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Results for “Oligonucleotide Array Sequence Analysis”

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PubMed2003

Monitoring DNA hybridization on alkyl modified silicon surface through capacitance measurement.

Single strand oligodeoxynucleotide is attached to the alkyl modified silicon surface through a peptide bond. The oligodeoxynucleotide-modified silicon substrate is used as a working electrode in an electrochemical cell system. After the electrode is treated by a solution containing strands of complementary oligodeoxynucleotide the Mott-Schottky measurements exhibit obvious negative shift in the flat band potential of the electrode, while in a control experiment treated with a solution of non-complementary oligodeoxynucleotide such a shift does not occur. The DNA hybridization is also manifested in a real time capacitance measurement. A DNA sensor based on the capacitance measurement could be more convenient than that based on a fluorescence detection.

Alkylation↗
PubMed2003

A new approach for immobilization of oligonucleotides onto piezoelectric quartz crystal for preparation of a nucleic acid sensor for following hybridization.

The aim of this study is to develop a nucleic acid sensor based on piezoelectric crystal microbalance system (QCM) for following hybridization. Piezoelectric quartz crystal surfaces were first treated in a glow-discharge apparatus with ethylene diamine (EDA) plasma at 15 W (discharge power), 2.5 min (incubation time) and 35 ml/min (monomer flow rate) to create amino groups on the crystal surfaces. The thickness of the EDA-plasma film formed was about 43+/-24 A. Then, the amino groups on the crystal surfaces were converted to aldehyde groups by reacting the amino groups with glutaraldehyde (GA) at different conditions. A GA concentration of 2.5% and an incubation time of 2 h were selected as optimal values at this step, corresponding to a GA surface concentration of about 270 ng/cm2. A double strand Oligonucleotides, having one extra base on 5'-end of one of the complementary strands, were immobilized through the amino groups of this base onto the GA-modified crystals. Optimal immobilization conditions were as follows: oligonucleotide concentration: 1 microg/ml; time: 3 h; pH: 9.2 carbonate buffer; ionic strength: 0.1; and temperature: 20 degrees C. The QCM sensor carrying the covalently bound strand was used in the hybridization experiments, which showed that equilibrium is achieved in about 5 min, and the frequency shift measured is related to the concentration of the target strand to be measured within the medium.

Adsorption↗
PubMed2002

In vitro antitumor activities of 2,6-di-[2-(heteroaryl)vinyl]pyridines and pyridiniums.

The in vitro antitumor activities of 2,6-di-[2-(heteroaryl)vinyl]pyridines versus the standard National Cancer Institute 60 cell lines panel and of 2,6-di-[2-(heteroaryl)vinyl] pyridinium cations versus MCF7 (human mammary carcinoma) and LNCap (prostate carcinoma) cell lines are reported. Antiproliferative effects in both series are particularly evident for MCF7 mammary adenocarcinoma cells. Multivariate analysis of DNA microarray data for responsive tumor cell lines suggest a mechanistic pathway involving polyamine biosynthesis and prolactin signal transduction.

Antineoplastic Agents↗
PubMed2000

Aptamers as therapeutic and diagnostic agents.

Aptamers are oligonucleotides derived from an in vitro evolution process called SELEX. Aptamers have been evolved to bind proteins which are associated with a number of disease states. Using this method, many powerful antagonists of such proteins have been found. In order for these antagonists to work in animal models of disease and in humans, it is necessary to modify the aptamers. First of all, sugar modifications of nucleoside triphosphates are necessary to render the resulting aptamers resistant to nucleases found in serum. Changing the 2'OH groups of ribose to 2'F or 2'NH2 groups yields aptamers which are long lived in blood. The relatively low molecular weight of aptamers (8000-12000) leads to rapid clearance from the blood. Aptamers can be kept in the circulation from hours to days by conjugating them to higher molecular weight vehicles. When modified, conjugated aptamers are injected into animals, they inhibit physiological functions known to be associated with their target proteins. A new approach to diagnostics is also described. Aptamer arrays on solid surfaces will become available rapidly because the SELEX protocol has been successfully automated. The use of photo-cross-linkable aptamers will allow the covalent attachment of aptamers to their cognate proteins, with very low backgrounds from other proteins in body fluids. Finally, protein staining with any reagent which distinguishes functional groups of amino acids from those of nucleic acids (and the solid support) will give a direct readout of proteins on the solid support.

Animals↗
PubMed2000

In vitro selection of nucleic acids for diagnostic applications.

In vitro selection methods have proven to be extraordinarily adept at generating a wide variety of nucleic acid-binding species (aptamers) and catalysts (ribozymes). To date, selected nucleic acids have primarily been of academic interest. However, just as antibodies have proven utility as 'universal receptors' that can be crafted against a huge variety of ligands and can be readily adapted to diagnostic assays, aptamers may yet find application in assays. A new class of research reagents, aptazymes, are not mere mimics of antibodies but in fact allow the direct transduction of molecular recognition to catalysis. Aptamers and aptazymes may prove to be uniquely useful for the development of chip arrays for the detection and quantitation of a wide range of molecules in organismal proteomes and metabolomes.

Animals↗
PubMedDate not supplied

Efficacy of 2-methoxyethoxy-modified antisense oligonucleotides for the study of mouse preimplantation development.

The advent of microarray technology, coupled with the availability of mouse cDNA collections derived specifically from preimplantation embryos, helps to provide global gene expression profiles for the earliest stages of development. However, to determine the functions of the large numbers of genes of interest, massive systematic functional assays such as gene 'knockdown' experiments are required. As a first step, the relative suppression of blastocyst formation by differentially-modified antisense oligonucleotides to E-cadherin was assayed. The injection of 2'-methoxyethoxy (2'-MOE)-modified oligonucleotides blocked the formation of blastocysts in two-thirds of embryos, whereas the injection of either control missense 2'-MOE-oligonucleotides, or oligonucleotides with a Morpholino modification, had no significant effect on embryonic development. Thus, the 2'-MOE-modified antisense oligonucleotides are candidates for effective examination of roles of large numbers of genes during early embryological development.

Animals↗
PubMed2003

Development of a universal probe for electronic microarray and its application in characterization of the Staphylococcus aureus polC gene.

Electronic microarray technology is an exceptionally accurate and effective technique for detecting and defining single nucleotide polymorphisms (SNPs) in DNA sequences. Target oligonucleotides are electronically addressed to a gel matrix containing streptavidin to which biotinylated polymerase chain reaction (PCR) amplicons are bound. Typically, a fluorescent-labeled reporter oligonucleotide specific for each locus of interest is hybridized and reported. We detail the development of a universal reporter system to replace the standard method that is used to detect many different sequences accurately. The universal reporter eliminates the need to synthesize specific labeled reporters for each SNP sequence thereby dramatically reducing the cost and time required for assay development. The feasibility of this approach was demonstrated by successfully analyzing eight SNPs distributed within a highly variable 1-kb region of the polC gene from six isolates of Staphylococcus aureus.

Bacterial Proteins↗
PubMed2005

Microarrays: new tools to unravel parasite transcriptomes.

The ability to monitor the expression levels of thousands of genes in a single microarray experiment is a huge progression from conventional Northern blot analysis or PCR-based techniques. Microarrays can play a pivotal role in the mass screening of genes in a wide range of fields including parasitology. The relatively few parasites that can be readily cultured or isolated from a host, as compared with cell lines or tissue sources, makes microarray technology ideal for maximizing experimental results from a limiting source of starting material. Khan et al. (1999 a) commented in an early review of microarray technology " With this system in place, one can anticipate a time when data from thousands of gene expression experiments will be available for meta-analysis........leading to more robust results and subtle conclusions". Now in 2005, microarrays represent a very powerful resource that can play an important role in the characterization and annotation of the transcriptomes of many parasites of medical and veterinary importance.

Animals↗
PubMed1999

Whole genome genetic-typing in yeast using high-density oligonucleotide arrays.

Genome sequence information in combination with new technologies has allowed researchers to approach genetic problems in new ways. High-density oligonucleotide arrays were used to probe the genome content of the yeast Saccharomyces cerevisiae. We show that these arrays, containing oligonucleotides complementary to the sequenced strain of S. cerevisiae, can be used to identify open reading frames that are missing or present in higher or lower copy number in related isolates of S. cerevisiae. We apply this method to the characterization of the genome of a strain derived from a clinical isolate of S. cerevisiae. Our results show that the telomeres are the regions with the most variability between the two strains.

Gene Deletion↗
PubMed1999

The folding of large RNAs studied by hybridization to arrays of complementary oligonucleotides.

Folding pathways of large RNAs are poorly understood. We have addressed this question by hybridizing in vitro transcripts, which varied in size, to an array of antisense oligonucleotides. All transcripts included a common sequence and all but one shared the same start-point; the other had a small deletion of the 5' end. Minimal free energy calculations predicted quite different folds for these transcripts. However, hybridization to the array showed predominant features that were shared by transcripts of all lengths, though some oligonucleotides that hybridized strongly to the short transcripts gave weak interaction with longer transcripts. A full-length RNA fragment that had been denatured by heating and allowed to cool slowly gave the same hybridization result as a shorter transcript. Taken together, these results support theories that RNA folding creates local stable states that are trapped early in the transcription or folding process. As the transcript elongates, interactions are added between regions that are transcribed early and those transcribed late. The method here described helps in identifying regions in the transcripts that take part in long-range interactions.

Hot Temperature↗
PubMed2002

Effect of oligonucleotide truncation on single-nucleotide distinction by solid-phase hybridization.

Oligonucleotide microarrays are used to analyze target sequences on the basis of differences in hybridization stability between matched and mismatched probe-target duplexes. DNA microarray manufacture via photolithographic synthesis generates a minority of full-length oligonucleotide probes along with a series of 5'-truncated contaminants. In a model experiment, we now investigate the effect of truncated oligonucleotides on the ability to distinguish target sequence variants that differ in a single nucleotide position. A series of oligonucleotides, mixed in proportions simulating stepwise synthetic yields of between 82 and 100%, were bound to a solid support and allowed to hybridize to a target molecule. The extent of hybridization was monitored over a range of temperatures via the fluorescence of a double-strand-specific dye. The discriminatory power of pure oligonucleotide probes was found to be significantly greater than that of a population of truncated probes, but only over a limited temperature interval. We conclude that at optimal temperatures greater oligonucleotide quality can improve the performance of oligonucleotide hybridization microarrays.

Animals↗
PubMed2004

Characterization of a polymeric adsorbed coating for DNA microarray glass slides.

A new method was developed to covalently attach target molecules onto the surface of glass substrates such as microwell plates, beads, tubes, and microscope slides, for hybridization assays with fluorescent targets. The innovative concept introduced by this work is to physically adsorb onto underivatized glass surfaces a functional copolymer, able to graft amino-modified DNA molecules. The polymer, obtained by radical copolymerization of N,N-dimethylacrylamide, N-acryloyloxysuccinimide, and 3-(trimethoxysilyl)propyl methacrylate, copoly(DMA-NAS-MAPS), self-adsorbs onto the glass surface very quickly, typically in 5-30 min. The film, formed on the surface, bears active esters, which react with amino-modified DNA targets. The surface layer is stable in an aqueous buffer containing various additives (SDS, urea, salt), even at boiling temperature. It should be emphasized that the coating is formed by the immersion of glass slides in a diluted aqueous solution of the polymer. Therefore, the procedure is fast, inexpensive, robust, and reliable, and it does not require time-consuming glass pretreatments. Slides, coated with copoly(DMA-NAS-MAPS), were profitably used as substrates for the preparation of low-density DNA microarrays. The density and the thickness of the films were evaluated by X-ray reflectivity measurements whereas the extent of reaction of functional groups with DNA molecules was determined by a functional test. The experiments indicate that half of the active groups present on the surface reacts with oligonucleotide probes.

Acrylamides↗
PubMed2005

Tuned graft copolymers as controlled coatings for DNA microarrays.

DNA microarrays have become a powerful tool for expression profiling and other genomics applications. A critical factor for their sensitivity is the interfacial coating between the chip substrate and the bound DNA. Such a coating has to embrace the divergent requirements of tightly binding the capture probe DNA during the spotting process and of minimizing the nonspecific binding of target DNA during the hybridization assay. To fulfill these conditions, most coatings require a passivation step. Here we demonstrate how the chain density of a graft copolymer with a polycationic backbone, poly(l-lysine)-graft-poly(ethylene glycol), can be tuned such that the binding capacity during capture probe deposition is maximized while the nonspecific binding during hybridization assays is kept to a minimum, thus alleviating the requirement for a separate passivation procedure. Evidence for the superior performance of such coatings in terms of signal-to-noise ratio and spot quality is presented using an evanescent field-based fluorescent sensing technique (the ZeptoREADER). The surface architecture is further characterized using optical waveguide lightmode spectroscopy and time-of-flight secondary ion mass spectrometry. Finally, in a model assay, we demonstrate that expression changes can be detected from 1 microg of total mRNA sample material with a limit of detectable differential expression of +/-1.5.

DNA↗
PubMed2006

Microarray glass slides coated with block copolymer brushes obtained by reversible addition chain-transfer polymerization.

The reversible addition-fragmentation chain-transfer polymerization was used to prepare microarray slides grafted with polymer brushes for DNA-based applications. Block copolymer brushes of N,N-dimethylacrylamide (DMA) and glycidyl methacrylate (GMA), poly(DMA-b-GMA) were prepared by extending living poly(dimethylacrylamide) chains. The functional surface was used as a substrate for oligonucleotide hybridization experiments. The results were compared to those provided by glass slides coated by a self-assembled monolayer made of (3-glycidyloxypropyl)trimethoxysilane. Surfaces coated with block polymer brushes bearing oxirane groups are more efficient as substrates for oligonucleotide hybridization than surfaces coated with nonpolymeric self-assembled monolayers containing the same functional group. The high probe grafting density and hybridization efficiency achieved with this polymeric coating reveal the importance of the block architecture to ensure good accessibility of the immobilized probe. The new surface was characterized by static angle measurements and diffuse reflectance FT-IR spectroscopy on a silica model system.

Acrylic Resins↗
PubMed2006

Polychromatic microarrays: simultaneous multicolor array hybridization of eight samples.

High-throughput microscale platforms have transformed modern analytical investigations. Traditional microarray analyses involve a comparative approach, with two samples, a known control and an unknown sample, hybridized side-by-side and then contrasted for genetic differences. The samples are labeled with separate dyes and hybridized together, providing a differential expression pattern based on the reporter intensities. In contrast, the fiber-optic microarray platform described herein is analyzed with a microscope, thereby enabling the use of virtually any reporter, including quantum dots. The instrumentation takes advantage of the narrow emission bands characteristic of quantum dots to perform multiplexed detection of Bacillus anthracis. Advancing beyond the standard red/green microarray experiment, a panel of eight reporters were linked to eight B. anthracis samples and simultaneously analyzed in a microarray format. The ability to employ an assortment of reporters, along with the capacity to simultaneously hybridize eight samples confers an unprecedented flexibility to array-based analyses, providing a 4-fold increase in throughput over standard two-color assays.

Bacillus anthracis↗