Dye-labeled DNA Probe Research Articles

Development of a multiplex one-tube loop-mediated isothermal amplification method for rapidly simultaneous detection of the mecA and nuc genes in methicillin-resistant Staphylococcus aureus

This study developed a multiplex one-tube loop-mediated isothermal amplification method using fluorescent dye-labeled DNA probes (mfLAMP) for the rapid simultaneous detection of the nuc (S. aureus-specific marker) and mecA genes (PBP2a-specific marker) in methicillin-resistant Staphylococcus aureus (MRSA). In the mfLAMP method, a graphene oxide (GO) was used as an effective adsorbent to eliminate free fluorescent dye-labeled DNA probes (mecAf-probe: Cy5-ssDNA-Biotin and nucf-probe: FAM-ssDNA-Biotin) present in negative results. The samples were then measured using a developed program that enable simultaneous detection of fluorescein (FAM) and Cyanine5 (Cy5). Green fluorescence represents the detection of the nuc gene, red fluorescence indicates the detection of the mecA gene, and yellow fluorescence signifies the detection of MRSA. The mfLAMP was specific to MRSA and could detect 10°CFU/mL of MRSA in standard buffer and artificially inoculated irrigation water. The recovery rate from contaminated irrigation water ranged from 100.41 to 109.85 %. This study is the first to report the mfLAMP using GO, which could be point-of-care testing for the rapid and simultaneous detection of MRSA's nuc and mecA genes. The results demonstrate that the mfLAMP will be a novel tool for simultaneously detecting more than two target genes or bacteria within 1 h without an enrichment step.

2D MOF Nanosensor-Integrated Digital Droplet Microfluidic Flow Cytometry for In Situ Detection of Multiple miRNAs in Single CTC Cells.

Current circulating tumor cells (CTCs) detection strategies based on surface epithelial markers suffer from low specificity in distinguishing between CTCs and epithelial cells in hematopoietic cell population. Tumor-associated miRNAs within CTCs are emerging as new biomarkers due to their high correlation with tumor development and progress. However, in-situ simultaneous analysis of multiple miRNAs in single CTC cell is still challenging. To overcome this limitation, a digital droplet microfluidic flow cytometry based on biofunctionalized 2D metal-organic framework nanosensor (Nano-DMFC) is developed for in situ detection of dual miRNAs simultaneously in single living breast cancer cells. Here, 2D MOF-based fluorescent resonance energy transfer (FRET) nanosensors are established by conjugating dual-color fluorescence dye-labeled DNA probes on MOF nanosheet surface. In the Nano-DMFC, 2D MOF-based nanoprobes are precisely microinjected into each single-cell encapsulated droplets to achieve dual miRNA characterization in single cancer cell. This Nano-DMFC platform successfully detects dual miRNAs at single-cell resolution in 10 mixed positive MCF-7 cells out of 10 000 negative epithelial cells in serum biomimic samples. Moreover, this Nano-DMFC platform shows good reproductivity in the recovery experiment of spiked blood samples, which demonstrate the high potential for CTC-based cancer early diagnosis and prognosis.

Fluorescence turn-on detection of miRNA-155 based on hybrid Ce-MOF/ PtNPs /graphene oxide serving as fluorescence quencher

Rapid, cost-effective, and specific detection of miRNAs is valuable for clinical diagnosis, pathogenesis, and various medical therapies. In this work, a simple and enzyme-free fluorescence probe was constructed for sensitive detection of miRNA-155 by using Ce-MOF @Pt NP@GO as a novel quencher. Our method is based on monitoring the fluorescence of cyanine (Cy5) in Cy5-tagged DNA probe in the presence of its specific target miRNA. Quenched dye labeled DNA probes are released from Ce-MOF@Pt NP@GO after the miRNA target hybridization. This is accompanied by the significant enhancement of fluorescence emission at 670 nm. Accuracy, sensitivity of the method can be affected by hybridization between the probe and target. This developed method offers a relatively rapid, sensitive, and inexpensive platform without enzyme or separation procedures toward the detection of miRNA155. Under optimal conditions, a detection limit of 5 pMand a lineardetection range of 0.01–2.5 nM were obtained for miRNA-155 determination. Meanwhile, thisapproach showed acceptable accuracy and precision for detecting miRNA in seurm samples.

Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor.

Single-molecule measurements provide detailed mechanistic insights into molecular processes, for example in genome regulation where DNA access is controlled by nucleosomes and the chromatin machinery. However, real-time single-molecule observations of nuclear factors acting on defined chromatin substrates are challenging to perform quantitatively and reproducibly. Here we present XSCAN (multiplexed single-molecule detection of chromatin association), a method to parallelize single-molecule experiments by simultaneous imaging of a nucleosome library, where each nucleosome type carries an identifiable DNA sequence within its nucleosomal DNA. Parallel experiments are subsequently spatially decoded, via the detection of specific binding of dye-labeled DNA probes. We use this method to reveal how the Cas9 nuclease overcomes the nucleosome barrier when invading chromatinized DNA as a function of PAM position.

A RuO2 Nanosheet as a Novel Quencher-free Platform for the Detection of Nucleic Acids in a Homogeneous Solution.

A fluorescent dye-labeled DNA probe was adsorbed and quenched on the monolayer of RuO2 nanosheets. Significant fluorescent recovery was observed upon the addition of complementary DNA due to desorption of the probe from the surface of the RuO2 nanosheet through duplex formation. The efficiency of fluorescence recovery was higher than that for graphene oxide, which was known as a quencher-free platform for the detection of nucleic acids in a homogeneous solution.

Exonuclease III-Regulated Target Cyclic Amplification-Based Single Nucleotide Polymorphism Detection Using Ultrathin Ternary Chalcogenide Nanosheets

Herein, we report that the ternary chalcogenide nanosheet exhibits different affinity toward oligonucleotides with different lengths and efficiently quenches the fluorescence of dye-labeled DNA probes. Based on these findings, as a proof-of-concept application, the ternary chalcogenide nanosheet is used as a target cyclic amplification biosensor, showing high specificity in discriminating single-base mismatch. This simple strategy is fast and sensitive for the single nucleotide polymorphism detection. Ultralow detection limit of unlabeled target (250 fM) and high discrimination ratio (5%) in the mixture of perfect match (mutant-type) and single-base mismatch (wild-type) target are achieved. This sensing method is extensively compatible for the single nucleotide polymorphism detection in clinical samples, making it a promising tool for the mutation-based clinical diagnostic and genomic research.

Scanning Electron Microscopic Aids for Identification of Larval and Post-Larval Bivalves

The identification of bivalve larvae and early postlarvae in plankton and benthic samples has long been a challenge, hampering both basic and applied research efforts in marine, estuarine, and freshwater environments. The usefulness of published optical micrographs of the early life-history stages of bivalves is limited because of the great morphological similarity of the imaged articulated shells, particularly at the early (straight-hinge) developmental stages. While a number of techniques have been refined in recent years and show promise for use in routine identifications of larval and post-larval bivalves (e.g., single-step nested multiplex polymerase chain reaction; in situ hybridization protocols through color coding with taxon-specific, dye-labeled DNA probes; coupled fluorescence in situ hybridization and cell sorting; and image analysis techniques using species-specific shell birefringence patterns under polarized light), no adequate comprehensive reference source exists that accurately depicts the morphology and morphometry of the shells of larval and post-larval stages of target bivalve species in a consistent format to assist in identification of such stages. To this end, scanning electron micrograph (SEM) sequences are presented of the disarticulated shell valves of laboratory-reared larval and post-larval stages of 56 species of bivalve molluscs from a wide spectrum of marine, estuarine, and freshwater habitats. Emphasis is placed on the usefulness of the morphology and morphometrics of consistently-oriented, disarticulated shell valves and associated hinge structures in discriminating the early life-history stages of these various bivalve species. Although the scanning electron micrograph sequences presented accurately depict the gross morphologies/ morphometrics and hinge structures of the disarticulated shell valves of the larvae and/or postlarvae of the 56 species of bivalves, it is important to emphasize that a scanning electron microscope is not necessary to observe even fine hinge structures associated with the early ontogenetic stages of these species. Such structures are readily visible using a wide range of optical compound microscopes equipped with high-intensity reflected light sources, although the disarticulated shell valves must be viewed in several planes of focus to discern the often subtle details seen clearly in the scanning electron micrographs. These morphological characters provide researchers with invaluable aids for the routine identification of the early life-history stages of these species isolated from plankton and benthic samples.

Quantitative super-resolution imaging with qPAINT.

Current super-resolution techniques offer unprecedented spatial resolution, but quantitative counting of spatially unresolvable molecules remains challenging. Here, we use the programmable and specific binding of dye-labeled DNA probes to count integer numbers of targets. This method, called quantitative Points Accumulation In Nanoscale Topography (qPAINT), avoids the challenging task of analyzing the environmentally sensitive hard-to-predict photophysics of dyes, and enables robust counting by analyzing the predictable binding kinetics of dye-labeled DNA probes. We benchmarked qPAINT in vitro and in situ by counting strands on DNA nanostructures, Nup98 protein clusters in the nuclear pore complex, Bruchpilot proteins in Drosophila, and finally the number of fluorescence in situ hybridization probes on single mRNA targets in fixed cells. We achieved high accuracy (~98–99 %), high precision (~80–95 %), and multiplexed detection over a large dynamic range.

Label-free and sensitive detection of T4 polynucleotide kinase activity via coupling DNA strand displacement reaction with enzymatic-aided amplification.

Several fluorescence signal amplification strategies have been developed for sensitive detection of T4 polynucleotide kinase (T4 PNK) activity, but they need fluorescence dye labeled DNA probe. We have addressed the limitation and report here a label-free strategy for sensitive detection of PNK activity by coupling DNA strand displacement reaction with enzymatic-aided amplification. A hairpin oligonucleotide (hpDNA) with blunt ends was used as the substrate for T4 PNK phosphorylation. In the presence of T4 PNK, the stem of hpDNA was phosphorylated and further degraded by lambda exonuclease (λ exo) from 5' to 3' direction to release a single-stranded DNA as a trigger of DNA strand displacement reaction (SDR). The trigger DNA can continuously displace DNA P2 from P1/P2 hybrid with the help of specific cleavage of nicking endonuclease (Nt.BbvCI). Then, DNA P2 can form G-quadruplex in the presence of potassium ions and quadruplex-selective fluorphore, N-methyl mesoporphyrin IX (NMM), resulting in a significant increase in fluorescence intensity of NMM. Thus, the accumulative release of DNA P2 led to fluorescence signal amplification for determining T4 PNK activity with a detection limit of 6.6×10(-4) U/mL, which is superior or comparative with established approaches. By ingeniously utilizing T4 PNK-triggered DNA SDR, T4 PNK activity can be specifically and facilely studied in homogeneous solution containing complex matrix without any external fluorescence labeling. Moreover, the influence of different inhibitors on the T4 PNK activity revealed that it also can be explored to screen T4 PNK inhibitors. Therefore, this label-free amplification strategy presents a facile and cost-effective approach for nucleic acid phosphorylation related research.

Target-Induced Conjunction of Split Aptamer Fragments and Assembly with a Water-Soluble Conjugated Polymer for Improved Protein Detection

Rapid and sensitive detection of proteins is crucial to biomedical research as well as clinical diagnosis. However, so far, most detection methods rely on antibody-based assays and are usually laborious and time-consuming, with poor sensitivity. Herein, we developed a simple and sensitive fluorescence-based strategy for protein detection by using split aptamer fragments and a water-soluble polycationic polymer (poly{[9,9-bis(6'-(N,N,N-diethylmethylammonium)hexyl)-2,7-fluorenylene ethynylene]-alt-co-[2,5-bis(3'-(N,N,N-diethylmethylammonium)-1'-oxapropyl)-1,4-phenylene] tetraiodide} (PFEP)). The thrombin-binding DNA aptamer was split into two fragments for target recognition. The PFEP with high fluorescence emission was used as energy donor to amplify the signal of dye-labeled DNA probe. In the absence of target, three DNA/PFEP complexes were formed via strong electrostatic interactions, resulting in efficient Föster resonance energy transfer (FRET) between two fluorophores. While the presence of target induces a conjunction of two split aptamer fragments to form G-quadruplex, and subsequent assemble with PFEP leading to the formation of G-quadruplex/thrombin/PFEP complex. The distance between the PFEP and dye increased due to protein's large size, leading to a remarkable decrease of the FRET signal. Compared with the intact aptamer, the use of shorter split aptamer fragments increases the possibility of forming G-quadruplex upon target. Thus, the rate of change of FRET signal before and after the addition of target improved significantly and a higher sensitivity (limit of detection (LOD) = 2 nM) was obtained. This strategy is superior in that it is rapid, has low cost and homogeneous detection, and does not need heating to avoid an unfavorable secondary structure of DNA probe. With further efforts, this method could be extended to a universal way for simple and sensitive detection of a variety of biomolecules.

Carbon nanotube signal amplification for ultrasensitive fluorescence polarization detection of DNA methyltransferase activity and inhibition

A versatile sensing platform based on multiwalled carbon nanotube (MWCNT) signal amplification and fluorescence polarization (FP) is developed for the simple and ultrasensitive monitoring of DNA methyltransferase (MTase) activity and inhibition in homogeneous solution. This method uses a dye-labeled DNA probe that possess a doubled-stranded DNA (dsDNA) part for Mtase and its corresponding restriction endonuclease recognition, and a single-stranded DNA part for binding MWCNTs. In the absence of MTase, the dye-labeled DNA is cleaved by restriction endonuclease, and releases very short DNA carrying the dye that cannot bind to MWCNTs, which has relatively small FP value. However, in the presence of MTase, the specific recognition sequence in the dye-labeled DNA probe is methylated and not cleaved by restriction endonuclease. Thus, the dye-labeled methylated DNA product is adsorbed onto MWCNTs via strong π–π stacking interactions, which leads to a significant increase in the FP value due to the enlargement of the molecular volume of the dye-labeled methylated DNA/MWCNTs complex. This provides the basic of a quantitative measurement of MTase activity. By using the MWCNT signal amplification approach, the detection sensitivity can be significantly improved by two orders of magnitude over the previously reported methods. Moreover, this method also has high specificity and a wide dynamic range of over five orders of magnitude. Additionally, the suitability of this sensing platform for MTase inhibitor screening has also been demonstrated. This approach may serve as a general detection platform for sensitive assay of a variety of DNA MTases and screening potential drugs.

High-Resolution Epifluorescence and Time-of-Flight Secondary Ion Mass Spectrometry Chemical Imaging Comparisons of Single DNA Microarray Spots

DNA microarray assay performance is commonly compromised by spot-spot probe and signal variations as well as heterogeneity within printed microspots. Accurate metrics for captured DNA target signal rely upon uniform spot distribution of both probe and target DNA to yield reliable hybridized signal. While often presumed, this is neither easily achieved nor often proven experimentally. High-resolution imaging techniques were used to determine spot heterogeneity in identical DNA array microspots comprising varied ratios of unlabeled and dye-labeled DNA probes contact-printed onto commercial arraying surfaces. Epifluorescence imaging data for individual array microspots were correlated with time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical state imaging of the same spots. Epifluorescence imaging intensity distinguished varying DNA density distributed both within a given spot and from spot to spot. TOF-SIMS chemical analysis confirmed these heterogeneous printed DNA distributions by tracking bound Cy3 dye, DNA base, and phosphate specific ion fragments often correlating to fluorescence patterns within identical spots. TOF-SIMS ion fragments originating from probe DNA and Cy3 dye are enriched in microspot centers, correlating with high fluorescence intensity regions. Both TOF-SIMS and epifluorescence support Marangoni flow effects on spot drying, with high-density DNA-Cy3 located in spot centers and nonhomogeneous DNA distribution within printed spots. Microspot image dimensional analysis results for DNA droplet spreading show differing DNA densities across printed spots. The study directly supports different DNA probe chemical and spatial microenvironments within spots that yield spot-spot signal variations known to affect DNA target hybridization efficiencies and kinetics. These variations critically affect probe-target duplex formation and DNA array signal generation.

Comparative microRNA detection from precursor‐microRNA‐transfected hepatocellular carcinoma cells by capillary electrophoresis with dual‐color laser‐induced fluorescence

A dual-LIF (dLIF) setup combined with CE for microRNA (miRNA) detection is proposed in this study. An argon ion laser (488 nm) and a solid state laser (640 nm) were chosen to excite the fluorescent dye-labeled DNA probe after splinted ligation of miRNA. The crosstalk of emission spectrum of Alex Fluor 488 and Alex Fluor 647 is minimized with a zero crosstalk matrix for Alex Fluor 647 to 488 channels. The linear ranges of the device for the fluorescent dye-labeled DNA probe were both from 1.0 nM to 0.1 pM. The limits of detection for Alexa Fluor 488-labeled DNA and Alex Fluor 647-labeled DNA were 9.3 and 31 fM, respectively. The detection of specific miRNA has been accomplished by combining splinted ligation with the fluorescent dye-labeled oligonucleotides. The linear range for the synthetic miRNA is from 1.0 nM to 1.0 pM. Without PCR amplification, CE-dLIF was applied to discriminate a pre-miR-10b*-transfected cells (contains precursor miR-10b*) from hepatocellular carcinoma cell (control cells). Therefore, this result indicates CE-dLIF has great potential to provide a rapid comparative assay for miRNAs detection.

Direct energy transfer from conjugated polymer to DNA intercalated dye: Label-free fluorescent DNA detection

The development of methods for DNA detection is of importance in disease diagnosis, gene-targeted drug discovery and molecular biology field. In this paper, we synthesize a new cationic water-soluble CP containing fluorene moiety and flexible ethylenic moiety in the backbone (PFV) for label-free DNA detection. The conformational freedom of PFV provides stronger interactions with double-stranded DNA (dsDNA) and optimizes the orientation of transition moments between PFV and ethidium bromide (EB) intercalated in dsDNA. The efficient FRET from PFV (donor) to EB (acceptor) intercalated in dsDNA is observed and the emission of EB is amplified by the good light-harvesting ability of conjugated polymers. The interactions between PFV and DNA can also be probed by measuring the FRET ratio between PFV and EB intercalated in DNA. In comparison to other DNA detection assays based on FRET and conjugated polymers, synthesis of dye-labeled DNA probe is avoided in our method, which significantly reduces the cost and the synthetic complexity. The PFV/dsDNA/EB system provides promising applications on DNA detection with a simply, fast and label-free manner.

FISH-CS—a rapid method for counting and sorting species of marine zooplankton

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 410:1-11 (2010) - DOI: https://doi.org/10.3354/meps08654 FEATURE ARTICLE FISH-CS—a rapid method for counting and sorting species of marine zooplankton Christine M. Henzler1,2,*, Elizabeth A. Hoaglund1,2, Steven D. Gaines1,2,3 1Marine Science Institute, and 2Department of Ecology, Evolution and Marine Biology, University of California Santa Barbara, Santa Barbara, California 93106, USA 3Present address: Bren School of Environmental Resource Management, University of California Santa Barbara, Santa Barbara, California 93106, USA *Email: henzler@msi.ucsb.edu ABSTRACT: Understanding population dynamics in marine species has long been hindered by the inherent difficulties of studying species in which all or part of the life cycle is planktonic. Plankton sample processing is laborious and, due to morphological similarity between disparate taxa, often identifies zooplankton only to higher taxonomic levels. As a consequence, many scientific issues that require identification to species level are impossible to explore adequately. Several in situ hybridization protocols show promise for identifying marine larvae by color-coding them with taxon-specific, dye-labeled DNA probes. We adapted these protocols and coupled them with recent cell sorting technology to rapidly and accurately identify bivalve larvae from diverse plankton samples. We developed probes for 2 bivalve taxa: Musculista senhousia and the species complex Mytilus edulis/galloprovincialis/trossulus. Coupled fluorescence in situ hybridization and cell sorting (FISH-CS) separated M. galloprovincialis larvae from both oyster Crassostrea gigas larvae and from a mixed plankton/M. galloprovincialis sample. The number of false positives and false negatives was assessed by a PCR assay. Our FISH-CS method is robust to plankton autofluorescence and can be easily adapted to work with nearly any planktonic species or life stage of appropriate size. KEY WORDS:Fluorescence in situ hybridization · Oligonucleotide probe · 18S rRNA · Marine larvae · Species identification Full text in pdf format Information about this Feature Article NextCite this article as: Henzler CM, Hoaglund EA, Gaines SD (2010) FISH-CS—a rapid method for counting and sorting species of marine zooplankton. Mar Ecol Prog Ser 410:1-11. https://doi.org/10.3354/meps08654 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 410. Online publication date: July 14, 2010 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2010 Inter-Research.

Real-time Fluorogenic PCR Assays for the Detection of entA, the Gene Encoding Staphylococcal Enterotoxin A

Staphylococcal enterotoxin A (SEA) is among the most potent of the growing list of known enterotoxins produced by Staphylococcus aureus. SEA, a 27 kDa monomeric protein, is encoded by the entA gene. We have developed two real-time fluorogenic PCR assays for the detection of nucleic acid sequences in entA. The assays are useful in detecting and identifying strains of S. aureus that produce SEA and can serve a confirmatory role in determining the presence of SEA in food samples. The assays were tested in two real-time PCR formats, using either dye-labeled DNA probes corresponding to each primer set that are degraded by the 5' exonuclease activity of Taq polymerase, or a PCR master mix that contains the DNA-binding dye SYBR Green. In both formats the assays have a limit of detection of between 1 and 13 copies of a S. aureus genome that contains a copy of entA. Neither assay cross-reacted with genomic DNA isolated from other strains of S. aureus or other species.

Direct gene expression analysis.

The direct analysis of single biological molecules is getting increasingly important in basic as well as pharmaceutical research (e.g. for gene expression analysis). In particular single-molecule fluorescence detection provides exciting new opportunities to probe biochemical processes in unprecedented detail. Currently several academic and industrial research groups work on the development of single molecule detection based technologies in order to directly detect and analyze RNA and DNA molecules. As these developed methods are characterized as homogenous assays and obviate any amplification of the target or the signal, they provide clear advantages compared to methods like real-time PCR or DNA- arrays. In the following we describe a recently developed approach based on fluorescence correlation spectroscopy (FCS). This expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target molecule (either DNA or RNA) in solution. The subsequent dual color cross-correlation analysis allows the quantification of the bio-molecule of interest in absolute numbers. Target concentrations of less than 10(-12) M can be easily monitored, covering the direct analysis of the expression levels of high, medium and low abundant genes.

Gene expression analysis using single molecule detection.

Recent developments of single molecule detection techniques and in particular the introduction of fluorescence correlation spectroscopy (FCS) led to a number of important applications in biological research. We present a unique approach for the gene expression analysis using dual-color cross-correlation. The expression assay is based on gene-specific hybridization of two dye-labeled DNA probes to a selected target gene. The counting of the dual-labeled molecules within the solution allows the quantification of the expressed gene copies in absolute numbers. As detection and analysis by FCS can be performed at the level of single molecules, there is no need for any type of amplification. We describe the gene expression assay and present data demonstrating the capacity of this novel technology. In order to prove the gene specificity, we performed experiments with gene-depleted total cDNA. The biological application was demonstrated by quantifying selected high, medium and low abundant genes in cDNA prepared from HL-60 cells.

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

A capillary electrophoresis method has been developed to study DNA-protein complexes by mobility-shift assay. This method is at least 100 times more sensitive than conventional gel mobility-shift procedures. Key features of the technique include the use of a neutral coated capillary, a small amount of linear polymer in the separation medium, and use of covalently dye-labeled DNA probes that can be detected with a commercially available laser-induced fluorescence monitor. The capillary method provides quantitative data in runs requiring < 20 min, from which dissociation constants are readily determined. As a test case we studied interactions of a developmentally important sea urchin embryo transcription factor, SpP3A2. As little as 2-10 x 10(6) molecules of specific SpP3A2-oligonucleotide complex were reproducibly detected, using recombinant SpP3A2, crude nuclear extract, egg lysates, and even a single sea urchin egg lysed within the capillary column.