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Results for “Oligonucleotide Array Sequence Analysis”

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At least 379 records · Page 21Linked to original sources
PubMed2005

Shear stress regulates gene expression in vascular endothelial cells in response to tumor necrosis factor-alpha: a study of the transcription profile with complementary DNA microarray.

We investigate the role of shear stress in regulating the gene expression in endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNF-alpha). ECs were kept in static condition or pre-exposed to a high level (HSS, 20 dynes/cm2) or a low level of shear stress (LSS, 0.5 dynes/cm2) for 24 h, and TNF-alpha was added under static condition for 4 h. In static ECs, DNA microarray showed that TNF-alpha caused a significant increase in expression of 102 genes and a significant decrease in expression of 12 genes. Pre-shearing of ECs decreased the TNF-alpha-responsiveness of many pro-inflammatory, pro-coagulant, proliferative, and pro-apoptotic genes, whereas it increased the responsiveness of some antioxidant, anti-coagulant, and anti-apoptotic genes. LSS showed less regulatory effects than HSS on EC gene expression in response to TNF-alpha. The microarray data were confirmed by reverse-transcription polymerase chain reaction for 64 selected genes. Pre-shearing of ECs at HSS significantly inhibited the TNF-alpha-induced p65 and p50 mRNA expressions and nuclear factor-kappaB (NF-kappaB)-DNA binding activity. Inhibition of NF-kappaB activity with the p65-antisense or lactacystin under static condition blocked the expression of most of the genes that are TNF-alpha-inducible and shear stress-down-regulated. Our findings suggest that laminar shear stress serves protective functions against atherogenesis.

Cell Adhesion↗
PubMed2006

A computational method to geometric measure of biological particles and application to DNA microarray spot size estimation.

Geometric measures (volume, area and length) of biological particles are of fundamental interest for biological studies. Many times, the measures are at micro-/nano-scale, and based on images of the biological particles. This paper proposes a computational method to geometric measure of biological particles. The method has been applied to DNA microarray spot size estimation. Compared with existing algorithms for microarray spot size estimation, the proposed method is computational efficient and also provides confidence probability on the measure. The contributions of this paper include a generic computational method to geometric measure of biological particles and application to DNA microarray spot size estimation.

Gene Expression Profiling↗
PubMed2004

Hydrogel drop microchips with immobilized DNA: properties and methods for large-scale production.

Although gel-based microchips offer significant advantages over two-dimensional arrays, their use has been impeded by the lack of an efficient manufacturing procedure. Here we describe two simple, fast, and reproducible methods of fabrication of DNA gel drop microchips. In the first, copolymerization method, unsaturated groups are chemically attached to immobilized molecules, which are then mixed with gel-forming monomers. In the second, simpler polymerization-mediated immobilization method, aminated DNA without prior modification is added to a polymerization mixture. Droplets of polymerization mixtures are spotted by a robot onto glass slides and the slides are illuminated with UV light to induce copolymerization of DNA with gel-forming monomers. This results in immobilization of DNA within the whole volume of semispherical gel drops. The first method can be better controlled while the second one is less expensive, faster, and better suited to large-scale production. The microchips manufactured by both methods are similar in properties. Gel elements of the chip are porous enough to allow penetration of DNA up to 500 nucleotides long and its hybridization with immobilized oligonucleotides. As shown with confocal microscope studies, DNA is hybridized uniformly in the whole volume of gel drops. The gels are mechanically and thermally stable and withstand 20 subsequent hybridizations or 30-40 PCR cycles without decrease in hybridization signal. A method for quality control of the chips by staining with fluorescence dye is proposed. Applications of hydrogel microchips in research and clinical diagnostics are summarized.

Acrylamides↗
PubMed2004

Avoidance of nonspecific hybridization by employing oligonucleotide micro-arrays generated from hydrolysis polymerase chain reaction probe sequences.

We have developed a low-density oligonucleotide-based micro-array where 5'-end-tethered capture probe sequences were derived from Primer Express software. The capture probes represent hydrolysis probe sequences devoid of any fluorochromes and were shown to retain hybridization binding specificity to their amplicons; hybridization specificity was retained independent to probe sequences. This procedure allowed the specificity of each capture probe to be verified using real-time polymerase chain reaction (PCR) in the presence of nucleic acid sequences typically expected to be present within a sample and therefore has reduced possibility of nonspecific hybridization when used in a micro-array format. We propose that specificity-validated probes are applied to form a micro-array for the purpose of general target screening, with incumbent multiparallelization and cost and time savings. However, if required, the subset of probe sequences of interest can be used for quantitative assessment in conventional real-time PCR.

Bacterial Proteins↗
PubMed2006

Comparison of commercial probe labeling kits for microarray: towards quality assurance and consistency of reactions.

Microarray technology is readily available to scientists interested in gene expression. Commensurate with this availability is the growing market in accessory products offering convenience but potentially variable performance. Here we evaluate seven commercial kits for probe labeling against a human apoptosis oligonucleotide array. All kits were found to label probes successfully using the manufacturers' instructions. The Stratagene Fairplay Microarray Labeling Kit was the most sensitive, with an overall call rate of 74% and the lowest rate of indeterminant calls for the HEK and HepG2 cell lines. The Invitrogen SuperScript Indirect cDNA Labeling System showed the most reproducible gene expression pattern and the least technical variation, both in terms of signal strength and between replicates on each array. The Promega Pronto! Plus System showed the least dye bias however, a higher level of variation between replicates was observed. Pairwise comparisons revealed that the Promega Pronto! Plus System and Invitrogen SuperScript Indirect cDNA Labeling System had the most similarity in their patterns of gene expression. Results obtained suggest variability in the performance of commercial kits between different manufacturers. This study supports the need to conduct comparative evaluations of commercial microarray probe labeling kits and the need for validation prior to use.

Apoptosis↗
PubMed2006

Cartridge-based high-throughput purification of oligonucleotides for reliable oligonucleotide arrays.

A novel, cartridge-based procedure for the efficient and irreversible detritylation of oligonucleotides is reported. This method, combined with a process for the elimination of depurinated fragments produces, in a highly parallel fashion, oligonucleotides with better purity than those traditionally obtained using reversed-phase high-performance liquid chromotography purification. Our combined detritylation and purification methodology compares favorably with commercial cartridge-based purification systems. The benefits of working with pure oligonucleotides, with regard to higher signal and better signal linearity, are shown in array-based hybridization experiments.

Hydrophobic and Hydrophilic Interactions↗
PubMed2006

N-(3-Triethoxysilylpropyl)-6-(N-maleimido)-hexanamide: An efficient heterobifunctional reagent for the construction of oligonucleotide microarrays.

Synthesis of a new heterobifunctional reagent, N-(3-triethoxysilylpropyl)-6-(N-maleimido)-hexanamide (TPMH), for the preparation of oligonucleotide microarrays is described. Its triethoxysilyl function is specific toward virgin glass surface and maleimide function undergoes conjugate addition to 3'- or 5'-thiol-modified oligonucleotides. The construction of microarrays using TPMH has been realized via two routes. In Route A, TPMH was reacted first with 3'- or 5'-thiol-modified oligonucleotide under microwaves, thereby producing oligonucleotide-triethoxysilyl conjugate in 15min, which was then brought in contact with virgin glass microslide, resulting in immobilization of an oligonucleotide sequence. In Route B, immobilization involves generation of maleimide functions on virgin glass surface by the reaction with TPMH, followed by coupling with thiol-modified oligonucleotides under microwaves in 15 min to produce surface-bound oligonucleotides. The microarrays constructed using both routes were analyzed by hybridizing with tetrachlorofluorescein-labeled complementary oligonucleotide. Subsequently, these microarrays were successfully used in the discrimination of single and double nucleotide mismatches based on fluorescence intensity.

Amides↗
PubMed2006

A microbial diagnostic microarray technique for the sensitive detection and identification of pathogenic bacteria in a background of nonpathogens.

A major challenge in microbial diagnostics is the parallel detection and identification of low-bundance pathogens within a complex microbial community. In addition, a high specificity providing robust, reliable identification at least at the species level is required. A microbial diagnostic microarray approach, using single nucleotide extension labeling with gyrB as the marker gene, was developed. We present a novel concept applying competitive oligonucleotide probes to improve the specificity of the assay. Our approach enabled the sensitive and specific detection of a broad range of pathogenic bacteria. The approach was tested with a set of 35 oligonucleotide probes targeting Escherichia coli, Shigella spp., Salmonella spp., Aeromonas hydrophila, Vibrio cholerae, Mycobacterium avium, Mycobacterium tuberculosis, Helicobacter pylori, Proteus mirabilis, Yersinia enterocolitica, and Campylobacter jejuni. The introduction of competitive oligonucleotides in the labeling reaction successfully suppressed cross-reaction by closely related sequences, significantly improving the performance of the assay. Environmental applicability was tested with environmental and veterinary samples harboring complex microbial communities. Detection sensitivity in the range of 0.1% has been demonstrated, far below the 5% detection limit of traditional microbial diagnostic microarrays.

Bacteria↗
PubMed2004

Multivariate selection of genetic markers in diagnostic classification.

Analysis of gene expression data obtained from microarrays presents a new set of challenges to machine learning modeling. In this domain, in which the number of variables far exceeds the number of cases, identifying relevant genes or groups of genes that are good markers for a particular classification is as important as achieving good classification performance. Although several machine learning algorithms have been proposed to address the latter, identification of gene markers has not been systematically pursued. In this article, we investigate several algorithms for selecting gene markers for classification. We test these algorithms using logistic regression, as this is a simple and efficient supervised learning algorithm. We demonstrate, using 10 different data sets, that a conditionally univariate algorithm constitutes a viable choice if a researcher is interested in quickly determining a set of gene expression levels that can serve as markers for disease. We show that the classification performance of logistic regression is not very different from that of more sophisticated algorithms that have been applied in previous studies, and that the gene selection in the logistic regression algorithm is reasonable in both cases. Furthermore, the algorithm is simple, its theoretical basis is well established, and our user-friendly implementation is now freely available on the internet, serving as a benchmarking tool for the development of new algorithms.

Algorithms↗
PubMed2003

Administration of the soluble complement inhibitor, Crry-Ig, reduces inflammation and aquaporin 4 expression in lupus cerebritis.

Changes in brain water and cerebral volume can lead to brain edema that may be one of the underlying causes of death in many neurological diseases. Cerebral water content is regulated by aquaporin 4 (AQ4) present in astrocytic end feet and around blood vessels. In systemic lupus erythematosus (SLE), magnetic resonance imaging (MRI) studies of the brain have demonstrated lesions with the prominent appearance of edema. Activation of complement may play a significant role in the pathogenesis of lupus cerebritis by causing inflammation that can lead to edema. In this study, the well-established MRL/lpr lupus mouse model was used to evaluate the role of complement in lupus cerebritis. IgG and C1q colocalized in perivascular deposits indicating that the blood-brain barrier was compromised. Both RNA and protein expressions of AQ4 were significantly increased in brains of MRL/lpr mice. Chronic administration of the soluble complement inhibitor, Crry-Ig, reduced inflammation as measured by decreased accumulation of IgG. In contrast to control MRL/lpr mice, AQ4 expression in complement inhibited MRL/lpr mice was not changed relative to untreated congenic controls. These results illustrate that complement activation in brains of lupus mice leads to enhanced AQ4 expression and inflammation. It is conceivable that increased AQ4 expression results in cerebral edema and hence complement inhibition may provide a new therapeutic option in inflammatory cerebral disorders such as lupus cerebritis.

Animals↗
PubMed2004

Oligonucleotide ligation assay-based DNA chip for multiplex detection of single nucleotide polymorphism.

An oligonucleotide ligation assay-based DNA chip has been developed to detect single nucleotide polymorphism. Synthesized nonamers, complementary to the flanking sequences of the mutation sites in target DNA, were immobilized onto glass slides through disulfide bonds on their 5' terminus. Allele-specific pentamers annealed adjacent to the nonamers on the complementary target DNA, containing 5'-phosphate groups and biotin labeled 3'-ends, were mixed with the target DNA in tube. Ligation reactions between nonamers and pentamers were carried out on chips in the presence of T4 DNA ligase. Ligation products were directly visualized on chips through enzyme-linked assay. The effect of G:T mismatch at different positions of pentamers on the ligation were evaluated. The results showed that any mismatch between pentamer and the target DNA could lead to the decrease of ligation, which can be detected easily. The established approach was further used for multiplex detection of mutations in rpoB gene of rifampin-resistant Mycobacterium tuberculosis clinical isolates.

DNA↗
PubMed2004

Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays.

Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO(2) surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 micros, respectively, about 10(9) times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits.

Adsorption↗
PubMed2004

Identification and characterization of Bacillus anthracis by multiplex PCR on DNA chip.

Bacillus anthracis can be identified by detecting virulence factor genes located on two plasmids, pXO1 and pXO2. Combining multiplex PCR with arrayed anchored primer PCR and biotin-avidin alkaline phosphatase indicator system, we developed a qualitative DNA chip method for characterization of B. anthracis, and simultaneous confirmation of the species identity independent of plasmid contents. The assay amplifies pag gene (in pXO1), cap gene (in pXO2) and Ba813 gene (a B. anthracis specific chromosomal marker), and the results were indicated by an easy-to-read profile based on the color reaction of alkaline phosphatase. About 1 pg of specific DNA fragments on the chip wells could be detected after PCR. With the proposed method, the avirulent (pXO1+/2-, pXO1-/2+ and pXO1-/2-) strains of B. anthracis and distinguished 'anthrax-like' strains from other B. cereus group bacteria were unambiguously identified, while the genera other than Bacillus gave no positive signal.

Alkaline Phosphatase↗
PubMed2005

Label-free electrochemical DNA sensor based on functionalised conducting copolymer.

A simple and label-free electrochemical sensor for recognition of the DNA hybridization event was prepared based on a new functionalised conducting copolymer, poly[pyrrole-co-4-(3-pyrrolyl) butanoic acid]. This precursor copolymer can be easily electrodeposited on the electrode surface and shows high electroactivity in an aqueous medium. An amino-substituted oligonucleotide (ODN) probe was covalently grafted onto the surface of the copolymer in a one step procedure and tested on hybridization with complementary ODN segments. The cyclic voltammogram of ODN probe-modified copolymer showed very little change when incubated in presence of non-complementary ODN, while a significant, and reproducible, modification of the voltammogram was observed after addition of complementary ODN. The AC impedance spectrum showed an increased charge transfer resistance (Rct) and double layer capacitance of the sensor film after hybridisation. Sensors with thinner films showed higher sensitivity than thicker films, suggesting that hybridisation at or near the surface of the film produces a larger change in electrical properties than that within the body of the film.

Biosensing Techniques↗
PubMed2005

Cold-plasma modification of oxide surfaces for covalent biomolecule attachment.

While many processes have been developed to modify the surface of glass and other oxides for biomolecule attachment, they rely primarily upon wet chemistry and are costly and time-consuming. We describe a process that uses a cold plasma and a subsequent in vacuo vapor-phase reaction to terminate a variety of oxide surfaces with epoxide chemical groups. These epoxide groups can react with amine-containing biomolecules, such as proteins and modified oligonucleotides, to form strong covalent linkages between the biomolecules and the treated surface. The use of a plasma activation step followed by an in vacuo vapor-phase reaction allows for the precise control of surface functional groups, rather than the mixture of functionalities normally produced. By maintaining the samples under vacuum throughout the process, adsorption of contaminants is effectively eliminated. This process modifies a range of different oxide surfaces, is fast, consumes a minimal amount of reagents, and produces attachment densities for bound biomolecules that are comparable to or better than commercially available substrates.

Adsorption↗
PubMed2006

An efficient and versatile approach for the construction of oligonucleotide microarrays.

A new immobilization chemistry for covalent attachment of phosphorylated oligonucleotides on epoxy-activated glass surface via opening of oxirane ring is described. The proposed strategy results in excellent immobilization efficiency, spot homogeneity, and morphology. The constructed microarray was successfully demonstrated for discrimination of nucleotide mismatches.

Base Pair Mismatch↗
PubMed2005

Effects of degenerate oligonucleotide-primed polymerase chain reaction amplification and labeling methods on the sensitivity and specificity of metaphase- and array-based comparative genomic hybridization.

Degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) is often applied to small amounts of DNA from microdissected tissues in the analyses of chromosomal copy number with comparative genomic hybridization (CGH). The sensitivity and specificity in CGH analyses largely depend on the unbiased amplification and labeling of probe DNA, and the sensitivity and specificity should be high enough to detect one-copy changes in aneuploid cancer cells when accurate assessment of chromosomal instability is needed. The present study was designed to assess the effects of DOP-PCR and labeling method on the sensitivity of metaphase- and array-based CGHs in the detection of one-copy changes in near-tetraploid Kato-III cells. By focusing on several chromosomes whose absolute copy numbers were determined by FISH, we first compared the green-to-red ratio profiles of metaphase- and array-based CGH to the absolute copy numbers using the DNA diluted with varying proportions of lymphocyte DNA, with and without prior DOP-PCR amplification, and found that the amplification process scarcely affected the sensitivity but gave slightly lower specificity. Second, we compared random priming (RP) labeling with nick translation (NT) labeling and found that the RP labeling gave fewer false-positive gains and fewer false-negative losses in the detection of one-copy changes. In array CGH, locus-by-locus concordance between the DNAs with and without DOP-PCR amplification was high (nearly 100%) in the gain of three copies or more and the loss of two copies or more. This suggests that we could pinpoint the candidate genes within large-shift losses-gains that are detected with array CGH in microdissected tissues.

Cell Line, Tumor↗
PubMed2004

Effect of linoleic acid on proliferation and gene expression in the breast cancer cell line T47D.

Human and animal studies have linked n-6 polyunsaturated fatty acids with mammary carcinogenesis. We investigated the cellular and molecular effects of linoleic acid on the human breast cancer cell line T47D. Linoleic acid had a stimulatory effect on the growth of T47D cells, associated with an increase in the proportion of cells in the S phase of the cell cycle. Microarray, functional group and quantitative PCR analyses indicate that linoleic acid may affect T47D cell growth by modulation of the estrogen receptor (ERalpha), the G13alpha G protein, and p38 MAP kinase gene expression as well genes involved in RNA transcription and cell cycle regulation.

Cell Division↗