b04048 -
The Journal of Physical Chemistry. B Sep 2019An efficient turn-on fluorescence probe for biomolecules not only helps in its sensitive detection but is also useful to understand the different interactions that are...
An efficient turn-on fluorescence probe for biomolecules not only helps in its sensitive detection but is also useful to understand the different interactions that are operating between biomolecules and probes. Polycyclic aromatic molecules are known to be strong interacting ligand for DNA and extensively studied as a model cancer drug. However, these molecules show large decrease in their emission intensity, i.e., a turn-off probe for DNA. In the present work, we have synthesized a benzothiazole-based anthracene derivative and studied its interaction with a natural DNA with the aim of having a turn-on DNA probe with a polycyclic aromatic moiety. Our detailed spectroscopic studies show that the new probe strongly interacts with DNA molecules and results in a significant increase in its emission yield. Time-resolved studies show a large increase in probe's excited-state lifetime in DNA solution. Detailed experiments have been performed to understand its mode of interaction with DNA molecules. The mode of interaction has also been supported by the blind molecular docking studies. Further, the viscosity-dependent photophysical properties and detailed quantum chemical calculations confirm that the new probe belongs to molecular rotor class of molecules. Association with DNA molecules results in a significant retardation in the nonradiative deactivation process due to the torsional motion in the excited state of the probe and leads to a significant increase in its emission yield. Thus, due its molecular rotor nature, despite with a turn-off fluorophore unit, anthracene, the new probe, acts as a sensitive turn-on fluorescence probe for DNA molecules.
Topics: Anthracenes; Benzothiazoles; DNA; DNA Probes; Density Functional Theory; Molecular Structure
PubMed: 31402670
DOI: 10.1021/acs.jpcb.9b05570