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A theoretical analysis was developed to predict molecular hybridization rates for microarrays where samples flow through microfluidic channels and for conventional microarrays where samples remain stationary during hybridization. The theory was validated by using a multiplexed microfluidic microarray where eight samples were hybridized simultaneously against eight probes using 60-mer DNA strands. Mass transfer coefficients ranged over three orders of magnitude where either kinetic reaction rates or molecular diffusion rates controlled overall hybridization rates. Probes were printed using microfluidic channels and also conventional spotting techniques. Consistent with the theoretical model, the microfluidic microarray demonstrated the ability to print DNA probes in less than 1 min and to detect 10-pM target concentrations with hybridization times in less than 5 min. 相似文献
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We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on. 相似文献
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The effects of mismatches on hybridization in DNA microarrays: determination of nearest neighbor parameters
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Quantifying interactions in DNA microarrays is of central importance for a better understanding of their functioning. Hybridization thermodynamics for nucleic acid strands in aqueous solution can be described by the so-called nearest neighbor model, which estimates the hybridization free energy of a given sequence as a sum of dinucleotide terms. Compared with its solution counterparts, hybridization in DNA microarrays may be hindered due to the presence of a solid surface and of a high density of DNA strands. We present here a study aimed at the determination of hybridization free energies in DNA microarrays. Experiments are performed on custom Agilent slides. The solution contains a single oligonucleotide. The microarray contains spots with a perfect matching (PM) complementary sequence and other spots with one or two mismatches (MM) : in total 1006 different probe spots, each replicated 15 times per microarray. The free energy parameters are directly fitted from microarray data. The experiments demonstrate a clear correlation between hybridization free energies in the microarray and in solution. The experiments are fully consistent with the Langmuir model at low intensities, but show a clear deviation at intermediate (non-saturating) intensities. These results provide new interesting insights for the quantification of molecular interactions in DNA microarrays. 相似文献
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The use of cross-species hybridization (CSH) to DNA microarrays, in which the target RNA and microarray probe are from different species, has increased in the past few years. CSH is used in comparative, evolutionary and ecological studies of closely related species, and for gene-expression profiling of many species that lack a representative microarray platform. However, unlike species-specific hybridization, CSH is still considered a non-standard use of microarrays. Here, we present the recent developments in the field of CSH for cDNA and oligomer microarray platforms. We discuss issues that influence the quality of CSH results, including platform choice, experiment design and data analysis, and suggest strategies that can lead to improvement of CSH studies to investigate species diversity. 相似文献
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Experiments show that deflections of microcantilever-DNA chip can be induced by many factors, such as grafting density, hybridization
efficiency, concentration, length and sequence of DNA molecules, buffer salt concentration, time, and temperature variation.
However, there are few theoretical works on microcantilever-DNA chips. The present paper is aimed to study the influence of
counterion effects of single-stranded DNA (ssDNA) polyelectrolyte solution on the nanomechanical behaviors of microcantilever-based
ssDNA chips during packing process. First, the effect of osmotic pressure induced by ingress of counterions into DNA brush
structures is studied with Hagan’s model for a cylindrical polyelectrolyte brush system on the basis of Poisson-Boltzmann
distribution hypothesis. Second, Zhang’s two-variable method for a laminated cantilever is used to formulate a four-layer
energy model for ssDNA chips with weak interactions. Third, the influence of grafting density, ssDNA chain length, and salt
concentration on packing deflection is investigated using the principle of minimum energy. The predictive tendency is qualitatively
similar to those observed in some related ssDNA chip experiments. The difference between the four-layer model and the simplified
two-layer model for ssDNA chips is also discussed. 相似文献
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The hybridization of oligomeric DNA was investigated using the frequency dependent techniques of quartz crystal microbalance (QCM) and electrochemical impedance spectroscopy (EIS). Synthetic 5'-amine-terminated single stranded oligonucleotides (ssDNA) were immobilized on the surface of the oxidized platinum driving electrodes of AT-cut quartz QCM crystals using 3-glycidoxypropyl-trimethoxysilane. Similar ssDNA coupling was accomplished on the exposed glass surface between the metallic digits of microlithographically fabricated interdigitated microsensor electrodes (IMEs). Confirmation of this covalent coupling surface chemistry was achieved using Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR). Substantial changes in resonant frequency values (0.012% decrease) and electrochemical impedance values (both real and imaginary components) (35.4 and 42.1% increase in impedance magnitude at 1.0 Hz in buffer and deionized water, respectively) were observed resulting from hybridization of the attached ssDNA upon exposure to its complement under appropriate hybridization conditions. Non-complementary (random) oligomer sequence demonstrated a modest change in resonant frequency and a non-detectable change in impedance. Microarray glass slide surfaces modified with 3-glycidoxypropyltrimethoxysilane (GPS), shown to be advantageous in the design and use of microarrays of amine-terminated ssDNA, is confirmed to arise from direct covalent coupling of the DNA to the surface with little non-specific adsorption. The possibility to detect the binding state of DNA in the vicinity of an electrode, without a direct connection between the measurement electrode and the DNA is hereby reported. The potential for development of label-free, low-density DNA microarrays is demonstrated and is being pursued. 相似文献
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In the course of exploring the hybridization properties of glass DNA microarrays, multi-stranded DNA structures were observed that could not be accounted for by classical Watson-Crick base pairing. Non-denatured double-stranded DNA array elements were shown to hybridize to single-stranded (ss)DNA probes. Similarly, ssDNA array elements were shown to bind duplex DNA probes. This led to a series of experiments demonstrating the formation of multi-stranded DNA structures on the surface of microarrays. These structures were observed with a number of heterogeneous sequences, including both purine and pyrimidine bases, with shared sequence identity between the ssDNA and one of the duplex strands. Furthermore, we observed a strong binding preference near the ends of duplexes containing a 3'-homologous strand. We suggest that such binding interactions on cationic solid surfaces could serve as a model for a number of biological processes mediated through multi-stranded DNA. 相似文献
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The manufacture and use of a whole-genome microarray is a complex process and it is essential that all data surrounding the process is stored, is accessible and can be easily associated with the data generated following hybridization and scanning. As part of a program funded by the Wellcome Trust, the Bacterial Microarray Group at St. George's Hospital Medical School (BmuG@S) will generate whole-genome microarrays for 12 bacterial pathogens for use in collaboration with specialist research groups. BmuG@S will collaborate with these groups at all levels, including the experimental design, methodology and analysis. In addition, we will provide informatic support in the form of a database system (BmuG@Sbase). BmuG@Sbase will provide access through a web interface to the microarray design data and will allow individual users to store their data in a searchable, secure manner. Tools developed by BmuG@S in collaboration with specific research groups investigating analysis methodology will also be made available to those groups using the arrays and submitting data to BmuG@Sbase. 相似文献
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Xinyuan Chong Le Liu Zhiyi Liu Suihua Ma Jun Guo Yanhong Ji Yonghong He 《Plasmonics (Norwell, Mass.)》2013,8(2):1185-1191
We investigate the ability of a parallel scan spectral surface plasmon resonance imaging system in analyzing hybridization of single-stranded DNA (ssDNA) on microarray. The ssDNA probes are modified by a thiol group and thereby can be immobilized onto the gold film. Hybridization experiments are carried out by using both complementary and noncomplementary sequence to confirm the specificity of interaction. We also demonstrate that the data analysis is very important in reducing the noise and improving the resolution by comparing polynomial fitting method with the combination of polynomial fitting and centroid method. Finally, the results demonstrate that the parallel scan spectral surface plasmon resonance imaging system can be used for high-throughput analysis of the hybridization of the ssDNA. 相似文献
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A model of base-call resolution on broad-spectrum pathogen detection resequencing DNA microarrays 总被引:1,自引:0,他引:1
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Oligonucleotide microarrays offer the potential to efficiently test for multiple organisms, an excellent feature for surveillance applications. Among these, resequencing microarrays are of particular interest, as they possess additional unique capabilities to track pathogens’ genetic variations and perform detailed discrimination of closely related organisms. However, this potential can only be realized if the costs of developing the detection microarray are kept at a manageable level. Selection and verification of the probes are key factors affecting microarray design costs that can be reduced through the development and use of in silico modeling. Models created for other types of microarrays do not meet all the required criteria for this type of microarray. We describe here in silico methods for designing resequencing microarrays targeted for multiple organism detection. The model development presented here has focused on accurate base-call prediction in regions that are applicable to resequencing microarrays designed for multiple organism detection, a variation from other uses of a predictive model in which perfect prediction of all hybridization events is necessary. The model will assist in simplifying the design of resequencing microarrays and in reduction of the time and costs required for their development for new applications. 相似文献
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Mocanu D Kolesnychenko A Aarts S Troost-Dejong A Pierik A Vossenaar E Stapert H 《Journal of biotechnology》2009,139(2):179-185
The goal of this study is to assess the influence of mass transfer phenomena on DNA hybridization kinetics in a flow-through, porous microarray for fast molecular testing. We present a scaled mathematical model of coupled convection, diffusion and reaction in porous media, which was used to simulate hybridization kinetics and to analyze the influence of convective transport on the reaction rate. In addition to computer simulations, we also present experimental data of hybridization collected on our microarray system for different flow rates. The results reported in this paper provide for a better understanding of the interaction between reaction and mass transfer processes during flow-through hybridization and suggest criteria for system design and optimization. 相似文献
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Microarrays have been used extensively in gene expression profiling and genotyping studies. To reduce the high cost and enhance the consistency of microarray experiments, it is often desirable to strip and reuse microarray slides. Our genome-wide analysis of microRNA expression involves the hybridization of fluorescently labeled nucleic acids to custom-made, spotted DNA microarrays based on GAPSII-coated slides. We describe here a simple and effective method to regenerate such custom microarrays that uses a very low-salt buffer to remove labeled nucleic acids from microarrays. Slides can be stripped and reused multiple times without significantly compromising data quality. Moreover, our analyses of the performance of regenerated slides identifies parameters that influence the attachment of oligonucleotide probes to GAPSII slides, shedding light on the interactions between DNA and the microarray surface and suggesting ways in which to improve the design of oligonucleotide probes. 相似文献
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The web application D-Maps provides a user-friendly interface to researchers performing studies based on microarrays. The program was developed to manage and process one- or two-color microarray data obtained from several platforms (currently, GeneTAC, ScanArray, CodeLink, NimbleGen and Affymetrix). Despite the availability of many algorithms and many software programs designed to perform microarray analysis on the internet, these usually require sophisticated knowledge of mathematics, statistics and computation. D-maps was developed to overcome the requirement of high performance computers or programming experience. D-Maps performs raw data processing, normalization and statistical analysis, allowing access to the analyzed data in text or graphical format. An original feature presented by D-Maps is GEO (Gene Expression Omnibus) submission format service. The D-MaPs application was already used for analysis of oligonucleotide microarrays and PCR-spotted arrays (one- and two-color, laser and light scanner). In conclusion, D-Maps is a valuable tool for microarray research community, especially in the case of groups without a bioinformatic core. 相似文献
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Ju Z Wells MC Walter RB 《Comparative biochemistry and physiology. Toxicology & pharmacology : CBP》2007,145(1):5-14
Toxicogenomics represents the merging of toxicology with genomics and bioinformatics to investigate biological functions of genome in response to environmental contaminants. Aquatic species have traditionally been used as models in toxicology to characterize the actions of environmental stresses. Recent completion of the DNA sequencing for several fish species has spurred the development of DNA microarrays allowing investigators access to toxicogenomic approaches. However, since microarray technology is thus far limited to only a few aquatic species and derivation of biological meaning from microarray data is highly dependent on statistical arguments, the full potential of microarray in aquatic species research has yet to be realized. Herein we review some of the issues related to construction, probe design, statistical and bioinformatical data analyses, and current applications of DNA microarrays. As a model a recently developed medaka (Oryzias latipes) oligonucleotide microarray was described to highlight some of the issues related to array technology and its application in aquatic species exposed to hypoxia. Although there are known non-biological variations present in microarray data, it remains unquestionable that array technology will have a great impact on aquatic toxicology. Microarray applications in aquatic toxicogenomics will range from the discovery of diagnostic biomarkers, to establishment of stress-specific signatures and molecular pathways hallmarking the adaptation to new environmental conditions. 相似文献
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Recently, long oligonucleotide (60- to 70-mer) microarrays for two-color experiments have been developed and are gaining widespread use. In addition, when there is limited availability of mRNA from tissue sources, RNA amplification can and is being used to produce sufficient quantities of cRNA for microarray hybridization. Taking advantage of the selective degradation of RNA under alkaline conditions, we have developed a method to "strip" glass-based oligonucleotide microarrays that use fluorescent RNA in the hybridization, while leaving the DNA oligonucleotide probes intact and usable for a second experiment. Replicate microarray experiments conducted using stripped arrays showed high reproducibility, however, we found that arrays could only be stripped and reused once without compromising data quality. The intraclass correlation (ICC) between a virgin array and a stripped array hybridized with the same sample showed a range of 0.90-0.98, which is comparable to the ICC of two virgin arrays hybridized with the same sample. Using this method, once-stripped oligonucleotide microarrays are usable, reliable, and help to reduce costs. 相似文献