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At least 235 records · Page 13Linked to original sources
PubMed2005

Determination of affinity constants of locked nucleic acid (LNA) and DNA duplex formation using label free sensor technology.

Locked nucleic acid (LNA) is a nucleic acid analogue containing 2'-O,4'-C-methylene-beta-D-ribofuranosyl nucleotides, which have a bicyclic furanose unit locked in a RNA mimicking sugar conformation. Oligonucleotides containing LNA monomers show an enhanced thermal stability and robustness against nuclease mediated cleavage. Therefore special tailored LNA is a versatile tool for gene array analysis and single nucleotide polymorphism (SNP) analysis. The higher melting temperatures result from a higher affinity between the LNA and its complementary base. This was verified by the determination of the affinity constants of the duplex formation of 3 oligonucleotides: DNA, L-DNA, in which all thymidines are substituted by LNA, and a fully modified LNA, to their complementary DNA strand. Affinity constants were calculated to be 1.5 x 10(9), 4.0 x 10(9) and >10(12) L mol(-1). This was done using the label free and time resolved sensing technology reflectometric interference spectroscopy (RIfS), in an assay format similar to a titration called binding inhibition assay.

Chromatography, Affinity↗
PubMed2003

Analysis of RNA-protein interactions by flow cytometry.

Flow cytometry, in combination with advances in bead coding technologies, is maturing as a powerful high-throughput approach for analyzing molecular interactions. Applications of this technology include antibody assays and single nucleotide polymorphism mapping. This review describes the recent development of a microbead flow cytometric approach to analyze RNA-protein interactions and discusses emerging bead coding strategies that together will allow genome-wide identification of RNA-protein complexes. The microbead flow cytometric approach is flexible and provides new opportunities for functional genomic studies and small-molecule screening.

Flow Cytometry↗
PubMed2005

The role of S100A6 in pancreatic cancer development and its clinical implication as a diagnostic marker and therapeutic target.

Recent microarray analyses showed that the S100 family contains members that are candidate diagnostic markers or therapeutic targets. In the present study, to evaluate the involvement of S100A6 in pancreatic cancer and its clinical usefulness for diagnosis, we examined S100A6 mRNA expression in pancreatic tissues and pancreatic juice from patients with different pancreatic diseases. To investigate the role of S100A6 in carcinogenesis of pancreatic cancer and the potential of S100A6 as a diagnostic marker for early detection of pancreatic cancer, we did immunohistochemistry and microdissection-based mRNA analysis of pancreatic normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. We also used in vitro experiments and microarray analysis with RNA interference to evaluate the functional role of S100A6 and its potential as a therapeutic target for pancreatic cancer. S100A6 mRNA levels were significantly higher in carcinoma specimens than in nonneoplastic tissues. In pancreatic juice, there was a significant difference in S100A6 expression between patients with carcinoma and those with nonneoplastic disease. Receiver operating characteristic curves revealed that S100A6 might be a useful marker for diagnosis of pancreatic cancer. Immunohistochemistry and microdissection-based analysis showed differential expression of S100A6 among normal ducts, pancreatic intraepithelial neoplasias, and invasive ductal carcinomas. In vitro data showed that inhibition of S100A6 decreased proliferation and invasiveness of cancer cells, and these findings were supported by microarray data. Our present results suggest that quantitation of S100A6 mRNA is a promising tool for diagnosis of pancreatic cancer, and that S100A6 may be a promising therapeutic target for pancreatic cancer.

Biomarkers, Tumor↗
PubMed2001

Discovery and analysis of antisense oligonucleotide activity in cell culture.

In the past decade antisense oligonucleotides (ASOs) have proven to be a useful tool for dissection of gene function in molecular cell biology (Koller, E., Gaarde, W. A., and Monia, B. P. (2000) Trends Pharm. Sci., 21, 142-148), and validation of gene targets in animal models (Crooke, S. T. (1998) Biotechnol. Gen. Eng. Rev. 15, 121-157), as well as a means for therapeutic treatment of human diseases (Bennett, C. F. (1999) Exp. Opin. Invest. Drugs 8, 237-253). An important step toward usage of ASOs in the described applications is identification of an active ASO. This article describes the underlying basis and means for achieving this goal in cell culture.

Animals↗
PubMed2002

Gene expression profiling by DNA microarrays and its application to dental research.

UNLABELLED: DNA microarray technology has been used for genome-wide gene expression studies that incorporate molecular genetics and computer science skills on massive levels. The technology permits the simultaneous analysis of tens of thousands of genes for the purposes of gene discovery, disease diagnosis. improved drug development, and therapeutics tailored to specific disease processes. OBJECTIVE: In this review, the two most common microarray technologies and their potential application to dental research will be discussed. The authors review current articles pertaining to the technologies and analysis of mRNA expression using DNA micro-arrays and its application to dental research. Since many genes contribute to normal functioning, research efforts are moving from the search for a disease specific gene to the understanding of the biochemical and molecular functioning of a variety of genes and how complicated networks of interaction can lead to a disease state, such as oral cancer. With the incorporation of DNA micro-array based research, we can look forward to more accurate diagnosis and surgical treatment/drug-delivery therapy based on an individual patient's genetic profile.

Carcinoma, Squamous Cell↗
PubMed2000

Surface immobilized biochemical macromolecules studied by scanning Kelvin microprobe.

The measurement of work function is a particularly effective method for the characterization of surfaces because of the sensitivity of the parameter to interfacial structure, modification and overall chemistry. Accordingly, techniques for the analysis of work function offer a powerful tool for monitoring surface chemical changes, especially for situations involving the immobilization of new moieties at the interface. In the present paper, we describe the performance of a new, modified scanning Kelvin microprobe which is capable of the tandem measurement of contact potential and surface topography with resolutions of 1 mV and 10 nm, respectively. The lateral resolution is 1 micron. The instrument has been applied to the study of substrates modified by the attachment of biochemical macromolecules such as oligonucleotides and DNA. This preliminary work confirms the great potential of the technique in the study of biocompatibility, macromolecular structure and microarray devices.

Algorithms↗
PubMed2005

Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription.

Bacteremia↗
PubMed2005

A flexible array format for large-scale, rapid blood group DNA typing.

BACKGROUND: Typing for blood group antigens is currently performed by hemagglutination. The necessary reagents are becoming costly and limited in availability, and the methods are labor-intensive. The purpose of this study was to determine the feasibility of the use of large-scale DNA analysis in a microarray as a substitute for blood group typing. STUDY DESIGN AND METHODS: DNA, extracted from blood samples that had been phenotyped for some of the red blood cell antigens, was analyzed for selected blood group alleles by bead array (BeadChip, (BioArray Solutions Ltd., Warren, NJ) Illumina) [corrected] and by manual polymerase chain reaction (PCR)-based assays. Selected alleles were identified by enzyme-mediated elongation of probes, which were on color-encoded beads assembled into arrays on silicon chips. The performance of a prototype BeadChip (BioArray Solutions Ltd., Warren, NJ) [corrected] (BLOOD-1) containing single-nucleotide polymorphisms (SNPs) for FYA/B, FY-GATA, DOA/B, COA/B, LWA/B, DIA/B, and SC1/SC2 was verified with DNA from serologically characterized donors. It was then used to analyze more than 400 samples of partially defined phenotype. Samples from Chinese, Ashkenazi, and Thai donors (total n = 227) were tested with BLOOD-1. An expanded BeadChip (BioArray Solutions Ltd., Warren, NJ) [corrected] with a total of 18 SNPs (36 alleles; SNPs in BLOOD-1 and M/N, S/s, Lu(a)/Lu(b), K/k, FY265[for the Fy(X) polymorphism], Jk(a)/Jk(b), DO323[for Hy], DO350[for Jo(a)], and HgbS) was then evaluated with a subset of previously tested samples from Chinese, Ashkenazi, and New York blood donors (127) and an additional set of samples from Israeli donors (total n = 188). RESULTS: Results obtained by BeadChip (BioArray Solutions Ltd., Warren, NJ) [corrected] analysis were concordant with those obtained with the manual PCR-restriction fragment length polymorphism, allele-specific PCR, and hemagglutination assays. The frequencies of the alleles in the samples from different ethnic panels were within the expected ranges; however, two new DO alleles were discovered. CONCLUSION: It has been shown that microarray technology can be used to type DNA and detect new alleles in donor cohorts.

Alleles↗
PubMed2004

Multiplex PCR allows simultaneous detection of pathogens in ships' ballast water.

There is enormous potential for global transfer of microorganisms, including pathogens, in ships' ballast water. We contend that a major advancement in the study of ballast-water microorganisms in particular, and of aquatic pathogens in general, will be expedited sample analysis, such as provided by the elegant technology of DNA microarrays. In order to use DNA microarrays, however, one must establish the appropriate conditions to bind target sequences in samples to multiple probes on the microarrays. We conducted proof-of-concept experiments to optimize simultaneous detection of multiple microorganisms using polymerase chain reaction (PCR) and Southern hybridization. We chose three target organisms, all potentially found in ballast water: a calicivirus, the bacterium Vibrio cholerae, and the photosynthetic protist Aureococcus anophagefferens. Here, we show simultaneous detection of multiple pathogens is possible, a result supporting the promising future use of microarrays for simultaneous detection of pathogens in ballast water.

Blotting, Southern↗
PubMed2005

Identification of direct genomic targets downstream of the nuclear factor-kappaB transcription factor mediating tumor necrosis factor signaling.

Tumor necrosis factor (TNF) is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the nuclear factor-kappaB (NF-kappaB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kappaB-dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kappaB inhibitor to systematically identify NF-kappaB-dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kappaB signaling was analyzed. We identified 50 unique genes that were regulated by TNF (Pr(F)<0.001) and demonstrated a change in signal intensity of+/-3-fold relative to control. Of these, 28 were NF-kappaB-dependent, encoding proteins involved in diverse cellular activities. Quantitative real-time PCR assays of eight characterized NF-kappaB-dependent genes and five genes not previously known to be NF-kappaB-dependent (Gro-beta and-gamma, IkappaBepsilon, interleukin (IL)-7R, and Naf-1) were used to determine whether they were directly or indirectly NF-kappaB regulated. Expression of constitutively active enhanced green fluorescent.NF-kappaB/Rel A fusion protein transactivated all but IL-6 and IL-7R in the absence of TNF stimulation. Moreover, TNF strongly induced all 12 genes in the absence of new protein synthesis. High probability NF-kappaB sites in novel genes were predicted by binding site analysis and confirmed by electrophoretic mobility shift assay. Chromatin immunoprecipitation assays show the endogenous IkappaBalpha/epsilon, Gro-beta/gamma, and Naf-1 promoters directly bound NF-kappaB/Rel A in TNF-stimulated cells. Together, these studies systematically identify the direct NF-kappaB-dependent gene network downstream of TNF signaling, extending our knowledge of biological processes regulated by this pathway.

Binding Sites↗
PubMed2002

Gene expression analysis using oligonucleotide arrays produced by maskless photolithography.

Microarrays containing 195,000 in situ synthesized oligonucleotide features have been created using a benchtop, maskless photolithographic instrument. This instrument, the Maskless Array Synthesizer (MAS), uses a digital light processor (DLP) developed by Texas Instruments. The DLP creates the patterns of UV light used in the light-directed synthesis of oligonucleotides. This digital mask eliminates the need for expensive and time-consuming chromium masks. In this report, we describe experiments in which we tested this maskless technology for DNA synthesis on glass surfaces. Parameters examined included deprotection rates, repetitive yields, and oligonucleotide length. Custom gene expression arrays were manufactured and hybridized to Drosophila melanogaster and mouse samples. Quantitative PCR was used to validate the gene expression data from the mouse arrays.

Animals↗
PubMed2000

A novel microarray strategy for detecting genes and pathways in microbes with unsequenced genomes.

Expression profile analysis of genes provides valuable information concerning the genetic response of cells to stimuli. We describe an adaptation of this technology that can be used to probe for the expression of specific families of genes in microbial species. In our method a combination of sets of oligonucleotide probes representing fingerprint sequences specific to protein families is used to identify the presence and expression levels of family homologs in a microbial cell. We demonstrate computationally, using exemplars, that when the cDNA complement from an organism is sequentially screened against a set of specific motif oligonucleotides, statistically significant information can be obtained concerning the expression of the corresponding genes. This method can be used to identify specific genes and pathways simultaneously in several organisms of interest even in the absence of sequence information from the organisms.

Computational Biology↗
PubMed2006

Microarray oligonucleotide probes.

Oligonucleotide probes are increasingly the method of choice for many modern DNA microarray applications. They provide higher target specificity, probe selection gives improved experimental control of hybridization properties, and targeting of specific gene subsequences allows better discrimination of highly similar targets such as splice variants or gene families. Only recently has there been substantial progress in dealing with the complexities of probe set design and probe-specific signal interpretation. After a discussion of advantages and disadvantages of oligonucleotide probes in comparison to amplicons, this chapter focuses on recent advances and remaining key challenges in probe design and computational data analysis for spotted and in situ-synthesized oligonucleotide microarray technologies. Both experimental questions and computational aspects are addressed. Experimental issues discussed include the choice of an optimal number of probes per target and probe lengths and their influence on bias and random measurement noise, effects of different probe or substrate modifications, and laboratory protocols on signal specificity and sensitivity. Computational topics include practical considerations and a case study in probe sequence design, the exploitation of probing multiple target regions, and the modeling of probe sequence-specific signals. The current state of the art of the field is examined, and principled thermodynamic probe design criteria are proposed that are based on the free energy of the probe-target complex at the hybridization temperature rather than its melting temperature. Finally, this chapter notes and discusses an emerging trend in recent computational work toward a focus on signal interpretation rather than probe sequence design.

Animals↗
PubMed2002

Evaluating the specificity of antisense oligonucleotide conjugates. A DNA array analysis.

Antisense oligonucleotides are potentially powerful tools for selective control of cellular and viral gene expression. Crucial to successful application of this approach is the specificity of the oligonucleotide for the chosen RNA target. Here we apply DNA array technology to examine the specificity of antisense oligonucleotide treatments. The molecules used in these studies consisted of phosphorothioate oligomers linked to the Antennapedia (Ant) delivery peptide. The antisense oligonucleotide component was complementary to a site flanking the AUG of the MDR1 message, which codes for P-glycoprotein, a membrane ATPase associated with multidrug resistance in tumor cells. Using a DNA array of 2059 genes, we analyzed cellular responses to molecules comprised of Ant peptide-oligonucleotide conjugates, as well as to the Ant peptide alone. Besides the expected reduction in MDR1 message level, 37 other genes (approximately 2% of those tested) showed changes of comparable magnitude. The validity of the array results was confirmed for selected genes using Northern blots to assess messenger RNA levels. These results suggest that studies using antisense oligonucleotide technology to modulate gene expression need to be interpreted with caution.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2003

OligoDesign: Optimal design of LNA (locked nucleic acid) oligonucleotide capture probes for gene expression profiling.

We report the development of new software, OligoDesign, which provides optimal design of LNA (locked nucleic acid) substituted oligonucleotides for functional genomics applications. LNAs constitute a novel class of bicyclic RNA analogs having an exceptionally high affinity and specificity toward their complementary DNA and RNA target molecules. The OligoDesign software features recognition and filtering of the target sequence by genome-wide BLAST analysis in order to minimize cross-hybridization with non-target sequences. Furthermore it includes routines for prediction of melting temperature, self-annealing and secondary structure for LNA substituted oligonucleotides, as well as secondary structure prediction of the target nucleotide sequence. Individual scores for all these properties are calculated for each possible LNA oligonucleotide in the query gene and the OligoDesign program ranks the LNA capture probes according to a combined fuzzy logic score and finally returns the top scoring probes to the user in the output. We have successfully used the OligoDesign tool to design a Caenorhabditis elegans LNA oligonucleotide microarray, which allows monitoring of the expression of a set of 120 potential marker genes for a variety of stress and toxicological processes and toxicologically relevant pathways. The OligoDesign program is freely accessible at http://lnatools.com/.

Animals↗
PubMed2006

Recirculating flow accelerates DNA microarray hybridization in a microfluidic device.

A recirculating microfluidic device fabricated by laminating Mylar and glass was developed for the analysis of hybridization of oligonucleotides to DNA microarrays. The device is part of a system that provides controlled hybridization to DNA probes immobilized in a microarray of polyacrylamide gel pads using recirculation and temperature control. The system was used to obtain real-time kinetics of DNA hybridization and more accurate melting profiles of target-probe duplexes than possible using a static hybridization format. Recirculation shortened the time of perfect match target-probe hybridization from 6 hours to 2 hours and shifted the Td by 1.54 degrees C, relative to static conditions. The experimental results were consistent with a three-dimensional simulation of hybridization using a recirculating buffer system.

Buffers↗
PubMed2004

TEL/ETV6 is a signal transducer and activator of transcription 3 (Stat3)-induced repressor of Stat3 activity.

The signal transducer and activator of transcription 3 (Stat3) transcription factor is required for the antiproliferative effects induced by cytokines, such as the interleukin-6 type. In order to investigate the role of Stat3 in inhibition of cell proliferation, we have used an inducible Stat3 construct in A375 melanoma cells. We found that activation of Stat3 to moderate levels was sufficient to repress A375 proliferation, by slowing cell transit through the cell cycle. Enhanced and prolonged Stat3 activity led to cell cycle arrest and apoptosis. Genes whose expression was altered by Stat3 activation were identified by oligonucleotide microarray analysis. We found that TEL (ETV6), a novel Stat3 target identified in this study, is a negative regulator of Stat3 activity. Small interfering RNA-mediated inhibition of TEL expression resulted in increased Stat3-dependent transcriptional activity and stronger Stat3 antiproliferative activity. Confirming these results, overexpression of TEL repressed Stat3 transcriptional activity. Intriguingly, Stat3 repression did not require TEL DNA binding and appeared to proceed via recruitment of TEL to Stat3. Inhibition of Stat3 activity by TEL represents a novel mechanism regulating the Stat3 signaling pathway.

Blotting, Western↗
PubMed2004

Microarray-based AMASE as a novel approach for mutation detection.

Alterations in the p53 tumor suppressor gene are important events in many cases of human cancers. We have developed a novel microarray based approach for re-sequencing and mutation detection of the p53 gene. The method facilitates rapid and simple scanning of the target gene sequence and could be expanded to include other candidate cancer genes. The methodology employs the previously described apyrase-mediated allele-specific extension reaction (AMASE). In order to re-sequence the selected region, four extension oligonucleotides with different 3'-termini were used for each base position and they were covalently attached to the glass slide's surface. The amplified single-stranded DNA templates were then hybridized to the array followed by in situ extension with fluorescently labeled dNTPs in the presence of apyrase. The model system used was based on analysis of a 15 bp stretch in exon 5 of the p53 gene. Mutations were scored as allelic fractions calculated as (wt)/(wt + mut) signals. When apyrase was included in the extension reactions of wild type templates, the mean allelic fraction was 0.96. When apyrase was excluded with the same wild type templates, significantly lower allelic fractions were obtained. Two 60-mer synthetic oligonucleotides were used to establish the detectable amount of mutations with AMASE and a clear distinction between all the points could be made. Several samples from different stages of skin malignancies were also analyzed. The results from this study imply the possibility to efficiently and accurately re-sequence the entire p53 gene with AMASE technology.

Alleles↗