Competitive Hybridization Research Articles

Quantitative and selective DNA detection with portable personal glucose meter using loop-based DNA competitive hybridization strategy

A portable, quantitative and selective DNA detection biosensor requiring only one step for target recognition and signal reporter generation using a loop-based DNA competitive hybridization assay and personal glucose meter (PGM) was developed. In the presence of target DNA, due to its competitive binding, the invertase-DNA conjugate was released which could be collected with the help of a magnet subsequently. The released invertase-DNA was used to catalyze the hydrolysis of sucrose into glucose with millions of turnovers which was target concentration dependent. We found that there was a linear relationship between the PGM signals and the target DNA concentration in the range of 100 pM to 100 nM with a detection limit of 100 pM. In addition, the sensor exhibits excellent sequence selectivity, being able to differentiate a single mismatch in the target DNA, and excellent anti-interference ability, having almost no effect in serum on the detection performance. The biosensor reported here is easier to operate, owning great potential in point of care testing in environments with limited resources and skilled personnel for rapid and sensitive detection of specific DNA sequence in real biological samples.

The establishment of hybrids of the Daphnia longispina complex explained by a mathematical model incorporating different overwintering life history strategies.

Interspecific hybridization (i.e. mating between species) occurs frequently in animals. Among cyclical parthenogens, hybrids can proliferate and establish through parthenogenetic reproduction, even if their sexual reproduction is impaired. In water fleas of the Daphnia longispina species complex, interspecific hybrids hatch from sexually produced dormant eggs. However, fewer hybrid genotypes contribute to the dormant egg bank and their hatching rate from dormant eggs is reduced, compared to eggs resulting from intraspecific crosses. Therefore, Daphnia hybrids would benefit from adaptations that increase their survival over winter as parthenogenetic lineages, avoiding the need to re-establish populations after winter from sexually produced dormant eggs. Here, we constructed a mathematical model to examine the conditions that could explain the frequently observed establishment of hybrids in the D. longispina species complex. Specifically, we compared the outcome of hybrid and parental taxa competition given a reduced contribution of hybrids to hatchlings from the sexually produced dormant egg bank, but their increased ability to survive winter as parthenogenetic lineages. In addition, different growth rates of parental species and differences in average annual temperatures were evaluated for their influence on hybrid production and establishment. Our model shows that increased overwinter performance as parthenogenetic females can compensate for reduced success in sexual reproduction, across all tested scenarios for varying relative growth rates of parental species. This pattern holds true for lower annual temperatures, but at higher temperatures hybrids were less successful. Consequently, hybrids might become less abundant as temperatures rise due to climate change, resulting in reduced diversity and faster differentiation of the parental species.

Tuning the ferromagnetism of a single layered titanium dioxide nanosheet with hole doping and uniaxial strain

The effects of hole doping and strain on the electronic and magnetic properties of a single layered TiO2 nanosheet were investigated here. It is found that the spontaneous magnetism can be introduced in all systems doped with low valence metal, indicating holes are the key fact to trigger local magnetic moments. Especially, a half-metal magnetism takes place when Li substitutes a Ti atom. As for the stability of dopants, the Al doping case shows lower formation energy than those of Li and Mg doping under O-rich conditions. Tuning the hole concentration, a phase transition from nonmagnetic to half-metal ferromagnetic ground state can emerge after the average spin magnetic moment reaches 1.0 µB/hole. Furthermore, the uniaxial strain effectively adjusts the magnetism by shifting the main peak of density of states near the fermi level. The anisotropic transition of magnetic state under uniaxial tensile strain was observed due to the competition of orbital hybridization between Ti and O atoms along different crystal directions.

Stationary distribution and ergodicity of a stochastic hybrid competition model with Lévy jumps

Taking white noises, Markovian switching and Lévy jumps into account, an n−species Lotka–Volterra competitive model with random perturbations is proposed and studied. Sufficient conditions for the existence and uniqueness of an ergodic stationary distribution of the model are established. These sufficient conditions are sharp in some cases. Some interesting results are revealed: the white noises and Lévy jumps could make the stationary distribution disappear as well as appear; the Markovian switching could make the stationary distribution appear. Some numerical simulations are also introduced to illustrate the main results.

ChIP-chip.

ChIP-chip can be used to analyze protein-DNA interactions in a region-wide and genome-wide manner. DNA microarrays contain PCR products or oligonucleotide probes that are designed to represent genomic sequences. Identification of genomic sites that interact with a specific protein is based on competitive hybridization of the ChIP-enriched DNA and the input DNA to DNA microarrays. The ChIP-chip protocol can be divided into two main sections: Amplification of ChIP DNA and hybridization of ChIP DNA to arrays. A large amount of DNA is required to hybridize to DNA arrays, and hybridization to a set of multiple commercial arrays that represent the entire human genome requires two rounds of PCR amplifications. The relative hybridization intensity of ChIP DNA and that of the input DNA is used to determine whether the probe sequence is a potential site of protein-DNA interaction. Resolution of actual genomic sites bound by the protein is dependent on the size of the chromatin and on the genomic distance between the probes on the array. As with expression profiling using gene chips, ChIP-chip experiments require multiple replicates for reliable statistical measure of protein-DNA interactions.

A facile one-step fluorescence method for the quantitation of low-content single base deamination impurity in synthetic oligonucleotides

Oligonucleotides are being researched and developed as potential drug candidates for the treatment of a broad spectrum of diseases. The characterization of antisense oligonucleotide (ASO) impurities caused by base mutations (e.g. deamination) which are closely related to the target ASO is a significant analytical challenge. Herein, we describe a novel one-step method, utilizing a strategy that combines fluorescence-ON detection with competitive hybridization, to achieve single base mutation quantitation in extensively modified synthetic ASOs. Given that this method is highly specific and sensitive (LoQ = 4 nM), we envision that it will find utility for screening other impurities as well as sequencing modified oligonucleotides.

Overexpressing Exogenous 5-Enolpyruvylshikimate-3-Phosphate Synthase (EPSPS) Genes Increases Fecundity and Auxin Content of Transgenic Arabidopsis Plants

Transgenic glyphosate-tolerant plants overproducing EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) may exhibit enhanced fitness in glyphosate-free environments. If so, introgression of transgenes overexpressing EPSPS into wild relative species may lead to increased competitiveness of crop-wild hybrids, resulting in unpredicted environmental impact. Assessing fitness effects of transgenes overexpressing EPSPS in a model plant species can help address this question, while elucidating how overproducing EPSPS affects the fitness-related traits of plants. We produced segregating T2 and T3 Arabidopsis thaliana lineages with or without a transgene overexpressing EPSPS isolated from rice or Agrobacterium (CP4). For each of the three transgenes, we compared glyphosate tolerance, some fitness-related traits, and auxin (indole-3-acetic acid) content in transgene-present, transgene-absent, empty vector (EV), and parental lineages in a common-garden experiment. We detected substantially increased glyphosate tolerance in T2 plants of transgene-present lineages that overproduced EPSPS. We also documented significant increases in fecundity, which was associated with increased auxin content in T3 transgene-present lineages containing rice EPSPS genes, compared with their segregating transgene-absent lineages, EV, and parental controls. Our results from Arabidopsis with nine transgenic events provide a strong support to the hypothesis that transgenic plants overproducing EPSPS can benefit from a fecundity advantage in glyphosate-free environments. Stimulated biosynthesis of auxin, an important plant growth hormone, by overproducing EPSPS may play a role in enhanced fecundity of the transgenic Arabidopsis plants. The obtained knowledge is useful for assessing environmental impact caused by introgression of transgenes overproducing EPSPS from any GE crop into populations of its wild relatives.

Nucleic Acid Self-Assembly Circuitry Aided by Exonuclease III for Discrimination of Single Nucleotide Variants.

Robust and rapid discrimination of one base mutations in nucleic acid sequences is important in clinical applications. Here, we report a hybridization-based assay exploiting nucleic acid self-assembly circuitry and enzyme exonuclease III (Exo III) for the differentiation of single nucleotide variants (SNVs). This one-step approach combines the merits of discrimination power of competitive DNA hybridization probes (probe + sink) with catalytic amplification assisted by Exo III. The phosphorothioate bonds modified on a wild-type (WT) specific sink inhibit the Exo III digestion; thus, subsequent catalytic amplification magnifies only the intended SNV targets. The integrated assay exhibits improved SNV discrimination rather than hybridization probes relying solely on competition or amplification and enables SNV detection at 1% abundance. Two frequent cancer-driver mutation sequences (EGFR-L861Q, NRAS-Q61K) were tested. Our strategy allows simple sequence design and can easily adapt to multianalyte SNV detections.

Endogenous MicroRNA-Triggered and Real-Time Monitored Drug Release via Cascaded Energy Transfer Payloads.

It is a great challenge to design a drug delivery system with a controlled manner, especially one triggered by an exclusive endogenous disease marker and with an easily tracked release process. Herein, we developed a drug delivery platform of carbon dots which were connected to a stem-loop molecular beacon loaded with doxorubicin and polyethylene glycol modified folic acid. Such a platform enables one to release drugs on demand under the stimuli of endogenous microRNA-21, and turn on the fluorescence of carbon dots and doxorubicin, which allows one to monitor the drug release process. The intracellular experiment indicated that folic acid could mediate endocytosis of the nanocarrier, and the overexpressed endogenous microRNA-21 served as a unique key to unlock the drug nanocarrier by competitive hybridization with the molecular beacon, which finally resulted in fluorescence recovery and realized a chemotherapeutic effect within human breast cancer cells. The nanocarrier may have potential application in personalized treatment of different cancer subtypes in which the corresponding miRNAs are overexpressed.

Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification.

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don't allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.

Effect of ligands binding on the isotherm of hybridization of the DNA-chip

The factors are analyzed, which influence both the hybridization thermodynamics and the DNA/DNA-duplex stability. The non-competitive hybridization of the DNA as well as the competitive hybridization on the surface are investigated. It is shown that the binding to the intercalating ligands results to an increase in the selectivity and sensitivity of the DNA chips.

An Effective Harmony Search Algorithm for Solving a No-Wait Hybrid Flow Shop Scheduling Problem with Machine Availability Constraint

This research investigates a no-wait hybrid flow shop scheduling problem. Minimizing the mean tardiness is considered as the objective to develop the optimal scheduling algorithm. Characteristics of our considered problem leads to the complexity of problem. First, no-wait operations. Second, setup time of each job is separated from its processing time and depends upon its preceding job. Third, all of jobs aren’t available at the first of scheduling. In other word, each job has individual ready time. Finally, machines are not continuously available due to the preventive maintenance. An effective harmony search algorithm is used to tackle the mentioned problem. A series of computational experiments is conducted by comparing our algorithm with previous meta-heuristic algorithms like population based simulated annealing (PBSA), Adopted imperialist competitive algorithm (ICA) and hybridization of PBSA and ICA (ICA+PBSA). To achieve reliable results, Taguchi approach is used to define robust parameters’ values for our proposed algorithm. The computational results with random test problems suggest that our proposed harmony search outperforms the three foregoing algorithms.

Analysis of product return rate and price competition in two supply chains

This article compares a normal and a reverse supply chain in the Betrand Competition. Each supply chain consists of a retailer and an exclusive supplier with stable partnership. The two chains compete with each other in three competition structures: the Centralized Competition Game, the Hybrid Competition Game (including two cases), and the Decentralized Competition Game. In different competition structures, we examine how the degree of competition intensity between the two chains and product return rate of the reverse chain influence the equilibrium decision of market price, profits of two chains and the choice of centralization. The article differs from the study of the traditional supply chain model as follows. Firstly, we analyze the normal and the reverse chain related to the same product in the Betrand competition. Secondly, the data show that the market price decreases with the rising of the product return rate and the falling of the competition intensity. Thirdly, it is found that the total profit of the normal chain decreases with the rising of the product return rate, while the total profit of the reverse chain increases with rising of the product return rate. Profits of two chains increase with the rise of the competition intensity. Finally, this article shows that centralization is an optimal strategy for one chain whereas centralization may be the best for the other chain.

Kinetic discrimination of DNA single-base mutations by localized surface plasmon resonance

Clinical application of DNA microarrays used for screening of single nucleotide polymorphisms (SNPs) are very important for diagnosis of diseases and appropriate treatment of patients. In this paper localized surface plasmon resonance (LSPR) technique has been used to study the DNA hybridization process for binary solutions of respectively perfectly matching (PM) and single base mismatching (MM) 93-mer ssDNA from KRAS codon 12. 5′-thiol modified 35-mer ssDNA has been linked to the Au nanodisks array as probe with a surface coverage of 2.8±0.1×1012/cm2. Probe’s binding properties was investigated in details, obtaining a sensitivity down to 10nMand 13nM, respectively for PM and MM, showing that the hybridization process occurs at a lower rate for MM with respect to PM target. The competitive hybridization is accounted for by an inhibition model, where the non-complementary sequences kinetically hinder the hybridization of the perfect matching sequences, owing to their above mentioned affinity constant differences for the same probe. Accordingly, the single nucleotide polymorphisms can therefore be revealed in a single step and label free mode with high sensitivity and specificity by LSPR measurements.

Competitive Assays of Label-Free DNA Hybridization with Single-Molecule Fluorescence Imaging Detection.

Single-molecule imaging of fluorescently labeled biomolecules is a powerful technique for measuring association interactions; however, care must be taken to ensure that the fluorescent labels do not influence the system being probed. Label-free techniques are needed to understand biomolecule interactions free from the influence of an attached label, but these techniques often lack sensitivity and specificity. To solve these challenges, we have developed a competitive assay that uses single-molecule detection to track the population of unlabeled target single-stranded DNA (ssDNA) hybridized with probe DNA immobilized at a glass interface by detecting individual duplexes with a fluorescently labeled "tracer" ssDNA. By labeling a small fraction (<0.2%) of target molecules, the "tracer" DNA tracks the available probe DNA sites without significant competition with the unlabeled target population. Single-molecule fluorescence imaging is a good read-out scheme for competitive assays, as it is sufficiently sensitive to detect tracer DNA on substrates with relatively low densities of probe DNA, ∼10(-3) of a monolayer, so that steric interactions do not hinder DNA hybridization. Competitive assays are used to measure the association constant of complementary strand DNA hybridization of 9- and 10-base pair targets, where the tracer assay predicts the same association constant as a traditional displacement competitive assay. This methodology was used to compare the Ka of hybridization for identical DNA strands differing only by the presence of a fluorescent label tethered to the 5' end of the solution-phase target. The addition of the fluorescent label significantly stabilizes the DNA duplex by 3.6 kJmol(-1), adding more stability than an additional adenine-thymine base-pairing interaction, 2.7 kJmol(-1). This competitive tracer assay could be used to screen a number of labeled and unlabeled target DNA strands to measure the impact of fluorescent labeling on duplex stability. This single-molecule competitive hybridization scheme could be easily adapted into a sensitive assay, where competition between tracer and target oligonucleotides for probe sites could be used to measure concentrations of unlabeled DNA or RNA.

Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection.

Epigenetic programming alterations in alligators from environmentally contaminated lakes

Previous studies examining the reproductive health of alligators in Florida lakes indicate that a variety of developmental and health impacts can be attributed to a combination of environmental quality and exposures to environmental contaminants. The majority of these environmental contaminants have been shown to disrupt normal endocrine signaling. The potential that these environmental conditions and contaminants may influence epigenetic status and correlate to the health abnormalities was investigated in the current study. The red blood cell (RBC) (erythrocyte) in the alligator is nucleated so was used as an easily purified marker cell to investigate epigenetic programming. RBCs were collected from adult male alligators captured at three sites in Florida, each characterized by varying degrees of contamination. While Lake Woodruff (WO) has remained relatively pristine, Lake Apopka (AP) and Merritt Island (MI) convey exposures to different suites of contaminants. DNA was isolated and methylated DNA immunoprecipitation (MeDIP) was used to isolate methylated DNA that was then analyzed in a competitive hybridization using a genome-wide alligator tiling array for a MeDIP-Chip analysis. Pairwise comparisons of alligators from AP and MI to WO revealed alterations in the DNA methylome. The AP vs. WO comparison identified 85 differential DNA methylation regions (DMRs) with ⩾3 adjacent oligonucleotide tiling array probes and 15,451 DMRs with a single oligo probe analysis. The MI vs. WO comparison identified 75 DMRs with the ⩾3 oligo probe and 17,411 DMRs with the single oligo probe analysis. There was negligible overlap between the DMRs identified in AP vs. WO and MI vs. WO comparisons. In both comparisons DMRs were primarily associated with CpG deserts which are regions of low CpG density (1–2CpG/100bp). Although the alligator genome is not fully annotated, gene associations were identified and correlated to major gene class functional categories and pathways of endocrine relevance. Observations demonstrate that environmental quality may be associated with epigenetic programming and health status in the alligator. The epigenetic alterations may provide biomarkers to assess the environmental exposures and health impacts on these populations of alligators.

An Engineered Kinetic Amplification Mechanism for Single Nucleotide Variant Discrimination by DNA Hybridization Probes.

Even a single-nucleotide difference between the sequences of two otherwise identical biological nucleic acids can have dramatic functional consequences. Here, we use model-guided reaction pathway engineering to quantitatively improve the performance of selective hybridization probes in recognizing single nucleotide variants (SNVs). Specifically, we build a detection system that combines discrimination by competition with DNA strand displacement-based catalytic amplification. We show, both mathematically and experimentally, that the single nucleotide selectivity of such a system in binding to single-stranded DNA and RNA is quadratically better than discrimination due to competitive hybridization alone. As an additional benefit the integrated circuit inherits the property of amplification and provides at least 10-fold better sensitivity than standard hybridization probes. Moreover, we demonstrate how the detection mechanism can be tuned such that the detection reaction is agnostic to the position of the SNV within the target sequence. in contrast, prior strand displacement-based probes designed for kinetic discrimination are highly sensitive to position effects. We apply our system to reliably discriminate between different members of the let-7 microRNA family that differ in only a single base position. Our results demonstrate the power of systematic reaction network design to quantitatively improve biotechnology.

Impact of recent breeding history on the competitiveness of Chinese maize hybrids

The competitiveness of crop cultivars has been observed to decrease along with breeding progress. Changes in the competitiveness of maize (Zea mays L.) as a consequence of breeding over past decades have not been reported. The main objectives of this study were to (1) test Donald’s concept of communal ideotype in maize, and (2) determine the specific changes in competitiveness of maize hybrids due to breeding. Field experiments were conducted in the 2013 and 2014 growing seasons, using a de Wit replacement series design. Three maize hybrids, ZD2 (1972), YD13 (1992) and ZD909 (2011), which differed in release dates, were used in this study. Each hybrid was grown in multiple row replacement series (0:6, 1:5, 2:4, 3:3, 4:2, 5:1, and 6:0), with a total plant density of 67,500seeds/ha. Inter-cultivar competition reduced dry matter accumulation (DMA) and grain yield per plant of the cultivars in the replacement series. In each replacement series, plant DMA, grain yield and harvest index (HI) of hybrids decreased with increasing proportion of the older hybrid. Population grain yield exhibited the same trend. Based on competitive outcome and aggressivity (AG), maize hybrids differed in their competitiveness. Breeding reduced the competitiveness of maize hybrids, which ranked ZD2>YD13>ZD909, but the varying characteristics of plant traits were not at all consistent with competitiveness. The competitiveness of maize hybrids decreased during the past few decades, supporting Donald’s communal ideotype, which states that modern high yielding hybrids should not perform well in competition with other genotypes. Shorter plants with erectophile leaves and lower root:shoot ratios could contribute to weak competitiveness. Based on an analysis of aggressivity (AG), the AG of ZD2 relative to YD13 (AGZD2→YD13) was 0.121, the AG of YD13 relative to ZD909 (AGYD13→ZD909) was 0.091, the AG of ZD2 relative to ZD909 (AGZD2→ZD909) was 0.153, and AGZD2→YD13+AGYD13→ZD909≠AGZD2→ZD909, indicating that the changes in competitiveness were not consistent with the breeding process. Our results show that the competitiveness of maize bybrids was reduced by breeding, but that variation in architectural plant traits among genotypes was not completely consistent with the changes in their competitiveness.

Label-Free MicroRNA Detection Based on Fluorescence Quenching of Gold Nanoparticles with a Competitive Hybridization.

MicroRNAs (miRNAs), critical biomarkers of acute and chronic diseases, play key regulatory roles in many biological processes. As a result, there is great demand for robust assay platforms to enable an accurate and efficient detection of low-level miRNAs in complex biological samples. In this work, a label-free and Au nanoparticles (NPs) quenching-based competition assay system was developed. In the designed system, Au NPs with diameter sizes of 10 and 20 nm displayed fluorescence quenching efficiencies of 84% and 82% for Cy3 dye on slide surface, whereas the quenching efficiency of commercial BHQ2 quencher was roughly 50%. Assay conditions were optimized for miRNA-205 detection. A limit of detection of 3.8 pM and a detection range covering from 3.8 pM to 10 nM were achieved. Furthermore, the proposed system was capable of specifically discriminating miRNAs with slight variations in their nucleotide sequence and was also qualified for assessing miRNA levels in human serum. Our strategy has the potential to provide new perspectives in profiling the pattern of miRNA expression and biomedical utilizations.