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BioTechniques Oct 2019Several approaches for miRNA expression analysis have been developed in recent years. In this article, we provide an updated and comprehensive review of available... (Review)
Review
Several approaches for miRNA expression analysis have been developed in recent years. In this article, we provide an updated and comprehensive review of available qPCR-based methods for miRNA expression analysis and discuss their advantages and disadvantages. Existing techniques involve the use of stem-loop reverse transcriptase-PCR, polyadenylation of RNAs, ligation of adapters or RT with complex primers, using universal or miRNA-specific qPCR primers and/or probes. Many of these methods are oriented towards the expression analysis of mature miRNAs and few are designed for the study of pre-miRNAs and pri-miRNAs. We also discuss findings from articles that compare results from existing methods. Finally, we suggest key points for the improvement of available techniques and for the future development of additional methods.
Topics: Computer Simulation; DNA Primers; Gene Expression; High-Throughput Nucleotide Sequencing; MicroRNAs; Polyadenylation; Real-Time Polymerase Chain Reaction; Software
PubMed: 31560239
DOI: 10.2144/btn-2019-0065 -
Proceedings of the National Academy of... Mar 2022Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current...
Real-time PCR is the most utilized nucleic acid testing tool in clinical settings. However, the number of targets detectable per reaction are restricted by current modes. Here, we describe a single-step, multiplex approach capable of detecting dozens of targets per reaction in a real-time PCR thermal cycler. The approach, termed MeltArray, utilizes the 5'-flap endonuclease activity of DNA polymerase to cleave a mediator probe into a mediator primer that can bind to a molecular beacon reporter, which allows for the extension of multiple mediator primers to produce a series of fluorescent hybrids of different melting temperatures unique to each target. Using multiple molecular beacon reporters labeled with different fluorophores, the overall number of targets is equal to the number of the reporters multiplied by that of mediator primers per reporter. The use of MeltArray was explored in various scenarios, including in a 20-plex assay that detects human Y chromosome microdeletions, a 62-plex assay that determines serovars, a 24-plex assay that simultaneously identifies and quantitates respiratory pathogens, and a minisequencing assay that identifies mutations, and all of these different assays were validated with clinical samples. MeltArray approach should find widespread use in clinical settings owing to its combined merits of multiplicity, versatility, simplicity, and accessibility.
Topics: Chromosome Deletion; Chromosomes, Human, Y; DNA Primers; Escherichia coli; Flap Endonucleases; Fluorescent Dyes; Humans; Limit of Detection; Multiplex Polymerase Chain Reaction; Taq Polymerase
PubMed: 35197282
DOI: 10.1073/pnas.2110672119 -
Brazilian Journal of Microbiology :... Jul 2019Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be...
Effective monitoring of Salmonella contamination in seafood processing to conform the requirements of HACCP is a great challenge today. Such challenges can be effectively addressed, if the conventional detection methods are replaced with DNA-based molecular methods. Accordingly, it was aimed to develop a robust PCR protocol for specific detection of Salmonella spp. Out of the different primers screened, one pair of primers developed in this study targeting invA gene demonstrated 100% inclusivity for a wide range of Salmonella serotypes and 100% exclusivity for wide range of non-target species. The in silico analysis of the nucleotide sequence obtained from the PCR product suggests its potential as a hybridization probe for genus specific detection of Salmonella spp. contamination. The PCR protocol was sensitive enough to detect 15 cells per reaction using crude DNA prepared within a short time directly from artificially contaminated shrimp tissue. The study demonstrated that the result of PCR reaction can come out on the same day of sample arrival. Incorporation of this pair of primers in a multiplex PCR designed for simultaneous detection of four common seafood-borne human pathogens yielded 147 bp, 302 bp, 403 bp, and 450 bp distinct DNA bands specifically targeting E. coli, toxigenic Vibrio cholerae, Salmonella spp., and V. parahaemolyticus, respectively in a single PCR tube. The PCR methods developed in this study has the potential to be used in the seafood processing plants for effective monitoring of CCPs required for implementation of HACCP-based quality assurance system.
Topics: Animals; DNA Primers; Food Contamination; Multiplex Polymerase Chain Reaction; Palaemonidae; Salmonella; Shellfish
PubMed: 31006836
DOI: 10.1007/s42770-019-00072-8 -
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 -
ACS Omega May 2022The strategies for nucleic acid sensing based on nucleic acid hybridization between the target sequence and the capture probe sequence are considered to be largely... (Review)
Review
The strategies for nucleic acid sensing based on nucleic acid hybridization between the target sequence and the capture probe sequence are considered to be largely successful as far as detection of a specific target of known sequence is concerned. However, when compared with other complementary methods, like direct sequencing, a number of results are still found to be either "false positives" or "false negatives". This suggests that modifications in these strategies are necessary to make them more accurate. In this minireview, we propose that one way toward improvement could be replacement of the DNA capture probes with the xeno nucleic acid or XNA capture probes. This is because the XNAs, especially the locked nucleic acid, the peptide nucleic acid, and the morpholino, have shown better single nucleobase mismatch discrimination capacity than the DNA capture probes, indicating their capacity for more precise detection of nucleic acid sequences, which is beneficial for detection of gene stretches having point mutations. Keeping the current trend in mind, this minireview will include the recent developments in nanoscale, fluorescent label-free applications, and present the cases where the XNA probes show clear advantages over the DNA probes.
PubMed: 35571783
DOI: 10.1021/acsomega.2c00581 -
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 -
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