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Methods in Molecular Biology (Clifton,... 2016Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in...
Quantitative real-time polymerase chain reaction is a technique for simultaneous amplification and product quantification of a target DNA as the process takes place in real time in a "closed-tube" system. Although this technique can provide an absolute quantification of the initial template copy number, quantification relative to a control sample or second sequence is typically adequate. The quantification process employs melting curve analysis and/or fluorescent detection systems and can provide amplification and genotyping in a relatively short time. Here we describe the properties and uses of various fluorescent detection systems used for quantification.
Topics: Animals; DNA Primers; DNA Probes; Humans; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity
PubMed: 26843055
DOI: 10.1007/978-1-4939-3360-0_15 -
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 -
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 -
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 Chemistry Nov 2022The investigation on electrochemiluminescence (ECL) multiplexing bioassays mainly focuses on simultaneously detecting either proteins or nucleic acids. To overcome the...
The investigation on electrochemiluminescence (ECL) multiplexing bioassays mainly focuses on simultaneously detecting either proteins or nucleic acids. To overcome the limitation of a short waveband for spectrum-resolved ECL multiplexing bioassays, herein, a highly monochromatic (FWHM <40 nm) and bandgap-engineered ECL luminophore, that is, mercaptopropionic acid-capped and Zn-mediated aggregation-induced emission (AIE) assembly of Au nanocrystals (NCs) (Zn-AIE-AuNCs), of strong emission and the maximum emission wavelength at 485 nm is developed. The highly monochromatic and bandgap-engineered ECL (485 nm) of Zn-AIE-AuNCs can multiplex with the single-waveband and surface-defect-involved ECL (775 nm) of dual-stabilizer-capped CuInS@ZnS NCs (CIS@ZnS-NCs), enabling a spectrum-resolved ECL multiplexing strategy with different NCs luminophores of a similar particle size as tags. This ECL multiplexing strategy can be utilized to simultaneously detect antigen and DNA probe together without any additional signal amplification procedure and obvious spectroscopic cross-talk, in which the highly monochromatic ECL from Zn-AIE-AuNCs is utilized to dynamically determine human carcinoembryonic antigen from 1 pg/mL to 50 ng/mL with a limit of detection (LOD) of 0.3 pg/mL, while the single-waveband ECL from CIS@ZnS-NCs is employed to linearly detect wild-type p53 from 1 pM to 50 nM with a LOD of 0.5 pM. The ECL immunoassay of the proposed strategy is free from the interference of the synchronously conducted DNA probe assay and vice versa, which would open an avenue to couple the immunoassay and DNA probe assay together for clinical colon and breast cancer identification.
Topics: Humans; Luminescent Measurements; Electrochemical Techniques; Immunoassay; Limit of Detection; DNA Probes; Biological Assay; Biosensing Techniques
PubMed: 36334096
DOI: 10.1021/acs.analchem.2c03579 -
RNA (New York, N.Y.) Dec 2018Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes....
Northern blot analysis detects RNA molecules immobilized on nylon membranes through hybridization with radioactive P-labeled DNA or RNA oligonucleotide probes. Alternatively, nonradioactive northern blot relies on chemiluminescent reactions triggered by horseradish peroxidase (HRP) conjugated probes. The use of regulated radioactive material and the complexity of chemiluminescent reactions and detection have hampered the adoption of northern blot techniques by the wider biomedical research community. Here, we describe a sensitive and straightforward nonradioactive northern blot method, which utilizes near-infrared (IR) fluorescent dye-labeled probes (irNorthern). We found that irNorthern has a detection limit of ∼0.05 femtomoles (fmol), which is slightly less sensitive than P-Northern. However, we found that the IR dye-labeled probe maintains the sensitivity after multiple usages as well as long-term storage. We also present alternative irNorthern methods using a biotinylated DNA probe, a DNA probe labeled by terminal transferase, or an RNA probe labeled during in vitro transcription. Furthermore, utilization of different IR dyes allows multiplex detection of different RNA species. Therefore, irNorthern represents a more convenient and versatile tool for RNA detection compared to traditional northern blot analysis.
Topics: Blotting, Northern; DNA Probes; Fluorescent Dyes; Nucleic Acid Hybridization; RNA; RNA Probes
PubMed: 30201850
DOI: 10.1261/rna.068213.118 -
The Journal of Membrane Biology Dec 2020Continuous, dynamic, and controlled membrane remodeling creates flow of information and materials across membranes to sustain life in all biological systems. Multiple... (Review)
Review
Continuous, dynamic, and controlled membrane remodeling creates flow of information and materials across membranes to sustain life in all biological systems. Multiple nanoscale phenomena of membranes regulate mesoscale processes in cells, which in turn control macro-scale processes in living organisms. Understanding the molecular mechanisms that cells use for membrane homeostasis, i.e., to generate, maintain, and deform the membrane structures has therefore been the mammoth's task in biology. Using the principles of DNA nanotechnology, researchers can now precisely recapitulate the functional interactions of the biomolecules that can now probe, program, and re-program membrane remodeling and associated phenomena. The molecular mechanisms for membrane dynamics developing in vitro conditions in which the membrane modulating components are precisely organized and modulated by DNA nanoscaffolds are adding new chapters in the field of DNA nanotechnology. In this review, we discuss DNA nanodevices-based membrane remodeling and trafficking machineries and their applications in biological systems.
Topics: Cell Membrane; DNA; DNA Probes; Molecular Dynamics Simulation; Nanostructures; Nanotechnology
PubMed: 33200237
DOI: 10.1007/s00232-020-00154-x -
Methods (San Diego, Calif.) Apr 2016In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid... (Review)
Review
In situ hybridization is the technique by which specific RNA or DNA molecules are detected in cytological preparations. Basically it involves formation of a hybrid molecule between an endogenous single-stranded RNA or DNA in the cell and a complementary single-stranded RNA or DNA probe. In its original form the probe was labeled with (3)H and the hybrid was detected by autoradiography. The first successful experiments in 1968 involved detection of the highly amplified ribosomal DNA in oocytes of the frog Xenopus, followed soon after by the reiterated "satellite DNA" in mouse and Drosophila chromosomes. Fluorescent probes were developed about ten years later.
Topics: Animals; Autoradiography; DNA; DNA Probes; Drosophila melanogaster; Fluorescent Dyes; History, 20th Century; History, 21st Century; In Situ Hybridization; Larva; Mice; Oocytes; Polytene Chromosomes; RNA; Salivary Glands; Tritium; Xenopus laevis
PubMed: 26655524
DOI: 10.1016/j.ymeth.2015.11.026 -
Analytical Methods : Advancing Methods... Dec 2022Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing...
Herein, a new allosteric DNA switch-mediated catalytic DNA circuit reaction strategy has been proposed for ratiometric and sensitive nucleic acid detection. The sensing system was based on two DNA hybrid probes, each of which was constructed by annealing a reconfigurable DNA hairpin with single-stranded DNA. Upon target recognition by the first DNA hybrid probe, a reconfigurable DNA switch was liberated, triggering a toehold-mediated strand displacement reaction (TSDR) with the second DNA hybrid probe, which was accompanied by the release of another reconfigurable DNA switch. This released allosteric DNA switch could further interact with the first hybrid DNA probe the TSDR strategy to form a reciprocal strand displacement network between the two DNA hybrid probes. Theoretically, this reciprocal strand displacement reaction would continue till the complete consumption of the reaction substrates. Thus, it provides a new signal amplification method leading toward target recognition. More interestingly, it creates a ratiometric signal response mode for target recognition, which involves the fluorescence increment of one fluorophore (Cy5) and concurrent decrement of another fluorophore (Cy3) accompanied by the target-triggered reciprocal strand displacement reaction. This process could achieve a low detection limit of about 0.1 pM toward the target nucleic acid and selective discrimination toward different mismatched targets. It could also be applied for detection in a serum sample. Thus, the developed catalytic DNA circuit reaction strategy together with ratiometric signal readout provides a new avenue for programmable, reliable and sensitive detection of nucleic acids and might also pave the way for developing more advanced DNA circuits or biosensors.
Topics: DNA, Catalytic; Nucleic Acid Hybridization; Limit of Detection; DNA; DNA Probes
PubMed: 36504112
DOI: 10.1039/d2ay01751b -
Analytical Chemistry Dec 2021Methods for producing DNA SAM-based sensors with improved thermal stability and control over the homogeneity of low DNA probe density will enable advanced sensor...
Methods for producing DNA SAM-based sensors with improved thermal stability and control over the homogeneity of low DNA probe density will enable advanced sensor development. The thermal stability of low-coverage DNA SAMs was studied for surfaces prepared using potential-assisted thiol exchange (E) and compared to DNA SAMs prepared without control over the substrate potential (OCP). Both surface preparation methods were studied using in situ fluorescence microscopy and electrochemistry with fluorophore or redox-modified DNA SAMs on a single-crystal gold bead electrode. Fluorescence microscopy showed that the influence of the underlying surface crystallography was important in both cases. The highest thermal stability was realized for square or rectangular surface atomic structure (e.g., surfaces from 110 to 100). The 111 and related surfaces were the least thermally stable. The low DNA coverage surfaces prepared by E had better thermal stability and higher DNA probe mobility as compared to OCP-prepared surfaces with the similar coverage. These results were correlated with methylene blue redox-tagged DNA probes, which directly measured the average DNA coverage. Both methods indicated that E DNA SAMs were more uniformly distributed across the electrode surface, while the surfaces prepared via OCP assembled into clusters with reduced mobility. The potential-assisted thiol-exchange approach to preparing low-coverage DNA SAMs was shown to quickly create modified surfaces that were consistent, had mobility characteristics which should yield superior DNA hybridization efficiencies, and having greater thermal stability which will translate into a longer shelf-life.
Topics: DNA; DNA Probes; Gold; Sulfhydryl Compounds; Surface Properties
PubMed: 34813297
DOI: 10.1021/acs.analchem.1c03353