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Analytical comparisons of SARS-COV-2 detection by qRT-PCR and ddPCR with multiple primer/probe sets.Emerging Microbes & Infections Dec 2020Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction...
Different primers/probes sets have been developed all over the world for the nucleic acid detection of SARS-CoV-2 by quantitative real time polymerase chain reaction (qRT-PCR) as a standard method. In our recent study, we explored the feasibility of droplet digital PCR (ddPCR) for clinical SARS-CoV-2 nucleic acid detection compared with qRT-PCR using the same primer/probe sets issued by Chinese Center for Disease Control and Prevention (CDC) targeting viral ORF1ab or N gene, which showed that ddPCR could largely minimize the false negatives reports resulted by qRT-PCR [Suo T, Liu X, Feng J, et al. ddPCR: a more sensitive and accurate tool for SARS-CoV-2 detection in low viral load specimens. medRxiv [Internet]. 2020;2020.02.29.20029439. Available from: https://medrxiv.org/content/early/2020/03/06/2020.02.29.20029439.abstract]. Here, we further stringently compared the performance of qRT-PCR and ddPCR for 8 primer/probe sets with the same clinical samples and conditions. Results showed that none of 8 primer/probe sets used in qRT-PCR could significantly distinguish true negatives and positives with low viral load (10 dilution). Moreover, false positive reports of qRT-PCR with UCDC-N1, N2 and CCDC-N primers/probes sets were observed. In contrast, ddPCR showed significantly better performance in general for low viral load samples compared to qRT-PCR. Remarkably, the background readouts of ddPCR are relatively lower, which could efficiently reduce the production of false positive reports.
Topics: Betacoronavirus; COVID-19; Coronavirus Infections; DNA Primers; DNA Probes; Humans; Multiplex Polymerase Chain Reaction; Pandemics; Pneumonia, Viral; Real-Time Polymerase Chain Reaction; SARS-CoV-2; Sensitivity and Specificity; Viral Load
PubMed: 32448084
DOI: 10.1080/22221751.2020.1772679 -
Methods in Molecular Biology (Clifton,... 2022High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to...
High-throughput DNA fluorescence in situ hybridization (hiFISH) combines multicolor combinatorial DNA FISH staining with automated image acquisition and analysis to visualize and localize tens to hundreds of genomic loci in up to millions of cells. hiFISH can be used to measure physical distances between pairs of genomic loci, radial distances from genomic loci to the nuclear edge or center, and distances between genomic loci and nuclear structures defined by protein or RNA markers. The resulting large datasets of 3D spatial distances can be used to study cellular heterogeneity in genome architecture and the molecular mechanisms underlying this phenomenon in a variety of cellular systems. In this chapter we provide detailed protocols for hiFISH to measure distances between genomic loci, including all steps involved in DNA FISH probe design and preparation, cell culture, DNA FISH staining in 384-well imaging plates, automated image acquisition and analysis, and, finally, statistical analysis.
Topics: Cell Nucleus; DNA; DNA Probes; Genome; In Situ Hybridization, Fluorescence
PubMed: 35867253
DOI: 10.1007/978-1-0716-2497-5_12 -
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 -
Chembiochem : a European Journal of... Apr 2021RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA... (Review)
Review
RNA molecules can fold into complex two- and three-dimensional shapes that are critical for their function. Chemical probes have long been utilized to interrogate RNA structure and are now considered invaluable resources in the goal of relating structure to function. Recently, the power of deep sequencing and careful chemical probe design have merged, permitting researchers to obtain a holistic understanding of how RNA structure can be utilized to control RNA biology transcriptome-wide. Within this review, we outline the recent advancements in chemical probe design for interrogating RNA structures inside cells and discuss the recent advances in our understanding of RNA biology through the lens of chemical probing.
Topics: DNA Adducts; DNA, Complementary; Molecular Probes; Nucleic Acid Conformation; RNA; RNA, Messenger; Transcriptome
PubMed: 32737940
DOI: 10.1002/cbic.202000340 -
Journal of Visualized Experiments : JoVE Feb 2022Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a...
Current single-cell epigenome analyses are designed for single use. The cell is discarded after a single use, preventing analysis of multiple epigenetic marks in a single cell and requiring data from other cells to distinguish signal from experimental background noise in a single cell. This paper describes a method to reuse the same single cell for iterative epigenomic analyses. In this experimental method, cellular proteins are first anchored to a polyacrylamide polymer instead of crosslinking them to protein and DNA, alleviating structural bias. This critical step allows repeated experiments with the same single cell. Next, a random primer with a scaffold sequence for proximity ligation is annealed to the genomic DNA, and the genomic sequence is added to the primer by extension using a DNA polymerase. Subsequently, an antibody against an epigenetic marker and control IgG, each labeled with different DNA probes, are bound to the respective targets in the same single cell. Proximity ligation is induced between the random primer and the antibody by adding a connector DNA with complementary sequences to the scaffold sequence of the random primer and the antibody-DNA probe. This approach integrates antibody information and nearby genome sequences in a single DNA product of proximity ligation. By enabling repeated experiments with the same single cell, this method allows an increase in data density from a rare cell and statistical analysis using only IgG and antibody data from the same cell. The reusable single cells prepared by this method can be stored for at least a few months and reused later to broaden epigenetic characterization and increase data density. This method provides flexibility to researchers and their projects.
Topics: DNA; DNA Probes; DNA-Directed DNA Polymerase; Epigenome; Epigenomics
PubMed: 35225292
DOI: 10.3791/63456 -
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 -
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 -
Proceedings of the National Academy of... Aug 2023Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical...
Electronic detection of DNA oligomers offers the promise of rapid, miniaturized DNA analysis across various biotechnological applications. However, known all-electrical methods, which solely rely on measuring electrical signals in transducers during probe-target DNA hybridization, are prone to nonspecific electrostatic and electrochemical interactions, subsequently limiting their specificity and detection limit. Here, we demonstrate a nanomechanoelectrical approach that delivers ultra-robust specificity and a 100-fold improvement in detection limit. We drive nanostructural DNA strands tethered to a graphene transistor to oscillate in an alternating electric field and show that the transistor-current spectra are characteristic and indicative of DNA hybridization. We find that the inherent difference in pliability between unpaired and paired DNA strands leads to the spectral characteristics with minimal influence from nonspecific electrostatic and electrochemical interactions, resulting in high selectivity and sensitivity. Our results highlight the potential of high-performance DNA analysis based on miniaturized all-electronic settings.
Topics: DNA; Nucleic Acid Hybridization; DNA Probes; Graphite; Hybridization, Genetic; Biosensing Techniques
PubMed: 37549255
DOI: 10.1073/pnas.2306130120 -
Small (Weinheim An Der Bergstrasse,... Jun 2019The ease of tailoring DNA nanostructures with sub-nanometer precision has enabled new and exciting in vivo applications in the areas of chemical sensing, imaging, and... (Review)
Review
The ease of tailoring DNA nanostructures with sub-nanometer precision has enabled new and exciting in vivo applications in the areas of chemical sensing, imaging, and gene regulation. A new emerging paradigm in the field is that DNA nanostructures can be engineered to study molecular mechanics. This new development has transformed the repertoire of capabilities enabled by DNA to include detection of molecular forces in living cells and elucidating the fundamental mechanisms of mechanotransduction. This Review first describes fundamental aspects of force-induced melting of DNA hairpins and duplexes. This is then followed by a survey of the currently available force sensing DNA probes and different fluorescence-based force readout modes. Throughout the Review, applications of these probes in studying immune receptor signaling, including the T cell receptor and B cell receptor, as well as Notch and integrin signaling, are discussed.
Topics: Animals; DNA; DNA Probes; Humans; Mechanotransduction, Cellular; Nanostructures; Nanotechnology
PubMed: 31069945
DOI: 10.1002/smll.201900961 -
Nature Protocols Mar 2021Chromatin conformation capture (3C) methods and fluorescent in situ hybridization (FISH) microscopy have been used to investigate the spatial organization of the genome....
Chromatin conformation capture (3C) methods and fluorescent in situ hybridization (FISH) microscopy have been used to investigate the spatial organization of the genome. Although powerful, both techniques have limitations. Hi-C is challenging for low cell numbers and requires very deep sequencing to achieve its high resolution. In contrast, FISH can be done on small cell numbers and capture rare cell populations, but typically targets pairs of loci at a lower resolution. Here we detail a protocol for optical reconstruction of chromatin architecture (ORCA), a microscopy approach to trace the 3D DNA path within the nuclei of fixed tissues and cultured cells with a genomic resolution as fine as 2 kb and a throughput of ~10,000 cells per experiment. ORCA can identify structural features with comparable resolution to Hi-C while providing single-cell resolution and multimodal measurements characteristic of microscopy. We describe how to use this DNA labeling in parallel with multiplexed labeling of dozens of RNAs to relate chromatin structure and gene expression in the same cells. Oligopaint probe design, primary probe making, sample collection, cryosectioning and RNA/DNA primary probe hybridization can be completed in 1.5 weeks, while automated RNA/DNA barcode hybridization and RNA/DNA imaging typically takes 2-6 d for data collection and 2-7 d for the automated steps of image analysis.
Topics: Cell Line; Cell Nucleus; Cells, Cultured; Chromatin; Chromatin Immunoprecipitation; Chromosomes; DNA; DNA Probes; Fluorescent Dyes; Genetic Techniques; Genome; Genomics; Humans; Image Processing, Computer-Assisted; In Situ Hybridization, Fluorescence; Microscopy, Fluorescence; Optical Restriction Mapping; RNA
PubMed: 33619390
DOI: 10.1038/s41596-020-00478-x