Competitive Hybridization Research Articles

A novel hybrid meta-heuristic algorithm for a no-wait flexible flow shop scheduling problem with sequence dependent setup times

In this paper, we contemplate the problem of scheduling a set of n jobs in a no-wait flexible flow shop manufacturing system with sequence dependent setup times to minimising the maximum completion time. With respect to NP-hardness of the considered problem, there seems to be no avoiding application of metaheuristic approaches to achieve near-optimal solutions for this problem. For this reason, three novel metaheuristic algorithms, namely population based simulated annealing (PBSA), adapted imperialist competitive algorithm (AICA) and hybridisation of adapted imperialist competitive algorithm and population based simulated annealing (AICA + PBSA), are developed to solve the addressed problem. Because of the sensitivity of our proposed algorithm to parameter's values, we employed the Taguchi method as an optimisation technique to extensively tune different parameters of our algorithm to enhance solutions accuracy. These proposed algorithms were coded and tested on randomly generated instances, then to validate the effectiveness of them computational results are examined in terms of relative percentage deviation. Moreover, some sensitive analyses are carried out for appraising the behaviour of algorithms versus different conditions. The computational evaluations manifestly support the high performance of our proposed novel hybrid algorithm against other algorithms which were applied in literature for related production scheduling problems.

A competitive displacement assay with quantum dots as fluorescence resonance energy transfer donors

The unique optoelectronic properties of semiconductor quantum dots (QDs) make them well-suited as fluorescent bioprobes for use in various biological applications. Modification of CdSe/ZnS QDs with biologically relevant molecules provides for multipotent probes that can be used for cellular labeling, bioassays, and localized optical interrogation by means of fluorescence resonance energy transfer (FRET). Herein, we demonstrate the use of red-emitting streptavidin-coated QDs (QD605) as donors in FRET to introduce a competitive displacement-based assay for the detection of oligonucleotides. Various QD–DNA bioconjugates featuring 25-mer probe sequences diagnostic of Hsp23 were prepared. The single-stranded oligonucleotide probes were hybridized to dye-labeled (Alexa Fluor 647) reporter sequences, which were provided for a FRET-sensitized emission signal due to proximity of the QD and dye. The dye-labeled sequence was designed to be partially complementary and include base-pair mismatches to facilitate displacement by a more energetically favorable, fully complementary recognition motif embedded within a 98-mer displacer sequence. Overall, this study demonstrates proof-of-concept at the nM level for competitive displacement hybridization assays in vitro by reduction of fluorescence intensity that directly correlates to the presence of oligonucleotides of interest. This work demonstrates an analytical method that could potentially be implemented for monitoring of intracellular gene expression in the future.

A Competitive Strategy Coupled with Endonuclease‐Assisted Target Recycling for DNA Detection Using Silver‐Nanoparticle‐Tagged Carbon Nanospheres as Labels

A simple competitive strategy was designed for the sensitive detection of sequence-specific DNA by combining endonuclease-assisted target recycling and electrochemical stripping analysis of silver nanoparticles (AgNPs). The AgNP-tagged carbon nanospheres were synthesized by means of in situ reduction of Ag(+) adsorbed onto a negatively charged polyelectrolyte layer and functionalized with streptavidin for binding biotin-labeled DNA strands. The labeled strand was captured on the DNA sensor surface by competitive hybridization of biotinated primer 1 and its cleaved product. The cleaved product could be amplified in homogeneous solution by endonuclease-assisted target recycling with a Y-shaped junction DNA structure, thus leading to the correlation of the stripping signal to the target concentration. The functionalized nanosphere was characterized with X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The proposed method showed a linear range from 0.1 to 1000 fM with a limit of detection of 0.066 fM (3σ) and good selectivity for base discrimination. The designed strategy provided a sensitive tool for DNA analysis and could be widely applied in bioanalysis and biomedicine.

First Report of Peanut stunt virus on Glycine max in Korea

Peanut stunt virus (PSV) is a member of the genus Cucumovirus inthe family Bromoviridae. Other members of the genus are its typemember Cucumber mosaic virus (CMV) and Tomato aspermy virus(TAV). PSV like other cucumoviruses has a tripartite genome ofpositive strand RNAs, designated RNA1, RNA2 and RNA3, inother decreasing size. PSV is an economically important pathogenand occurs worldwide in legume plants. Since PSV was firstdescribed in the United States in 1966, many strains of PSV havebeen characterized. In Korea, there has been previous record on PSVinfection of peanut, black locast, red goosefoot and white clover(The Korean Society of Plant Pathology 2009)., In Sangju County ofSouth Korea in 2005, a PSV (PSV-K1) was first detected using RT-PCR from naturally infected soybean showing yellow mosaic on theleaves and virus particles were isolated from these infected leaves(Fig. 1A). Purified virus preparations were revealed the presence ofsmall isometric virions of 28 nm in diameter (Fig. 1B).The nucleotide sequence of full-length RNA3 from PSV strainK1 was determined. PSV-K1 RNA3 is 2089 nucleotides (nt)organized into two putative ORFs and 5'- and 3'-untranslatedregions (UTRs) of 52 and 229nt, respectively. Two large openreading frames (ORFs) encoded a putative movement protein (MP)(nt 53 −925) and a coat protein (CP) (nt 1183 −1860) predicted to be a31-kDa protein (P31) and a 25-kDa protein (P25), respectively.ORF3a (MP) is separated from the next ORF3b (CP) by an IR non-coding region (internal region) 257nt in length (nt 926 −1182).Multiple sequence alignments were generated using DNAMAN7.0 (Lynnon Biosoft, Quebec, Canada) on the basis of completenucleotide sequences of PSV RNA3, and a phylogenetic tree wasconstructed by the neighbor-joining algorithm (Fig. 2). Bootstrapanalysis was performed with 1,000 replicates. The resulting tree ofRNA3 revealed that PSV-K1 RNA3 is more closely related to that ofPSV-ER (94.6%) than to those of other cucumoviruses. Percentnucleotide sequence identities of RNA3 from PSV-K1 ranged from58.7 to 94.6% with RNA3 sequences of members of the genusCucumovirus. PSV was first divided into two subgroups based on awide range of comparative analyses, including serology, competi-tion hybridization and sequence comparison (Hu et al., 1997).Recently, the complete nucleotide (nt) sequence of PSV-Mi andPSV-Rp strain was determined, and based on nt sequence diversity,the establishment of a subgroup and was proposed (Yan et al., 2005and Kiss et al., 2008).Phylogenetic analyses of PSV-K1 show clearly that this strain isrelated to representatives of subgroup containing PSV-ER B. ased onthese data, we conclude that PSV is closely related to PSV-ER strainfrom cowpea and it is the first report of PSV on soybean in Korea.

Direct quantification of microRNA at low picomolar level in sera of glioma patients using a competitive hybridization followed by amplified voltammetric detection.

MicroRNAs (miRNAs), acting as oncogenes or tumor suppressors in humans, play a key role in regulating gene expression and are believed to be important for developing novel therapeutic treatments and clinical prognoses. Due to their short lengths (17-25 nucleotides) and extremely low concentrations (typically < picomolar) in biological samples, quantification of miRNAs has been challenging to conventional biochemical methods, such as Northern blotting, microarray, and quantitative polymerase chain reaction (qPCR). In this work, a biotinylated miRNA (biotin-miRNA) whose sequence is the same as that of a miRNA target is introduced into samples of interest and allowed to compete with the miRNA target for the oligonucleotide (ODN) probe preimmobilized onto an electrode. Voltammetric quantification of the miRNA target was accomplished after complexation of the biotin-miRNA with ferrocene (Fc)-capped gold nanoparticle/streptavidin conjugates. The Fc oxidation current was found to be inversely proportional to the concentration of target miRNA between 10 fM and 2.0 pM. The method is highly reproducible (relative standard deviation (RSD) < 5%), regenerable (at least 8 regeneration/assay cycles without discernible signal decrease), and selective (with sequence specificity down to a single nucleotide mismatch). The low detection levels (10 fM or 0.1 attomoles of miRNA in a 10 μL solution) allow the direct quantification of miRNA-182, a marker correlated to the progression of glioma in patients, to be performed in serum samples without sample pretreatment and RNA extraction and enrichment. The concentration of miRNA-182 in glioma patients was found to be 3.1 times as high as that in healthy persons, a conclusion in excellent agreement with a separate qPCR measurement of the expression level. The obviations of the requirement of an internal reference in qPCR, simplicity, and cost-effectiveness are other additional advantages of this method for detection of nucleic acids in clinical samples.

Thermostable DNA Immobilization and Temperature Effects on Surface Hybridization

Monolayer films of nucleic acids on solid supports are encountered in a range of diagnostic and bioanalytical applications. These applications often rely on elevated temperatures to improve performance; moreover, studies at elevated temperatures can provide fundamental information on layer organization and functionality. To support such applications, this study compares thermostability of oligonucleotide monolayers immobilized to gold by first coating the gold with a nanometer-thick film (an "anchor layer") of a polymercaptosiloxane, to which DNA oligonucleotides are subsequently tethered through maleimide-thiol conjugation, with thermostability of monolayers formed via widely used attachment through a terminal thiol moiety on the DNA. The temperature range covered is from 25 to 90 °C. After confirming stability of immobilization and, more importantly, retention of hybridization activity even under the harshest conditions investigated, these thermostable films are used to demonstrate measurements of (1) reversible surface melting transitions and (2) temperature dependence of competitive hybridization, when fully matched and mismatched sequences compete for binding to immobilized DNA oligonucleotides. The competitive hybridization experiments reveal a pronounced impact of temperature on rates of approach to equilibrium, with kinetic freezing into nonequilibrium states close to room temperature and rapid approach to equilibrium at elevated temperatures. Modeling of competitive surface hybridization equilibria using thermodynamic parameters derived from surface melting transitions of the individual sequences is also discussed.

Competitive Reporter Monitored Amplification (CMA) - Quantification of Molecular Targets by Real Time Monitoring of Competitive Reporter Hybridization

BackgroundState of the art molecular diagnostic tests are based on the sensitive detection and quantification of nucleic acids. However, currently established diagnostic tests are characterized by elaborate and expensive technical solutions hindering the development of simple, affordable and compact point-of-care molecular tests.Methodology and Principal FindingsThe described competitive reporter monitored amplification allows the simultaneous amplification and quantification of multiple nucleic acid targets by polymerase chain reaction. Target quantification is accomplished by real-time detection of amplified nucleic acids utilizing a capture probe array and specific reporter probes. The reporter probes are fluorescently labeled oligonucleotides that are complementary to the respective capture probes on the array and to the respective sites of the target nucleic acids in solution. Capture probes and amplified target compete for reporter probes. Increasing amplicon concentration leads to decreased fluorescence signal at the respective capture probe position on the array which is measured after each cycle of amplification. In order to observe reporter probe hybridization in real-time without any additional washing steps, we have developed a mechanical fluorescence background displacement technique.Conclusions and SignificanceThe system presented in this paper enables simultaneous detection and quantification of multiple targets. Moreover, the presented fluorescence background displacement technique provides a generic solution for real time monitoring of binding events of fluorescently labelled ligands to surface immobilized probes. With the model assay for the detection of human immunodeficiency virus type 1 and 2 (HIV 1/2), we have been able to observe the amplification kinetics of five targets simultaneously and accommodate two additional hybridization controls with a simple instrument set-up. The ability to accommodate multiple controls and targets into a single assay and to perform the assay on simple and robust instrumentation is a prerequisite for the development of novel molecular point of care tests.

Abstract 4283: Cross-species hybridization of microarrays for studying tumor transcriptome of brain metastasis

Abstract Although the importance of the cellular microenvironment (soil) during invasion and metastasis of cancer cells (seed) has been well-recognized, technical challenges have limited the ability to assess the influence of the microenvironment on cancer cells at the molecular level. Here, we show that an experimental strategy, competitive cross-species hybridization of microarray experiments, can characterize the influence of different microenvironments on cancer cells by independently extracting gene expression data of cancer and host cells when human cancer cells were xenografted into different organ sites of immunocompromised mice. Surprisingly, the analysis of gene expression data showed that the brain microenvironment induces complete reprogramming of metastasized cancer cells, resulting in a gain of neuronal cell characteristics and mimicking neurogenesis during development. We also show that epigenetic changes coincide with transcriptional reprogramming in cancer cells. These observations provide proof of principle for competitive cross-species hybridization of microarray experiments to characterize the effect of the microenvironment on tumor cell behavior Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4283. doi:1538-7445.AM2012-4283

Repeated Template Switching: Obstacles in cDNA Libraries and Ways to Avoid Them

We screened zebra finch cDNA libraries with the plaque-based competitive hybridization technique for genes differentially expressed in the brains of courting and non-courting males. The cDNA libraries were generated by the SMART TM cDNA Library Construction Kit. Sixty-six percent (35/53) of the isolated clones possessed high homology to sequences present in the cDNA libraries of species ranging from Arabidopsis to humans. The clones had to be false positives since the corresponding sequences are absent in genomic libraries. The false positive clones were tandem repeats of different lengths of the SMART IV TM oligonucleotide and originated through repeated template switching during reverse transcription. We report here based on in silico data that multiple template switching and subsequent repeated incorporation of primers at 3`end of first strand cDNA is a common event during the generation of cDNA libraries created by the template switching strategy. We demonstrate that the repeat containing clones disturbs hybridization procedures aimed to identify differentially expressed genes. We propose an additional hybridization step with repeat containing clones to avoid the identification of false positives, and recommend the use of switch primers without palindrome sequences to avoid the formation of tandem repeats during cDNA library construction.

Label-free fluorescent assays based on aptamer–target recognition

Label-free fluorescent assays were developed based on the competition of intramolecular DNA hybridization and aptamer-target binding. Using small molecule adenosine triphosphate (ATP) and biomacro-molecule thrombin as model targets, our design was proved to be a general method with good sensitivity and high selectivity.

Tunable Magnetism and Half-Metallic Stability of Half-Heusler Compound NiMn&lt;sub&gt;1-x&lt;/sub&gt;Nb&lt;sub&gt;x&lt;/sub&gt;Sb

By using the first-principles calculations within the density functional theory (DFT), we investigate the electronic structure, magnetism and half-metallic stability of half-Heusler compound NiMn1-xNbxSb. The results showed that the lattice constants and magnetic moments per unit respectively follow the Vegard law and the Slater-Pauling rule well. The most stable half-metallicity occurs at doping concentration x=0.25 due to the Fermi level is situated the middle of the spin-down gap. Our studies also indicate that the competition of hybridization of d-electrons and RKKY-type magnetic coupling plays a dominating role in determining the magnetism.

Cross-species hybridization of microarrays for studying tumor transcriptome of brain metastasis

Although the importance of the cellular microenvironment (soil) during invasion and metastasis of cancer cells (seed) has been well-recognized, technical challenges have limited the ability to assess the influence of the microenvironment on cancer cells at the molecular level. Here, we show that an experimental strategy, competitive cross-species hybridization of microarray experiments, can characterize the influence of different microenvironments on cancer cells by independently extracting gene expression data of cancer and host cells when human cancer cells were xenografted into different organ sites of immunocompromised mice. Surprisingly, the analysis of gene expression data showed that the brain microenvironment induces complete reprogramming of metastasized cancer cells, resulting in a gain of neuronal cell characteristics and mimicking neurogenesis during development. We also show that epigenetic changes coincide with transcriptional reprogramming in cancer cells. These observations provide proof of principle for competitive cross-species hybridization of microarray experiments to characterize the effect of the microenvironment on tumor cell behavior.

Array CGH Phylogeny: How accurate are Comparative Genomic Hybridization-based trees?

BackgroundArray-based Comparative Genomic Hybridization (CGH) data have been used to infer phylogenetic relationships. However, the reliability of array CGH analysis to determine evolutionary relationships has not been well established. In most CGH work, all species and strains are compared to a single reference species, whose genome was used to design the array. In the accompanying work, we critically evaluated CGH-based phylogeny using simulated competitive hybridization data. This work showed that a limited number of conditions, principally the tree topology and placement of the reference taxon in the tree, had a strong effect on the ability to recover the correct tree topology. Here, we add to our simulation study by testing the use of CGH as a phylogenetic tool with experimental CGH data from competitive hybridizations between N. crassa and other Neurospora species. In the discussion, we add to our empirical study of Neurospora by reanalyzing of data from a previous CGH phylogenetic analysis of the yeast sensu stricto complex.ResultsArray ratio data for Neurospora and related species were normalized with loess, robust spline, and linear ratio based methods, and then used to construct Neighbor-Joining and parsimony trees. These trees were compared to published phylogenetic analyses for Neurospora based on multilocus sequence analysis (MLSA). For the Neurospora dataset, the best combination of methods resulted in recovery of the MLSA tree topology less than half the time. Our reanalysis of a yeast dataset found that trees identical to established phylogeny were recovered only by pruning taxa - including the reference taxon - from the analysis.ConclusionOur results indicate that CGH data can be problematic for phylogenetic analysis. Success fluctuates based on the methods utilized to construct the tree and the taxa included. Selective pruning of the taxa improves the results - an impractical approach for normal phylogenetic analysis. From the more successful methods we make suggestions on the normalization and post-normalization methods that work best in estimating genetic distance between taxa.

Research on Supply Chain Hybrid Competition and Channel Pervasion Effects in E-Commerce Age

Supply chain hybrid competition happens among two manufacturers and one traditional retailer in e-commerce age. Being a channel leader the first manufacturer’s role becomes a little sophisticated. That is, being the traditional retailer’s supplier and competing with the traditional retailer’s on the internet at the same time. Besides, as an online competitor the second manufacturer’s channel status could go steadily up as electronic market becomes more mature institutionally. This paper compares two kinds of game models corresponding to two different channel statuses in supply chain partners, which means a weakest traditional retailer or a weakest online manufacturer separately. It finds two models’ existence conditions of supply chain partners’ optimal revenues, optimal decision variables, etc. By numerical analysis it especially analyzes their channel pervasion effects, at the same time, it gives some useful managerial inspiration further.

Development of DNA sensor crustacean allergen analysis in food products

Crustaceans is one of the most common causes of food allergies. Its persistency is life-threatening therefore, method to detect the presence of crustacean constituent in food products would be indispensable to ensure the safety. We have developed a simple and rapid method to detect the presence of crustacean residues in food products through the allergenic Pen gene. Detection method was based on isothermal DNA amplification using primers specific to target Pen genes. Detection of DNA products was based on fluorescence visualization on UV light source after competitive DNA hybridization using fluorescence beta-pyrroridinyl peptide nucleic acid probe and its corresponding quencher. This method was crustacean specific and could detect shrimp, prawn, crab and lobster residues. Its sensitivity was sufficient to detect 20 copies of specific Pen DNA to Pen gene. Presence of shrimp, prawn, crab and lobster constituents were able to detect within 2 hours at DNA level without relying on laboratory facilities. This method should provide a benefit to food manufacturers, clinical doctors and allergenic patients by providing rapid information on the presence of crustacean contamination in foods.

Competitive Protection of Aptamer-Functionalized Gold Nanoparticles by Controlling the DNA Assembly

Functionalization and control of nanoparticle surface are critical to the desirable application of nanoparticles. We describe a DNA assembly method that dynamically protects aptamer-functionalized gold nanoparticles (apt-AuNP) from nondesirable binding to interfering molecules. Competitive protection of the apt-AuNP was achieved by using a carefully designed DNA that served two primary functions: reducing cross-reaction by competitive hybridization to the aptamer and overcoming nonspecific adsorption by restricted access to the AuNP surface. Tuning the length and sequence of the hybridization and the overhang components of the protection DNA allowed for control of the selectivity of apt-AuNP probes for the target protein. Alternating application of the protection DNA containing a polyA and polyT overhang enabled layer-by-layer formation of the supermolecular assembly. This directed assembly of apt-AuNP resulted in enhancement of signal for visual detection of minute amounts of proteins. This method was successfully demonstrated for Western blot analysis of thrombin, with an improved detection limit of 6 ng. This study demonstrates a unique dynamic protection strategy for achieving high specificity and sensitivity in the detection of protein.

Oligonucleotide Bioanalysis: Sensitivity Versus Specificity

Oligonucleotide Bioanalysis: Sensitivity Versus Specificity

When Genetic Instability Is a Good Thing

Many years of cell-cycle research have been devoted to understanding the mechanisms that replicate and repair the genome so that cells can proliferate without accumulating the genetic damage that drives the initiation and progression of cancer. A classic example of genetic instability in cancer cells is the amplification of segments of the genome in a normally diploid cell type that increases the copy number and thus the expression of oncogenes. However, those who study cell-cycle control in a developmental context know that these types of events are often a normal part of development. This paper by the Orr-Weaver laboratory furthered our understanding of developmentally regulated gene amplification that occurs in Drosophila ovaries. The authors used competitive genome hybridization to a cDNA microarray to identify new follicle cell amplicons required for egg production. This work set the stage for using modern genomic techniques to comprehensively identify gene amplification events that drive the synthesis of large amounts of gene product needed to produce certain structures during development. This PaperPick refers to Gene amplification as a developmental strategy: isolation of two developmental amplicons in Drosophila , by Julie M. Claycomb, Matt Benasutti, Giovanni Bosco, Douglas D. Fenger, and Terry L. Orr-Weaver, published in January 2004. Video Abstract Dr. Orr-Weaver discusses her group's work on regulatory programs that amplify (or suppress) overreplication of cell-type-appropriate genes in the Drosophila embryo.

Impact of DNA–Surface Interactions on the Stability of DNA Hybrids

The structure and stability of single- and double-stranded DNA hybrids immobilized on gold are strongly affected by nucleotide-surface interactions. To systematically analyze the effects of these interactions, a set of model DNA hybrids was prepared in conformations that ranged from end-tethered double-stranded to directly adsorbed single-stranded (hairpins) and characterized by surface plasmon resonance (SPR) imaging, X-ray photoelectron spectroscopy (XPS), fluorescence microscopy, and near edge X-ray absorption fine structure (NEXAFS) spectroscopy. The stabilities of these hybrids were evaluated by exposure to a series of stringency rinses in solutions of successively lower ionic strength and by competitive hybridization experiments. In all cases, directly adsorbed DNA hybrids are found to be significantly less stable than either free or end-tethered hybrids. The surface-induced weakening and the associated asymmetry in hybridization responses of the two strands forming hairpin stems are most pronounced for single-stranded hairpins containing blocks of m adenine (A) nucleotides and n thymine (T) nucleotides, which have high and low affinity for gold surfaces, respectively. The results allow a qualitative scale of relative stabilities to be developed for DNA hybrids on surfaces. Additionally, the results suggest a route for selectively weakening portions of immobilized DNA hybrids and for introducing asymmetric hybridization responses by using sequence design to control nucleotide-surface interactions--a strategy that may be used in advanced biosensors and in switches or other active elements in DNA-based nanotechnology.

Gene Expression Rhythms in the Mussel Mytilus galloprovincialis (Lam.) across an Annual Cycle

Seasonal environmental changes may affect the physiology of Mytilus galloprovincialis (Lam.), an intertidal filter-feeder bivalve occurring commonly in Mediterranean and Atlantic coastal areas. We investigated seasonal variations in relative transcript abundance of the digestive gland and the mantle (gonads) of males and females. To identify gene expression trends – in terms of relative mRNA abundance- we used a medium-density cDNA microarray (1.7 K probes) in dual-color competitive hybridization analyses. Hierarchical clustering of digestive gland microarray data showed two main branches, distinguishing profiles associated with the “hot” months (May–August) from the other months. Genes involved in chitin metabolism, associated with mussel nutrition and digestion showed higher mRNA levels during summer. Moreover, we found different gene transcriptomic patterns in the digestive glands of males when compared to females, during the four stages of mussel gonadal development. Microarray data from gonadal transcripts also displayed clear patterns during the different developmental phases respect to the resting period (stage I) with peak relative mRNA abundance at the ripe phase (stage III) for both sexes. These data showed a clear temporal pattern in transcriptomic profiles of mussels sampled over an annual cycle. Physiological response to thermal variation, food availability, and reproductive status across months may contribute to variation in relative mRNA abundance.