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Sample records for density oligonucleotide microarray

  1. Development of low-density oligonucleotide microarrays for detecting mutations causing Wilson's disease

    PubMed Central

    Mathur, Manjula; Singh, Ekta; Poduval, T.B.; Rao, Akkipeddi V.S.S.N.

    2015-01-01

    Background & objectives: Wilson's disease (WD) is an autosomal recessive disorder caused by mutations in ATP7B, a copper transporter gene, leading to hepatic and neuropsychiatric manifestations due to copper accumulation. If diagnosed early, WD patients can be managed by medicines reducing morbidity and mortality. Diagnosis of this disease requires a combination of tests and at times is inconclusive due to overlap of the symptoms with other disorders. Genetic testing is the preferred alternative in such cases particularly for individuals with a family history. Use of DNA microarray for detecting mutations in ATP7B gene is gaining popularity because of the advantages it offers in terms of throughput and sensitivity. This study attempts to establish the quality analysis procedures for microarray based diagnosis of Wilson's disease. Methods: A home-made microarrayer was used to print oligonucleotide based low-density microarrays for addressing 62 mutations causing Wilson's disease reported from Indian population. Inter- and intra- array comparisons were used to study quality of the arrays. The arrays were validated by using mutant samples generated by site directed mutagenesis. Results: The hybridization reaction were found to be consistent across the surface of a given microarray. Our results have shown that 52 °C post-hybridization wash yields better reproducibility across experiments compared to 42 °C. Our arrays have shown > 80 per cent sensitivity in detecting these 62 mutations. Interpretation & conclusions: The present results demonstrate the design and evaluation of a low-density microarray for the detection of 62 mutations in ATP7B gene, and show that a microarray based approach can be cost-effective for detecting a large number of mutations simultaneously. This study also provides information on some of the important parameters required for microarray based diagnosis of genetic disorders. PMID:25900953

  2. Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

    NASA Technical Reports Server (NTRS)

    Moseyko, Nick; Zhu, Tong; Chang, Hur-Song; Wang, Xun; Feldman, Lewis J.

    2002-01-01

    Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.

  3. Pigeons: A Novel GUI Software for Analysing and Parsing High Density Heterologous Oligonucleotide Microarray Probe Level Data

    PubMed Central

    Lai, Hung-Ming; May, Sean T.; Mayes, Sean

    2014-01-01

    Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip® probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC).

  4. Detection of microbial communities in continuous and discontinuous membrane bioreactor using high-density oligonucleotide Microarray

    NASA Astrophysics Data System (ADS)

    Duan, Liang; Song, Yonghui; Xia, Siqing; Hermanowicz, Slawomir W.

    2010-11-01

    This study compared the whole composition of microbial communities in continuous-flow (MBR) and batch-fed (discontinuous) (MSBR) aerobic membrane bioreactors using high-density universal 16S rRNA Microarray. The array includes 506,944 probes targeted to 8935 clusters in 16S rRNA gene sequences. The Microarray results showed that both MBR and MSBR had high microbial diversity. 1126 and 1002 bacterial subfamilies were detected and can separate as 37 and 32 phyla in MBR and MSBR, respectively. Proteobacteria was the predominant phylum, 703 and 597 subfamilies were found in two systems, which constituted 62.4% and 59.6% of the whole bacteria. Gamma- and Alpha-were the dominant classes in Proteobacteria. It occupied 38.1% and 26.3%, 31.2% and 39.2% for MBR and MSBR, respectively. Bacteroidetes, Firmicutes and Actinobacteria were the subdominant groups, occupying around 9.4% and 7.6%, 6.1% and 6.5%, 6.0% and 9.0% of the total bacteria in two reactors. Some bacterial groups such as Acidobacteria, Chloroflexi, Cyanobacteria, Verrucomicrobia and Spirochaetes also found more than 15 subfamilies. All the results indicated that the MBR system had more bacteria community diversity than MSBR's. Moreover, it was very interested that MBR and MSBR had almost the same bacterial composition except Enterobacteriaceae. 63 OTUs of Enterobacteriaceae were detected in MBR, while just 10 OTUs were found in MSBR. That's one of the reasons leading to the difference of the bacterial diversity between two bioreactors.

  5. An imputation approach for oligonucleotide microarrays.

    PubMed

    Li, Ming; Wen, Yalu; Lu, Qing; Fu, Wenjiang J

    2013-01-01

    Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as "bright spots", "dark clouds", and "shadowy circles", etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request. PMID:23505547

  6. PERFORMANCE CHARACTERISTICS OF 65-MER OLIGONUCLEOTIDE MICROARRAYS

    PubMed Central

    Lee, Myoyong; Xiang, Charlie C.; Trent, Jeffrey M.; Bittner, Michael L.

    2009-01-01

    Microarray fabrication using pre-synthesized long oligonucleotide is becoming increasingly important, but a study of large-scale array productions is not published yet. We addressed the issue of fabricating oligonucleotide microarrays by spotting commercial, pre-synthesized 65-mers with 5′ amines representing 7500 murine genes. Amine-modified oligonucleotides were immobilized on glass slides having aldehyde groups via transient Schiff base formation followed by reduction to produce a covalent conjugate. When RNA derived from the same source was used for Cy3 and Cy5 labeling and hybridized to the same array, signal intensities spanning three orders of magnitude were observed, and the coefficient of variation between the two channels for all spots was 8–10%. To ascertain the reproducibility of ratio determination of these arrays, two triplicate hybridizations (with fluorochrome reversal) comparing RNAs from a fibroblast (NIH3T3) and a breast cancer (JC) cell line were carried out. The 95% confidence interval for all spots in the six hybridizations was 0.60 – 1.66. This level of reproducibility allows use of the full range of pattern finding and discriminant analysis typically applied to cDNA microarrays. Further comparative testing was carried out with oligonucleotide microarrays, cDNA microarrays and RT-PCR assays to examine the comparability of results across these different methodologies. PMID:17617369

  7. Application of a High-Density Oligonucleotide Microarray Approach To Study Bacterial Population Dynamics during Uranium Reduction and Reoxidation†

    PubMed Central

    Brodie, Eoin L.; DeSantis, Todd Z.; Joyner, Dominique C.; Baek, Seung M.; Larsen, Joern T.; Andersen, Gary L.; Hazen, Terry C.; Richardson, Paul M.; Herman, Donald J.; Tokunaga, Tetsu K.; Wan, Jiamin M.; Firestone, Mary K.

    2006-01-01

    Reduction of soluble uranium U(VI) to less-soluble uranium U(IV) is a promising approach to minimize migration from contaminated aquifers. It is generally assumed that, under constant reducing conditions, U(IV) is stable and immobile; however, in a previous study, we documented reoxidation of U(IV) under continuous reducing conditions (Wan et al., Environ. Sci. Technol. 2005, 39:6162-6169). To determine if changes in microbial community composition were a factor in U(IV) reoxidation, we employed a high-density phylogenetic DNA microarray (16S microarray) containing 500,000 probes to monitor changes in bacterial populations during this remediation process. Comparison of the 16S microarray with clone libraries demonstrated successful detection and classification of most clone groups. Analysis of the most dynamic groups of 16S rRNA gene amplicons detected by the 16S microarray identified five clusters of bacterial subfamilies responding in a similar manner. This approach demonstrated that amplicons of known metal-reducing bacteria such as Geothrix fermentans (confirmed by quantitative PCR) and those within the Geobacteraceae were abundant during U(VI) reduction and did not decline during the U(IV) reoxidation phase. Significantly, it appears that the observed reoxidation of uranium under reducing conditions occurred despite elevated microbial activity and the consistent presence of metal-reducing bacteria. High-density phylogenetic microarrays constitute a powerful tool, enabling the detection and monitoring of a substantial portion of the microbial population in a routine, accurate, and reproducible manner. PMID:16957256

  8. Long synthetic oligonucleotides for microarray expression measurement

    NASA Astrophysics Data System (ADS)

    Li, Jiong; Wang, Hong; Liu, Heping; Zhang, M.; Zhang, Chunxiu; Lu, Zu-Hong; Gao, Xiang; Kong, Dong

    2001-09-01

    There are generally two kinds of DNA microarray used for genomic-scale gene expression profiling of mRNA: cDNA and DNA chip, but both of them suffer from some drawbacks. To meet more requirements, another oligonucleotide microarray with long was produced. This type of microarray had the advantages of low cost, minimal Cross-hybridization, flexible and easy to make, which is most fit for small laboratories with special purposes. In this paper, we devised different probes with different probe lengths, GC contents and gene positions to optimization the probe design. Experiments showed 70 mer probes are suitable for both sufficient sensitivity and reasonable costs. Higher G-C content produces stronger signal intensity thus better sensitivity and probes designed at 3 untranslated region of gene within the range of 300 pb should be best for both sensitivity and specificity.

  9. PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

    EPA Science Inventory

    Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

  10. Genome-wide approach to identify second gene targets for malignant rhabdoid tumors using high-density oligonucleotide microarrays

    PubMed Central

    Takita, Junko; Chen, Yuyan; Kato, Motohiro; Ohki, Kentaro; Sato, Yusuke; Ohta, Shigeru; Sugita, Kanji; Nishimura, Riki; Hoshino, Noriko; Seki, Masafumi; Sanada, Masashi; Oka, Akira; Hayashi, Yasuhide; Ogawa, Seishi

    2014-01-01

    Malignant rhabdoid tumor (MRT) is a rare and highly lethal cancer that mainly affects infants and young children. The majority of MRT are characterized by loss of function of SMARCB1 on chromosome 22q11.2. However, little is known about genetic changes other than SMARCB1 alterations that are responsible for the development and/or progression of MRT. To explore additional gene targets in MRT, we analyzed 21 MRT specimens (12 fresh tumors and 9 MRT-derived cell lines) using high-density single nucleotide polymorphism genotyping microarrays. Although MRT genomes are characterized by common 22q11.2 deletions, affecting the SMARCB1 locus with a frequency of 95.2% (20/21 specimens), other genetic changes have been less frequent. Of the 20 specimens with deletions of 22q11.2, eight specimens showed uniparental disomy of the SMARCB1 locus with homozygous deletions or gene mutations. High-resolution analysis also disclosed the recurrent hemizygous/homozygous deletions of 7q35–q36.1, involving the CNTNAP2 locus in three specimens. Mutations analysis of CNTNAP2 showed a novel R157C missense mutation in a primary case, and methylation analysis showed recurrent hypermethylation of CNTNAP2 in three of nine cell lines. These results demonstrated that CNTNAP2 is one of the additional gene targets, other than SMARCB1, in MRT. PMID:24418192

  11. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    SciTech Connect

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S.; Golova, Julia; Perov, Alexander N.; Protic, Miroslava; Robison, Richard; Shipma, Matthew; White, Amanda M.; Willse, Alan R.

    2006-01-01

    A diagnostic, genome-independent microbial fingerprinting method using DNA oligonucleotide microarrays was used for high-resolution differentiation between closely related Bacillus strains, including two strains of Bacillus anthracis that are monomorphic (indistinguishable) via amplified fragment length polymorphism fingerprinting techniques. Replicated hybridizations on 391-probe nonamer arrays were used to construct a prototype fingerprint library for quantitative comparisons. Descriptive analysis of the fingerprints, including phylogenetic reconstruction, is consistent with previous taxonomic organization of the genus. Newly developed statistical analysis methods were used to quantitatively compare and objectively confirm apparent differences in microarray fingerprints with the statistical rigor required for microbial forensics and clinical diagnostics. These data suggest that a relatively simple fingerprinting microarray and statistical analysis method can differentiate between species in the Bacillus cereus complex, and between strains of B. anthracis. A synthetic DNA standard was used to understand underlying microarray and process-level variability, leading to specific recommendations for the development of a standard operating procedure and/or continued technology enhancements for microbial forensics and diagnostics.

  12. Design and analysis of mismatch probes for long oligonucleotide microarrays

    SciTech Connect

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  13. A Comparative Study of Normalization Methods Used in Statistical Analysis of Oligonucleotide Microarray Data

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Normalization methods used in the statistical analysis of oligonucleotide microarray data were evaluated. The oligonucleotide microarray is considered an efficient analytical tool for analyzing thousands of genes simultaneously in a single experiment. However, systematic variation in microarray, ori...

  14. Particle-Based Microarrays of Oligonucleotides and Oligopeptides

    PubMed Central

    Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K.; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F. Ralf; Breitling, Frank; Loeffler, Felix F.

    2014-01-01

    In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

  15. Gene expression profiling in peanut using oligonucleotide microarrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently have a moderately significant number of ESTs been released into the public domain. Utilization of these ESTs for the oligonucleotide microarrays provides a means to investigate l...

  16. Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

    PubMed

    Francois, Patrice; Charbonnier, Yvan; Jacquet, Jean; Utinger, Dominic; Bento, Manuela; Lew, Daniel; Kresbach, Gerhard M; Ehrat, Markus; Schlegel, Werner; Schrenzel, Jacques

    2006-06-01

    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. PMID:16216356

  17. [Research progress of probe design software of oligonucleotide microarrays].

    PubMed

    Chen, Xi; Wu, Zaoquan; Liu, Zhengchun

    2014-02-01

    DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software. PMID:24804514

  18. Oligonucleotide microarray for subtyping of influenza A viruses

    NASA Astrophysics Data System (ADS)

    Klotchenko, S. A.; Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Chervyakova, O. V.; Smirnova, E. A.; Kushnareva, E. V.; Strochkov, V. M.; Taylakova, E. T.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Sansyzbay, A. R.

    2012-02-01

    Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

  19. Portable system for microbial sample preparation and oligonucleotide microarray analysis.

    PubMed

    Bavykin, S G; Akowski, J P; Zakhariev, V M; Barsky, V E; Perov, A N; Mirzabekov, A D

    2001-02-01

    We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager. PMID:11157263

  20. Portable system for microbial sample preparation and oligonucleotide microarray analysis.

    SciTech Connect

    Bavykin, S. G.; Akowski, J. P.; Zakhariev, V. M.; Barsky, V. E.; Mirzabekov, A. D.; Perov, A. N.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2001-02-01

    We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.

  1. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    PubMed Central

    2010-01-01

    Background Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. Results Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals) and Plasmodium falciparum (a related parasite responsible for severe human malaria), we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP)-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis) and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at pilot scale to inform

  2. Human papilloma virus strain detection utilising custom-designed oligonucleotide microarrays.

    PubMed

    Ayers, Duncan; Platt, Mark; Javad, Farzad; Day, Philip J R

    2011-01-01

    Within the past 15 years, the utilisation of microarray technology for the detection of specific pathogen strains has increased rapidly. Presently, it is possible to simply purchase a pre-manufactured "off the shelf " oligonucleotide microarray bearing a wide variety of known signature DNA sequences previously identified in the organism being studied. Consequently, a hybridisation analysis may be used to pinpoint which strain/s is present in any given clinical sample. However, there exists a problem if the study necessitates the identification of novel sequences which are not represented in commercially available microarray chips. Ideally, such investigations require an in situ oligonucleotide microarray platform with the capacity to synthesise microarrays bearing probe sequences designed solely by the researcher. This chapter will focus on the employment of the Combimatrix® B3 CustomArray™ for the synthesis of reusable, bespoke microarrays for the purpose of discerning multiple Human Papilloma Virus strains. PMID:20938834

  3. Simulation and visualization of flow pattern in microarrays for liquid phase oligonucleotide and peptide synthesis.

    PubMed

    O-Charoen, Sirimon; Srivannavit, Onnop; Gulari, Erdogan

    2007-01-01

    Microfluidic microarrays have been developed for economical and rapid parallel synthesis of oligonucleotide and peptide libraries. For a synthesis system to be reproducible and uniform, it is crucial to have a uniform reagent delivery throughout the system. Computational fluid dynamics (CFD) is used to model and simulate the microfluidic microarrays to study geometrical effects on flow patterns. By proper design geometry, flow uniformity could be obtained in every microreactor in the microarrays. PMID:17480053

  4. SIMULATION AND VISUALIZATION OF FLOW PATTERN IN MICROARRAYS FOR LIQUID PHASE OLIGONUCLEOTIDE AND PEPTIDE SYNTHESIS

    PubMed Central

    O-Charoen, Sirimon; Srivannavit, Onnop; Gulari, Erdogan

    2008-01-01

    Microfluidic microarrays have been developed for economical and rapid parallel synthesis of oligonucleotide and peptide libraries. For a synthesis system to be reproducible and uniform, it is crucial to have a uniform reagent delivery throughout the system. Computational fluid dynamics (CFD) is used to model and simulate the microfluidic microarrays to study geometrical effects on flow patterns. By proper design geometry, flow uniformity could be obtained in every microreactor in the microarrays. PMID:17480053

  5. High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

    PubMed Central

    Borovkov, Alex Y.; Loskutov, Andrey V.; Robida, Mark D.; Day, Kristen M.; Cano, Jose A.; Le Olson, Tien; Patel, Hetal; Brown, Kevin; Hunter, Preston D.; Sykes, Kathryn F.

    2010-01-01

    To meet the growing demand for synthetic genes more robust, scalable and inexpensive gene assembly technologies must be developed. Here, we present a protocol for high-quality gene assembly directly from low-cost marginal-quality microarray-synthesized oligonucleotides. Significantly, we eliminated the time- and money-consuming oligonucleotide purification steps through the use of hybridization-based selection embedded in the assembly process. The protocol was tested on mixtures of up to 2000 oligonucleotides eluted directly from microarrays obtained from three different chip manufacturers. These mixtures containing <5% perfect oligos, and were used directly for assembly of 27 test genes of different sizes. Gene quality was assessed by sequencing, and their activity was tested in coupled in vitro transcription/translation reactions. Genes assembled from the microarray-eluted material using the new protocol matched the quality of the genes assembled from >95% pure column-synthesized oligonucleotides by the standard protocol. Both averaged only 2.7 errors/kb, and genes assembled from microarray-eluted material without clonal selection produced only 30% less protein than sequence-confirmed clones. This report represents the first demonstration of cost-efficient gene assembly from microarray-synthesized oligonucleotides. The overall cost of assembly by this method approaches 5¢ per base, making gene synthesis more affordable than traditional cloning. PMID:20693531

  6. Traceback identification of plant components in commercial compound feed through an oligonucleotide microarray based on tubulin intron polymorphism.

    PubMed

    Ponzoni, Elena; Morello, Laura; Gianì, Silvia; Breviario, Diego

    2014-11-01

    According to EU Regulations, all components of commercial compound feed need to be declared on the label. Effective protection against fraud requires severe controls based on accurate analytical methods to ascertain what is declared by the producers. The aim of this work was to develop an oligonucleotide microarray for the molecular recognition of multiple plant components in commercial feeds. We tested the potential of the highly polymorphic first intron sequences from members of the plant β-tubulin gene family as a target for plant DNA identification. 23 oligonucleotide capture probes, targeting species-specific intron sequences, were assembled within a low density microarray for the identification of 10 plant species, selected from among those most commonly used in cattle feed formulation. The ability of the array to detect specific components in complex flour blends and in compound feed was evaluated. PMID:24874359

  7. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L.) gene expression oligonucleotide microarray.

    PubMed

    Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement. PMID:23110046

  8. Development, Characterization and Experimental Validation of a Cultivated Sunflower (Helianthus annuus L.) Gene Expression Oligonucleotide Microarray

    PubMed Central

    Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement. PMID:23110046

  9. POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer

    PubMed Central

    Lausted, Christopher; Dahl, Timothy; Warren, Charles; King, Kimberly; Smith, Kimberly; Johnson, Michael; Saleem, Ramsey; Aitchison, John; Hood, Lee; Lasky, Stephen R

    2004-01-01

    DNA arrays are valuable tools in molecular biology laboratories. Their rapid acceptance was aided by the release of plans for a pin-spotting microarrayer by researchers at Stanford. Inkjet microarraying is a flexible, complementary technique that allows the synthesis of arrays of any oligonucleotide sequences de novo. We describe here an open-source inkjet arrayer capable of rapidly producing sets of unique 9,800-feature arrays. PMID:15287980

  10. Oligonucleotide microarray expression profiling of contrasting invasive phenotypes in colorectal cancer.

    PubMed

    Thorn, Christopher C; Williams, Deborah; Freeman, Thomas C

    2011-01-01

    This chapter refers to the application of laser-capture microdissection with oligonucleotide microarray analysis. The protocol described has been successfully used to identify differential transcript expression between contrasting colorectal cancer invasive phenotypes. Tissue processing, RNA extraction, quality control, amplification, fluorescent labelling, purification, hybridisation, and elements of data analysis are covered. PMID:21761306

  11. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  12. Quantitative Oligonucleotide Microarray Fingerprinting of Salmonella enterica isolates

    SciTech Connect

    Willse, Alan R.; Straub, Tim M.; Wunschel, Sharon C.; Small, Jack A.; Call, Douglas R.; Daly, Don S.; Chandler, Darrell P.

    2004-03-22

    We report on a genome-independent microbial fingerprinting method using nucleic acid microarrays for microbial forensics and epidemiology applications. We demonstrate that the microarray method provides high-resolution differentiation between closely related microorganisms using Salmonella enterica strains. In replicate trials we used a simple 192-probe nonamer array to construct a fingerprint library of 25 closely related Salmonella isolates. Controlling false discovery rate for multiple testing at alpha =.05, at least 295 of 300 pairs of S. enterica isolate fingerprints were found to be statistically distinct using a modified Hotelling Tsquared test. Although we find most pairs of Salmonella fingerprints to be distinct, forensic applications will also require a protocol for library construction and reliable microbial classification against a fingerprint library. We outline additional steps required to produce a protocol for library construction and reliable classification of unknown organisms.

  13. Improved DNA microarray detection sensitivity through immobilization of preformed in solution streptavidin/biotinylated oligonucleotide conjugates.

    PubMed

    Mavrogiannopoulou, E; Petrou, P S; Koukouvinos, G; Yannoukakos, D; Siafaka-Kapadai, A; Fornal, K; Awsiuk, K; Budkowski, A; Kakabakos, S E

    2015-04-01

    A novel immobilization approach involving binding of preformed streptavidin/biotinylated oligonucleotide conjugates onto surfaces coated with biotinylated bovine serum albumin is presented. Microarrays prepared according to the proposed method were compared, in terms of detection sensitivity and specificity, with other immobilization schemes employing coupling of biotinylated oligonucleotides onto directly adsorbed surface streptavidin, or sequential coupling of streptavidin and biotinylated oligonucleotides onto a layer of adsorbed biotinylated bovine serum albumin. A comparison was performed employing biotinylated oligonucleotides corresponding to wild- and mutant-type sequences of seven single point mutations of the BRCA1 gene. With respect to the other immobilization protocols, the proposed oligonucleotide immobilization approach offered the highest hybridization signals (at least 5 times higher) and permitted more elaborative washings, thus providing considerably higher discrimination between complimentary and non-complementary DNA sequences for all mutations tested. In addition, the hybridization kinetics were significantly enhanced compared to two other immobilization protocols, permitting PCR sample analysis in less than 40 min. Thus, the proposed oligonucleotide immobilization approach offered improved detection sensitivity and discrimination ability along with considerably reduced analysis time, and it is expected to find wide application in DNA mutation detection. PMID:25805150

  14. Construction and evaluation of a Clostridium thermocellum ATCC 27405 whole-genome oligonucleotide microarray

    SciTech Connect

    Brown, Steven David; Raman, Babu; McKeown, Catherine K; Kale, Shubhangi P; He, Zhili; Mielenz, Jonathan R

    2007-04-01

    Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.

  15. Construction and Evaluation of a Clostridium thermocellum ATCC 27405 Whole-Genome Oligonucleotide Microarray

    NASA Astrophysics Data System (ADS)

    Brown, Steven D.; Raman, Babu; McKeown, Catherine K.; Kale, Shubha P.; He, Zhili; Mielenz, Jonathan R.

    Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.

  16. Innovative instrumentation for microarray scanning and analysis: application for characterization of oligonucleotide duplexes behavior.

    PubMed

    Khomyakova, E B; Dreval, E V; Tran-Dang, M; Potier, M C; Soussaline, F P

    2004-05-01

    Accuracy in microarray technology requires new approaches to microarray reader development. A microarray reader system (optical scanning array or OSA reader) based on automated microscopy with large field of view, high speed 3 axis scanning at multiple narrow-band spectra of excitation light has been developed. It allows fast capture of high-resolution, multi-fluorescence images and is characterized by a linear dynamic range and sensitivity comparable to commonly used photo-multiplier tube (PMT)-based laser scanner. Controlled by high performance software, the instrument can be used for scanning and quantitative analysis of any type of dry microarray. Studies implying temperature-controlled hybridization chamber containing a microarray can also be performed. This enables the registration of kinetics and melting curves. This feature is required in a wide range of on-chip chemical and enzymatic reactions including on-chip PCR amplification. We used the OSA reader for the characterization of hybridization and melting behaviour of oligonucleotide:oligonucleotide duplexes on three-dimensional Code Link slides. PMID:15209342

  17. Drop drying on surfaces determines chemical reactivity - the specific case of immobilization of oligonucleotides on microarrays

    PubMed Central

    2013-01-01

    Background Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface. Results We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5′-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface. Conclusions Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different

  18. Development and Validation of an Oligonucleotide Microarray for Detection of Multiple Virulence and Antimicrobial Resistance Genes in Escherichia coli†

    PubMed Central

    Bruant, Guillaume; Maynard, Christine; Bekal, Sadjia; Gaucher, Isabelle; Masson, Luke; Brousseau, Roland; Harel, Josée

    2006-01-01

    An oligonucleotide microarray detecting 189 Escherichia coli virulence genes or markers and 30 antimicrobial resistance genes was designed and validated using DNA from known reference strains. This microarray was confirmed to be a powerful diagnostic tool for monitoring emerging E. coli pathotypes and antimicrobial resistance, as well as for environmental, epidemiological, and phylogenetic studies including the evaluation of genome plasticity. PMID:16672535

  19. Selection of optimal oligonucleotide probes for microarrays using multiple criteria, global alignment and parameter estimation

    PubMed Central

    Li, Xingyuan; He, Zhili; Zhou, Jizhong

    2005-01-01

    The oligonucleotide specificity for microarray hybridization can be predicted by its sequence identity to non-targets, continuous stretch to non-targets, and/or binding free energy to non-targets. Most currently available programs only use one or two of these criteria, which may choose ‘false’ specific oligonucleotides or miss ‘true’ optimal probes in a considerable proportion. We have developed a software tool, called CommOligo using new algorithms and all three criteria for selection of optimal oligonucleotide probes. A series of filters, including sequence identity, free energy, continuous stretch, GC content, self-annealing, distance to the 3′-untranslated region (3′-UTR) and melting temperature (Tm), are used to check each possible oligonucleotide. A sequence identity is calculated based on gapped global alignments. A traversal algorithm is used to generate alignments for free energy calculation. The optimal Tm interval is determined based on probe candidates that have passed all other filters. Final probes are picked using a combination of user-configurable piece-wise linear functions and an iterative process. The thresholds for identity, stretch and free energy filters are automatically determined from experimental data by an accessory software tool, CommOligo_PE (CommOligo Parameter Estimator). The program was used to design probes for both whole-genome and highly homologous sequence data. CommOligo and CommOligo_PE are freely available to academic users upon request. PMID:16246912

  20. High-throughput detection of food-borne pathogenic bacteria using oligonucleotide microarray with quantum dots as fluorescent labels.

    PubMed

    Huang, Aihua; Qiu, Zhigang; Jin, Min; Shen, Zhiqiang; Chen, Zhaoli; Wang, Xinwei; Li, Jun-Wen

    2014-08-18

    Bacterial pathogens are mostly responsible for food-borne diseases, and there is still substantial room for improvement in the effective detection of these organisms. In the present study, we explored a new method to detect target pathogens easily and rapidly with high sensitivity and specificity. This method uses an oligonucleotide microarray combined with quantum dots as fluorescent labels. Oligonucleotide probes targeting the 16SrRNA gene were synthesized to create an oligonucleotide microarray. The PCR products labeled with biotin were subsequently hybridized using an oligonucleotide microarray. Following incubation with CdSe/ZnS quantum dots coated with streptavidin, fluorescent signals were detected with a PerkinElmer Gx Microarray Scanner. The results clearly showed specific hybridization profiles corresponding to the bacterial species assessed. Two hundred and sixteen strains of food-borne bacterial pathogens, including standard strains and isolated strains from food samples, were used to test the specificity, stability, and sensitivity of the microarray system. We found that the oligonucleotide microarray combined with quantum dots used as fluorescent labels can successfully discriminate the bacterial organisms at the genera or species level, with high specificity and stability as well as a sensitivity of 10 colony forming units (CFU)/mL of pure culture. We further tested 105 mock-contaminated food samples and achieved consistent results as those obtained from traditional biochemical methods. Together, these results indicate that the quantum dot-based oligonucleotide microarray has the potential to be a powerful tool in the detection and identification of pathogenic bacteria in foods. PMID:24927399

  1. Minimizing DNA microarrays to a single molecule per spot: using zero-mode waveguide technology to obtain kinetic data for a large number of short oligonucleotide hybridization reactions

    NASA Astrophysics Data System (ADS)

    Sobek, Jens; Rehrauer, Hubert; Kuhn, Gerrit; Schlapbach, Ralph

    2016-03-01

    We have shown recently that the hybridization of short oligonucleotides can be studied in a zero-mode waveguide nanostructure (ZMW) chip using a modified DNA sequencer.[1] Here we present an extension of this method enabling the parallel measurement of kinetic constants of a large number of hybridization reactions on a single chip. This can be achieved by immobilization of a mixture of oligonucleotides, which leads to a statistical and random distribution of single molecules in the 150'000 ZMWs of a SMRT™ cell. This setup is comparable to a classical microarray with ZMWs in place of spots but unknown allocation of probes. The probe surface density is reduced by a factor of ~1010 allowing the study of hybridization in the absence of interactions with neighboring probes. Hybridization with a dye labelled oligonucleotide results in trains of fluorescence pulses from which interpulse durations (IPDs) and pulse widths (PWs) can be extracted. Since the identity of a probe in a ZMW is unknown, the immobilized oligonucleotide is sequenced in a subsequent step. After mapping the fluorescence traces to the sequence, the association and dissociation rate constant for each oligonucleotide can be calculated. By selecting suitable probes, the method can be used to determine rate constants of hybridization for a large number of mismatch oligonucleotides in a single measurement and at single-molecule level.

  2. Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Tripathy, Sushant

    Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

  3. Multi-Gene Detection and Identification of Mosquito-Borne RNA Viruses Using an Oligonucleotide Microarray

    PubMed Central

    Grubaugh, Nathan D.; McMenamy, Scott S.; Turell, Michael J.; Lee, John S.

    2013-01-01

    Background Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae), Alphavirus (Togaviridae), Orthobunyavirus (Bunyaviridae), and Phlebovirus (Bunyaviridae). Methodology/Principal Findings The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. Conclusions/Significance We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish public health

  4. Assessment and integration of publicly available SAGE, cDNA microarray, and oligonucleotide microarray expression data for global coexpression analyses.

    PubMed

    Griffith, Obi L; Pleasance, Erin D; Fulton, Debra L; Oveisi, Mehrdad; Ester, Martin; Siddiqui, Asim S; Jones, Steven J M

    2005-10-01

    Large amounts of gene expression data from several different technologies are becoming available to the scientific community. A common practice is to use these data to calculate global gene coexpression for validation or integration of other "omic" data. To assess the utility of publicly available datasets for this purpose we have analyzed Homo sapiens data from 1202 cDNA microarray experiments, 242 SAGE libraries, and 667 Affymetrix oligonucleotide microarray experiments. The three datasets compared demonstrate significant but low levels of global concordance (rc<0.11). Assessment against Gene Ontology (GO) revealed that all three platforms identify more coexpressed gene pairs with common biological processes than expected by chance. As the Pearson correlation for a gene pair increased it was more likely to be confirmed by GO. The Affymetrix dataset performed best individually with gene pairs of correlation 0.9-1.0 confirmed by GO in 74% of cases. However, in all cases, gene pairs confirmed by multiple platforms were more likely to be confirmed by GO. We show that combining results from different expression platforms increases reliability of coexpression. A comparison with other recently published coexpression studies found similar results in terms of performance against GO but with each method producing distinctly different gene pair lists. PMID:16098712

  5. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    SciTech Connect

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

  6. ASSESSMENT OF THE SWINE PROTEIN-ANNOTATED OLIGONUCLEOTIDE MICROARRAY AND UTILITY OF THE ARRAYS FOR EQTL AND TRANSCRIPTIONAL PROFILING STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions com...

  7. Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays.

    PubMed

    Small, J; Call, D R; Brockman, F J; Straub, T M; Chandler, D P

    2001-10-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 microg of total RNA, representing approximately 7.5 x 10(6) Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  8. Development of oligonucleotide microarrays for simultaneous multi-species identification of Phellinus tree-pathogenic fungi.

    PubMed

    Tzean, Yuh; Shu, Po-Yao; Liou, Ruey-Fen; Tzean, Shean-Shong

    2016-03-01

    Polyporoid Phellinus fungi are ubiquitously present in the environment and play an important role in shaping forest ecology. Several species of Phellinus are notorious pathogens that can affect a broad variety of tree species in forest, plantation, orchard and urban habitats; however, current detection methods are overly complex and lack the sensitivity required to identify these pathogens at the species level in a timely fashion for effective infestation control. Here, we describe eight oligonucleotide microarray platforms for the simultaneous and specific detection of 17 important Phellinus species, using probes generated from the internal transcribed spacer regions unique to each species. The sensitivity, robustness and efficiency of this Phellinus microarray system was subsequently confirmed against template DNA from two key Phellinus species, as well as field samples collected from tree roots, trunks and surrounding soil. This system can provide early, specific and convenient detection of Phellinus species for forestry, arboriculture and quarantine inspection, and could potentially help to mitigate the environmental and economic impact of Phellinus-related diseases. PMID:26853539

  9. Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    Urakawa, Hidetoshi; El Fantroussi, Said; Smidt, Hauke; Smoot, James C.; Tribou, Erik H.; Kelly, John J.; Noble, Peter A.; Stahl, David A.

    2003-01-01

    The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of Staphylococcus epidermidis (38 nucleotides) and Nitrosomonas eutropha (39 nucleotides) were hybridized to perfect-match probes (18-mer and 19-mer) and to a set of probes having all possible single-base-pair mismatches. The melting profiles of all probe-target duplexes were determined in parallel by using an imposed temperature step gradient. We derived an optimum wash temperature for each probe and target by using a simple formula to calculate a discrimination index for each temperature of the step gradient. This optimum corresponded to the output of an independent analysis using a customized neural network program. These results together provide an experimental and analytical framework for optimizing mismatch discrimination among all probes on a DNA microarray.

  10. Development of Polymer-Coated Glass Slides as Optical Oligonucleotide Microarrays

    PubMed Central

    Pourjahed, Atefeh; Rabiee, Mohammad; Tahriri, Mohammadreza

    2013-01-01

    Background The microarray technology is in needed of cost-effective, low background noise and stable substrates for successful hybridization and analysis. Methods In this research, we developed a three-dimentional stable and mechanically reliable microarray substrates by coating of two polymeric layers on standard microscope glass slides. For fabrication of these substrates, a thin film of oxidized agarose was prepared on the Poly-L-Lysine (PLL) coated glass slides. Unmodified oligonucleotide probes were spotted and immobilized on these double layered thin films by adsorption on the porous structure of the agarose film. Some of the aldehyde groups of the activated agarose linked covalently to PLL amine groups; on the other side, they bound to amino groups of adsorbed tail of biomolecules. These linkages were fixed by UV irradiation at 254 nm using a CL-1000 UV. These prepared substrates were compared to only agarose-coated and PLL-coated slides Results Atomic Force Microscope (AFM) results demonstrated that agarose provided three-dimensional surface which had higher loading and bindig capacity for biomolecules than PLL-coated surface which had two-dimensional surface. The nano-indentation tests demonstrated the prepared double coating was more reliable and flexible for mechanical robotic spotting. In addition, the repeated indentation on different substrates showed uniformity of coatings. The stability of novel coating was sufficient for hybridization process. The signal-to-noise ratio in hybridization reactions performed on the agarose-PLL coated substrates increased two fold and four fold compared to agarose and PLL coated substrates, respectively. Conclusion Finally, the agarose-PLL microarrays had the highest signal (2920) and lowest background signal (205) in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes. PMID:24285999

  11. An oligonucleotide-tagged microarray for routine diagnostics of colon cancer by genotyping KRAS mutations.

    PubMed

    Liu, Yuliang; Gudnason, Haukur; Li, Yi-Ping; Bang, Dang Duong; Wolff, Anders

    2014-10-01

    Colorectal cancer (CRC) is one of the most prevalent types of cancer, causing significant morbidity and mortality worldwide. CRC is curable if diagnosed at an early stage. Mutations in the oncogene KRAS play a critical role in early development of CRC. Detection of activated KRAS is of diagnostic and therapeutic importance. In this study, KRAS gene fragments containing mutations in codon 12 were amplified by multiplex PCR using a 5'-Cy5-labeled reverse primer in combination with 3'-mutation-specific forward primers that were linked with four unique nucleotide-sequence tags at the 5'-end. The Cy5-labeled reverse primer was extended under PCR amplification to the 5'-end of the mutation-specific forward primers and thus included the complimentary sequence of the tag. PCR products were hybridized to tag-probes immobilized on various substrates and detected by a scanner. Our results indicate that all mutations at codon 12 of KRAS derived from cancer cells and clinical samples could be unambiguously detected. KRAS mutations were accurately detected when the mutant DNA was present only in 10% of the starting mixed materials including wild-type genomic DNA, which was isolated from either cancer cells or spiked fecal samples. The immobilized tag-probes were stable under multiple thermal cycling treatments, allowing re-use of the tag-microarray and further optimization to solid PCR. Our results demonstrated that a novel oligonucleotide-tagged microarray system has been developed which would be suitable to be used for detection of KRAS mutations and clinical diagnosis of CRC. PMID:25018048

  12. Use of Oligonucleotide Microarrays for Rapid Detection and Serotyping of Acute Respiratory Disease-Associated Adenoviruses

    PubMed Central

    Lin, Baochuan; Vora, Gary J.; Thach, Dzung; Walter, Elizabeth; Metzgar, David; Tibbetts, Clark; Stenger, David A.

    2004-01-01

    The cessation of the adenovirus vaccination program for military trainees has resulted in several recent acute respiratory disease (ARD) outbreaks. In the absence of vaccination, rapid detection methods are necessary for the timely implementation of measures to prevent adenovirus transmission within military training facilities. To this end, we have combined a fluorogenic real-time multiplex PCR assay with four sets of degenerate PCR primers that target the E1A, fiber, and hexon genes with a long oligonucleotide microarray capable of identifying the most common adenovirus serotypes associated with adult respiratory tract infections (serotypes 3, 4, 7, 16, and 21) and a representative member of adenovirus subgroup C (serotype 6) that is a common cause of childhood ARD and that often persists into adulthood. Analyses with prototype strains demonstrated unique hybridization patterns for representative members of adenovirus subgroups B1, B2, C, and E, thus allowing serotype determination. Microarray-based sensitivity assessments revealed lower detection limits (between 1 and 100 genomic copies) for adenovirus serotype 4 (Ad4) and Ad7 cell culture lysates, clinical nasal washes, and throat swabs and purified DNA from clinical samples. When adenovirus was detected from coded clinical samples, the results obtained by this approach demonstrated an excellent concordance with those obtained by the more established method of adenovirus identification as well as by cell culture with fluorescent-antibody staining. Finally, the utility of this method was further supported by its ability to detect adenoviral coinfections, contamination, and, potentially, recombination events. Taken together, the results demonstrate the usefulness of the simple and rapid diagnostic method developed for the unequivocal identification of ARD-associated adenoviral serotypes from laboratory or clinical samples that can be completed in 1.5 to 4.0 h. PMID:15243087

  13. Design and Verification of a Pangenome Microarray Oligonucleotide Probe Set for Dehalococcoides spp. ▿ †

    PubMed Central

    Hug, Laura A.; Salehi, Maryam; Nuin, Paulo; Tillier, Elisabeth R.; Edwards, Elizabeth A.

    2011-01-01

    Dehalococcoides spp. are an industrially relevant group of Chloroflexi bacteria capable of reductively dechlorinating contaminants in groundwater environments. Existing Dehalococcoides genomes revealed a high level of sequence identity within this group, including 98 to 100% 16S rRNA sequence identity between strains with diverse substrate specificities. Common molecular techniques for identification of microbial populations are often not applicable for distinguishing Dehalococcoides strains. Here we describe an oligonucleotide microarray probe set designed based on clustered Dehalococcoides genes from five different sources (strain DET195, CBDB1, BAV1, and VS genomes and the KB-1 metagenome). This “pangenome” probe set provides coverage of core Dehalococcoides genes as well as strain-specific genes while optimizing the potential for hybridization to closely related, previously unknown Dehalococcoides strains. The pangenome probe set was compared to probe sets designed independently for each of the five Dehalococcoides strains. The pangenome probe set demonstrated better predictability and higher detection of Dehalococcoides genes than strain-specific probe sets on nontarget strains with <99% average nucleotide identity. An in silico analysis of the expected probe hybridization against the recently released Dehalococcoides strain GT genome and additional KB-1 metagenome sequence data indicated that the pangenome probe set performs more robustly than the combined strain-specific probe sets in the detection of genes not included in the original design. The pangenome probe set represents a highly specific, universal tool for the detection and characterization of Dehalococcoides from contaminated sites. It has the potential to become a common platform for Dehalococcoides-focused research, allowing meaningful comparisons between microarray experiments regardless of the strain examined. PMID:21666017

  14. Quantitative genotyping of single-nucleotide polymorphisms by allele-specific oligonucleotide hybridization on DNA microarrays.

    PubMed

    Rickert, Andreas M; Ballvora, Agim; Matzner, Ulrich; Klemm, Manfred; Gebhardt, Christiane

    2005-08-01

    Genotyping of SNPs (single-nucleotide polymorphisms) has challenged the development of several novel techniques. Most of these methods have been introduced to discriminate binary SNPs in diploid species. In the present study, the quantitative genotyping of SNPs in natural DNA pools of a polyploid organism via DNA microarrays was analysed. Three randomly selected SNP loci were genotyped in the tetraploid species potato (Solanum tuberosum). For each SNP, 24 oligomers were designed, 12 with forward and 12 with reverse orientation. They contained the polymorphic site at one of the positions 11, 14 and 17. Several steps of optimizations were performed, including the 'materials' used and the establishment of hybridization conditions. Glass surfaces were either epoxy- or aldehyde-modified, and allele-specific oligonucleotides contained either SH or NH2 groups. Hybridization stringency conditions were established by varying the concentration of formamide in the hybridization buffer. For SNP BA213c14t7/403, the quantitative discrimination between all four different naturally occurring genotypes could be demonstrated. PMID:15847606

  15. Genome-wide expression analysis of Saccharomyces pastorianus orthologous genes using oligonucleotide microarrays.

    PubMed

    Horinouchi, Takaaki; Yoshikawa, Katsunori; Kawaide, Risa; Furusawa, Chikara; Nakao, Yoshihiro; Hirasawa, Takashi; Shimizu, Hiroshi

    2010-11-01

    The lager brewing yeast, Saccharomyces pastorianus, an allopolyploid species hybrid, contains 2 diverged sub-genomes; one derived from Saccharomyces cerevisiae (Sc-type) and the other from Saccharomyces bayanus (Sb-type). We analyzed the functional roles of these orthologous genes in determining the phenotypic features of S. pastorianus. We used a custom-made oligonucleotide microarray containing probes designed for both Sc-type and Sb-type ORFs for a comprehensive expression analysis of S. pastorianus in a pilot-scale fermentation. We showed a high degree of correlation between the expression levels and the expression changes for a majority of orthologous gene sets during the fermentation process. We screened the functional categories and metabolic pathways where Sc- or Sb-type genes have higher expression levels than the corresponding orthologous genes. Our data showed that, for example, pathways for sulfur metabolism, cellular import, and production of branched amino acids are dominated by Sb-type genes. This comprehensive expression analysis of orthologous genes can provide valuable insights on understanding the phenotype of S. pastorianus. PMID:20547377

  16. A high density COX1 barcode oligonucleotide array for identification and detection of species of Penicillium subgenus Penicillium.

    PubMed

    Chen, W; Seifert, K A; Lévesque, C A

    2009-05-01

    We developed a COX1 barcode oligonucleotide array based on 358 sequences, including 58 known and two new species of Penicillium subgenus Penicillium, and 12 allied species. The array was robotically spotted at near microarray density on membranes. Species and clade-specific oligonucleotides were selected using the computer programs SigOli and Array Designer. Robotic spotting allowed 768 spots with duplicate sets of perfect match and the corresponding mismatch and positive control oligonucleotides, to be printed on 2 × 6 cm(2) nylon membranes. The array was validated with hybridizations between the array and digoxigenin (DIG)-labelled COX1 polymerase chain reaction amplicons from 70 pure DNA samples, and directly from environmental samples (cheese and plants) without culturing. DNA hybridization conditions were optimized, but undesired cross-reactions were detected frequently, reflecting the relatively high sequence similarity of the COX1 gene among Penicillium species. Approximately 60% of the perfect match oligonucleotides were rejected because of low specificity and 76 delivered useful group-specific or species-specific reactions and could be used for detecting certain species of Penicillium in environmental samples. In practice, the presence of weak signals on arrays exposed to amplicons from environmental samples, which could have represented weak detections or weak cross reactions, made interpretation difficult for over half of the oligonucleotides. DNA regions with very few single nucleotide polymorphisms or lacking insertions/deletions among closely related species are not ideal for oligonucleotide-based diagnostics, and supplementing the COX1-based array with oligonucleotides derived from additional genes would result in a more robust hierarchical identification system. PMID:21564971

  17. Microarray-based long oligonucleotides probe designed for Brucella Spp. detection and identification of antibiotic susceptibility pattern

    PubMed Central

    Khazaei, Zahra; Najafi, Ali; Piranfar, Vahhab; Mirnejad, Reza

    2016-01-01

    Brucella spp. is a common zoonotic infection referred to as Brucellosis, and it is a serious public health problem around the world. There are currently six classical species (pathogenic species in both animals and humans) within the genus Brucella. The ability and practicality facilitated by a microarray experiment help us to recognize Brucella spp. and its antibiotic resistant gene. Rapid phenotypic determination of antibiotic resistance is not possible by disk diffusion methods. Thus, evaluating antibiotics pattern and Brucella detection appear necessary technique by molecular methods in brucellosis. So, the aim of this study was to design a microarray long oligonucleotides probe and primer for the complete diagnosis of Brucella spp. and obtaining genetic profiles for antibiotic resistance in bacteria at the same time. In this study, we designed 16 antibiotic-resistant gene solid-phase primers with similar melting temperatures of 60 °C and 16 long oligonucleotide probes. These primers and probes can identify tetracycline-, chloramphenicol-, and aminoglycoside-resistant genes, respectively. The design of microarray probes is a versatile process that be done in a wide range of selections. Since the long oligo microarray probes are the best choices for specific diagnosis and definite treatment, this group of probes was designed in the present survey. PMID:27280008

  18. Microarray-based long oligonucleotides probe designed for Brucella Spp. detection and identification of antibiotic susceptibility pattern.

    PubMed

    Khazaei, Zahra; Najafi, Ali; Piranfar, Vahhab; Mirnejad, Reza

    2016-04-01

    Brucella spp. is a common zoonotic infection referred to as Brucellosis, and it is a serious public health problem around the world. There are currently six classical species (pathogenic species in both animals and humans) within the genus Brucella. The ability and practicality facilitated by a microarray experiment help us to recognize Brucella spp. and its antibiotic resistant gene. Rapid phenotypic determination of antibiotic resistance is not possible by disk diffusion methods. Thus, evaluating antibiotics pattern and Brucella detection appear necessary technique by molecular methods in brucellosis. So, the aim of this study was to design a microarray long oligonucleotides probe and primer for the complete diagnosis of Brucella spp. and obtaining genetic profiles for antibiotic resistance in bacteria at the same time. In this study, we designed 16 antibiotic-resistant gene solid-phase primers with similar melting temperatures of 60 °C and 16 long oligonucleotide probes. These primers and probes can identify tetracycline-, chloramphenicol-, and aminoglycoside-resistant genes, respectively. The design of microarray probes is a versatile process that be done in a wide range of selections. Since the long oligo microarray probes are the best choices for specific diagnosis and definite treatment, this group of probes was designed in the present survey. PMID:27280008

  19. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens.

    PubMed

    Lee, Dae-Young; Lauder, Heather; Cruwys, Heather; Falletta, Patricia; Beaudette, Lee A

    2008-07-15

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10(3)A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater. PMID:18423816

  20. Differential analysis for high density tiling microarray data

    PubMed Central

    Ghosh, Srinka; Hirsch, Heather A; Sekinger, Edward A; Kapranov, Philipp; Struhl, Kevin; Gingeras, Thomas R

    2007-01-01

    Background High density oligonucleotide tiling arrays are an effective and powerful platform for conducting unbiased genome-wide studies. The ab initio probe selection method employed in tiling arrays is unbiased, and thus ensures consistent sampling across coding and non-coding regions of the genome. These arrays are being increasingly used to study the associated processes of transcription, transcription factor binding, chromatin structure and their association. Studies of differential expression and/or regulation provide critical insight into the mechanics of transcription and regulation that occurs during the developmental program of a cell. The time-course experiment, which comprises an in-vivo system and the proposed analyses, is used to determine if annotated and un-annotated portions of genome manifest coordinated differential response to the induced developmental program. Results We have proposed a novel approach, based on a piece-wise function – to analyze genome-wide differential response. This enables segmentation of the response based on protein-coding and non-coding regions; for genes the methodology also partitions differential response with a 5' versus 3' versus intra-genic bias. Conclusion The algorithm built upon the framework of Significance Analysis of Microarrays, uses a generalized logic to define regions/patterns of coordinated differential change. By not adhering to the gene-centric paradigm, discordant differential expression patterns between exons and introns have been identified at a FDR of less than 12 percent. A co-localization of differential binding between RNA Polymerase II and tetra-acetylated histone has been quantified at a p-value < 0.003; it is most significant at the 5' end of genes, at a p-value < 10-13. The prototype R code has been made available as supplementary material [see Additional file 1]. PMID:17892592

  1. Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses

    NASA Technical Reports Server (NTRS)

    Urakawa, Hidetoshi; Noble, Peter A.; El Fantroussi, Said; Kelly, John J.; Stahl, David A.

    2002-01-01

    The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (T(d)) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5' terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the T(d) and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the T(d) and signal intensity, and it decreased the variability of the signal. Although T(d)s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower T(d)s than those with mismatches in the first or second position. The trained NNs predicted the T(d) with high accuracies (R(2) = 0.93). However, the NNs predicted the signal intensity only moderately accurately (R(2) = 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in T(d)s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5' terminus plays a greater role in determining the T(d) and signal intensity of duplexes than the type of mismatch.

  2. Comparative Analysis of Human Conjunctival and Corneal Epithelial Gene Expression with Oligonucleotide Microarrays

    PubMed Central

    Turner, Helen C.; Budak, Murat T.; Murat Akinci, M. A.; Wolosin, J. Mario

    2010-01-01

    Purpose To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. Methods cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the β-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. Results The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA2-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Conclusions Comparative gene expression profiling leads to the identification of many biological processes

  3. Event-specific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Jia; Zhu, Shuifang; Miao, Haizhen; Huang, Wensheng; Qiu, Minyan; Huang, Yan; Fu, Xuping; Li, Yao

    2007-07-11

    With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes. PMID:17559227

  4. Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

    PubMed Central

    Girard, Laurie D.; Boissinot, Karel; Peytavi, Régis; Boissinot, Maurice; Bergeron, Michel G.

    2014-01-01

    The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design

  5. Evolutionary sequence comparisons using high-density oligonucleotide arrays.

    PubMed

    Hacia, J G; Makalowski, W; Edgemon, K; Erdos, M R; Robbins, C M; Fodor, S P; Brody, L C; Collins, F S

    1998-02-01

    We explored the utility of high-density oligonucleotide arrays (DNA chips) for obtaining sequence information from homologous genes in closely related species. Orthologues of the human BRCA1 exon 11, all approximately 3.4 kb in length and ranging from 98.2% to 83.5% nucleotide identity, were subjected to hybridization-based and conventional dideoxysequencing analysis. Retrospective guidelines for identifying high-fidelity hybridization-based sequence calls were formulated based upon dideoxysequencing results. Prospective application of these rules yielded base-calling with at least 98.8% accuracy over orthologous sequence tracts shown to have approximately 99% identity. For higher primate sequences with greater than 97% nucleotide identity, base-calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence. Using a second-tier confirmatory hybridization chip strategy, shown in several cases to confirm the identity of predicted sequence changes, the complete sequence of the chimpanzee, gorilla and orangutan orthologues should be deducible solely through hybridization-based methodologies. Analysis of less highly conserved orthologues can still identify conserved nucleotide tracts of at least 15 nucleotides and can provide useful information for designing primers. DNA-chip based assays can be a valuable new technology for obtaining high-throughput cost-effective sequence information from related genomes. PMID:9462745

  6. ASSESSING THE USE OF OLIGONUCLEOTIDE MICROARRAYS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) TO EXAMINE EXPOSURE VALUES

    EPA Science Inventory

    New microarray technologies to determine gene expression in animals exposed to chemical stressors promise to revolutionize the science of toxicology. The work described in this abstract concerns development and validation of a gene microarray for the fathead minnow designed to ai...

  7. Cassava (Manihot esculenta) transcriptome analysis in response to infection by the fungus Colletotrichum gloeosporioides using an oligonucleotide-DNA microarray.

    PubMed

    Utsumi, Yoshinori; Tanaka, Maho; Kurotani, Atsushi; Yoshida, Takuhiro; Mochida, Keiichi; Matsui, Akihiro; Ishitani, Manabu; Sraphet, Supajit; Whankaew, Sukhuman; Asvarak, Thipa; Narangajavana, Jarunya; Triwitayakorn, Kanokporn; Sakurai, Tetsuya; Seki, Motoaki

    2016-07-01

    Cassava anthracnose disease (CAD), caused by the fungus Colletotrichum gloeosporioides f. sp. Manihotis, is a serious disease of cassava (Manihot esculenta) worldwide. In this study, we established a cassava oligonucleotide-DNA microarray representing 59,079 probes corresponding to approximately 30,000 genes based on original expressed sequence tags and RNA-seq information from cassava, and applied it to investigate the molecular mechanisms of resistance to fungal infection using two cassava cultivars, Huay Bong 60 (HB60, resistant to CAD) and Hanatee (HN, sensitive to CAD). Based on quantitative real-time reverse transcription PCR and expression profiling by the microarray, we showed that the expressions of various plant defense-related genes, such as pathogenesis-related (PR) genes, cell wall-related genes, detoxification enzyme, genes related to the response to bacterium, mitogen-activated protein kinase (MAPK), genes related to salicylic acid, jasmonic acid and ethylene pathways were higher in HB60 compared with HN. Our results indicated that the induction of PR genes in HB60 by fungal infection and the higher expressions of defense response-related genes in HB60 compared with HN are likely responsible for the fungal resistance in HB60. We also showed that the use of our cassava oligo microarray could improve our understanding of cassava molecular mechanisms related to environmental responses and development, and advance the molecular breeding of useful cassava plants. PMID:27138000

  8. Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping

    NASA Technical Reports Server (NTRS)

    Royce, Thomas E.; Rozowsky, Joel S.; Bertone, Paul; Samanta, Manoj; Stolc, Viktor; Weissman, Sherman; Snyder, Michael; Gerstein, Mark

    2005-01-01

    Traditional microarrays use probes complementary to known genes to quantitate the differential gene expression between two or more conditions. Genomic tiling microarray experiments differ in that probes that span a genomic region at regular intervals are used to detect the presence or absence of transcription. This difference means the same sets of biases and the methods for addressing them are unlikely to be relevant to both types of experiment. We introduce the informatics challenges arising in the analysis of tiling microarray experiments as open problems to the scientific community and present initial approaches for the analysis of this nascent technology.

  9. Low-density microarray technologies for rapid human norovirus genotyping

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human noroviruses cause up to 21 million cases of foodborne disease in the United States annually and are the most common cause of acute gastroenteritis in industrialized countries. To reduce the burden of foodborne disease associated with viruses, the use of low density DNA microarrays in conjuncti...

  10. ASSESSING THE USE OF OLIGONUCLEOTIDE MICROARRAYS FOR FATHEAD MINNOW (PIMEPHALES PROMELAS) TO EXAMINE EXPOSURE VARIABLES

    EPA Science Inventory

    Microarray technology has proven to be a useful tool for analyzing the transcriptome of various organisms representing conditions such as disease states, developmental stages, and responses to chemical exposure. Although most commercially available arrays are limited to organism...

  11. Identification of SNPs and INDELS in swine transcribed sequences using short oligonucleotide microarrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Genome-wide detection of single feature polymorphisms (SFP) in swine using transcriptome profiling of day 25 placental RNA by contrasting probe intensities from either Meishan or an occidental composite breed with Affymetrix porcine microarrays is presented. A linear mixed model analysis was used to...

  12. Detection of Genes Involved in Biodegradation and Biotransformation in Microbial Communities by Using 50-Mer Oligonucleotide Microarrays

    PubMed Central

    Rhee, Sung-Keun; Liu, Xueduan; Wu, Liyou; Chong, Song C.; Wan, Xiufeng; Zhou, Jizhong

    2004-01-01

    To effectively monitor biodegrading populations, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the 2,402 known genes and pathways involved in biodegradation and metal resistance. This array contained 1,662 unique and group-specific probes with <85% similarity to their nontarget sequences. Based on artificial probes, our results showed that under hybridization conditions of 50°C and 50% formamide, the 50-mer microarray hybridization can differentiate sequences having <88% similarity. Specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was ∼5 to 10 ng of genomic DNA in the absence of background DNA and 50 to 100 ng of pure-culture genomic DNA in the presence of background DNA or 1.3 × 107 cells in the presence of background RNA. Strong linear relationships between the signal intensity and the target DNA and RNA were observed (r2 = 0.95 to 0.99). Application of this type of microarray to analyze naphthalene-amended enrichment and soil microcosms demonstrated that microflora changed differently depending on the incubation conditions. While the naphthalene-degrading genes from Rhodococcus-type microorganisms were dominant in naphthalene-degrading enrichments, the genes involved in naphthalene (and polyaromatic hydrocarbon and nitrotoluene) degradation from gram-negative microorganisms, such as Ralstonia, Comamonas, and Burkholderia, were most abundant in the soil microcosms. In contrast to general conceptions, naphthalene-degrading genes from Pseudomonas were not detected, although Pseudomonas is widely known as a model microorganism for studying naphthalene degradation. The real-time PCR analysis with four representative genes showed that the microarray-based quantification was very consistent with real-time PCR (r2 = 0.74). In addition, application of the arrays to both polyaromatic

  13. Quantitative profiling of housekeeping and Epstein-Barr virus gene transcription in Burkitt lymphoma cell lines using an oligonucleotide microarray

    PubMed Central

    Bernasconi, Michele; Berger, Christoph; Sigrist, Jürg A; Bonanomi, Athos; Sobek, Jens; Niggli, Felix K; Nadal, David

    2006-01-01

    Background The Epstein-Barr virus (EBV) is associated with lymphoid malignancies, including Burkitt's lymphoma (BL), and can transform human B cells in vitro. EBV-harboring cell lines are widely used to investigate lymphocyte transformation and oncogenesis. Qualitative EBV gene expression has been extensively described, but knowledge of quantitative transcription is lacking. We hypothesized that transcription levels of EBNA1, the gene essential for EBV persistence within an infected cell, are similar in BL cell lines. Results To compare quantitative gene transcription in the BL cell lines Namalwa, Raji, Akata, Jijoye, and P3HR1, we developed an oligonucleotide microarray chip, including 17 housekeeping genes, six latent EBV genes (EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1, LMP2), and four lytic EBV genes (BZLF1, BXLF2, BKRF2, BZLF2), and used the cell line B95.8 as a reference for EBV gene transcription. Quantitative polymerase chain reaction assays were used to validate microarray results. We found that transcription levels of housekeeping genes differed considerably among BL cell lines. Using a selection of housekeeping genes with similar quantitative transcription in the tested cell lines to normalize EBV gene transcription data, we showed that transcription levels of EBNA1 were quite similar in very different BL cell lines, in contrast to transcription levels of other EBV genes. As demonstrated with Akata cells, the chip allowed us to accurately measure EBV gene transcription changes triggered by treatment interventions. Conclusion Our results suggest uniform EBNA1 transcription levels in BL and that microarray profiling can reveal novel insights on quantitative EBV gene transcription and its impact on lymphocyte biology. PMID:16756670

  14. Evaluation of Gel-Pad Oligonucleotide Microarray Technology by Using Artificial Neural Networks†

    PubMed Central

    Pozhitkov, Alex; Chernov, Boris; Yershov, Gennadiy; Noble, Peter A.

    2005-01-01

    Past studies have suggested that thermal dissociation analysis of nucleic acids hybridized to DNA microarrays would improve discrimination among duplex types by scanning through a broad range of stringency conditions. To more fully constrain the utility of this approach using a previously described gel-pad microarray format, artificial neural networks (NNs) were trained to recognize noisy or low-quality data, as might derive from nonspecific fluorescence, poor hybridization, or compromised data collection. The NNs were trained to classify dissociation profiles (melts) into groups based on selected characteristics (e.g., initial signal intensity, area under the curve) using a data set of 21,044 profiles derived from 186 probes hybridized to a study set of RNA extracted from 32 microbes common to the human oral cavity. Three melt profile groups were identified: one group consisted mostly of ideal melt profiles; another group consisted mostly of poor melt profiles; and, the remainder were difficult to classify. Screening of melting profiles of perfect-match hybrids revealed inconsistencies in the form of melting profiles even for identical probes on the same microarray hybridized to same target rRNA. Approximately 18% of perfect-match duplex types were correctly classified as poor. Experimental variability and deviation from ideal melt behavior were shown to be attributable primarily to a method of local background subtraction that was very sensitive to displacement of the grid frames used for image capture (both determined by the image analysis system) and duplexes with low binding constants. Additional results showed that long RNA fragments limit the discriminating power among duplex types. PMID:16332861

  15. An Efficient Covalent Coating on Glass Slides for Preparation of Optical Oligonucleotide Microarrays

    PubMed Central

    Pourjahed, Atefeh; Rabiee, Mohammad; Tahriri, Mohammadreza

    2013-01-01

    Objective(s): Microarrays are potential analyzing tools for genomics and proteomics researches, which is in needed of suitable substrate for coating and also hybridization of biomolecules. Materials and Methods: In this research, a thin film of oxidized agarose was prepared on the glass slides which previously coated with poly-L-lysine (PLL). Some of the aldehyde groups of the activated agarose linked covalently to PLL amine groups; also bound to the amino groups of biomolecules. These linkages were fixed by UV irradiation. The prepared substrates were compared to only agarose-coated and PLL-coated slides. Results: Results on atomic force microscope (AFM) demonstrated that agarose provided three-dimensional surface which had higher loading and bindig capacity for biomolecules than PLL-coated surface which had two-dimensional surface. In addition, the signal-to-noise ratio in hybridization reactions performed on the agarose-PLL coated substrates increased two fold and four fold compared to agarose and PLL coated substrates, respectively. Conclusion: The agarose-PLL microarrays had the highest signal (2546) and lowest background signal (205) in hybridization, suggesting that the prepared slides are suitable in analyzing wide concentration range of analytes. PMID:24570832

  16. Use of a multiplexed CMOS microarray to optimize and compare oligonucleotide binding to DNA probes synthesized or immobilized on individual electrodes.

    PubMed

    Maurer, Karl; Yazvenko, Nina; Wilmoth, Jodi; Cooper, John; Lyon, Wanda; Danley, David

    2010-01-01

    The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode. PMID:22163607

  17. Use of a Multiplexed CMOS Microarray to Optimize and Compare Oligonucleotide Binding to DNA Probes Synthesized or Immobilized on Individual Electrodes

    PubMed Central

    Maurer, Karl; Yazvenko, Nina; Wilmoth, Jodi; Cooper, John; Lyon, Wanda; Danley, David

    2010-01-01

    The CombiMatrix microarray with 12,544 electrodes supports in situ electrochemical synthesis of user-defined DNA probes. As an alternative, we immobilized commercially synthesized DNA probes on individual electrodes coated with electropolymerized polypyrrole (Ppy). Hybridization was measured using a biotinylated target oligonucleotide and either Cy5-streptavidin and fluorescence detection or horseradish peroxidase-streptavidin and enzyme-enhanced electrochemical detection. Detection efficiencies were optimized by varying the deposition of the Ppy, the terminal groups on the DNA probes, and other factors that impacted fluorescence quenching and electrical conductivity. Optimized results were compared against those obtained using a microarray with the same DNA sequences synthesized in situ. Immobilized probes produced higher fluorescence signals, possibly by providing a greater stand off between the Cy5 on the target oligonucleotide and the quenching effects of the Ppy and the platinum electrode. PMID:22163607

  18. Comparative genomic analysis of Acidithiobacillus ferrooxidans strains using the A. ferrooxidans ATCC 23270 whole-genome oligonucleotide microarray.

    PubMed

    Luo, Hailang; Shen, Li; Yin, Huaqun; Li, Qian; Chen, Qijiong; Luo, Yanjie; Liao, Liqin; Qiu, Guanzhou; Liu, Xueduan

    2009-05-01

    Acidithiobacillus ferrooxidans is an important microorganism used in biomining operations for metal recovery. Whole-genomic diversity analysis based on the oligonucleotide microarray was used to analyze the gene content of 12 strains of A. ferrooxidans purified from various mining areas in China. Among the 3100 open reading frames (ORFs) on the slides, 1235 ORFs were absent in at least 1 strain of bacteria and 1385 ORFs were conserved in all strains. The hybridization results showed that these strains were highly diverse from a genomic perspective. The hybridization results of 4 major functional gene categories, namely electron transport, carbon metabolism, extracellular polysaccharides, and detoxification, were analyzed. Based on the hybridization signals obtained, a phylogenetic tree was built to analyze the evolution of the 12 tested strains, which indicated that the geographic distribution was the main factor influencing the strain diversity of these strains. Based on the hybridization signals of genes associated with bioleaching, another phylogenetic tree showed an evolutionary relationship from which the co-relation between the clustering of specific genes and geochemistry could be observed. The results revealed that the main factor was geochemistry, among which the following 6 factors were the most important: pH, Mg, Cu, S, Fe, and Al. PMID:19483787

  19. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    PubMed

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  20. Development and Assessment of Whole-Genome Oligonucleotide Microarrays to Analyze an Anaerobic Microbial Community and its Responses to Oxidative Stress

    SciTech Connect

    Scholten, Johannes C.; Culley, David E.; Nie, Lei; Munn, Kyle J.; Chow, Lely; Brockman, Fred J.; Zhang, Weiwen

    2007-06-29

    The application of DNA microarray technology to investigate multiple-species microbial community presents great challenges. In this study, we reported the design and quality assessment of four whole genome oligonucleotide microarrays for two syntroph bacteria, Desulfovibrio vulgaris and Syntrophobacter fumaroxidans, and two archaeal methanogens, Methanosarcina barkeri and Methanospirillum hungatei, and their application to analyze global gene expression of this four-species microbial community in response to oxidative stress. In order to minimize the possible cross-hybridization, cross-genome comparison was performed to assure all probes unique to each genome so that the microarrays could provide species-level resolution. Microarray quality was validated by the good reproducibility of experimental measurements of multiple biological and analytical replicates. Microarray analysis showed that S. fumaroxidans and M. hungatei responded to the stress with up-regulation of several genes known to be involved in ROS detoxification, such as catalase and rubrerythrin in S. fumaroxidans and thioredoxin and heat shock protein Hsp20 in M. hungatei. Consistent with previous study in pure culture, the microarray analysis showed that genes involved in methane production and energy metabolism were down-regulated by oxidative stress in M. barkeri. However, D. vulgaris seemed less sensitive to the oxidative stress when grown in a community, with almost no gene up-regulated. The study demonstrated the successful application of microarray technology to multiple-species microbial community, and our preliminary results indicated that the approach can provide novel insights on the metabolic and regulatory networks within microbial communities.

  1. A comparison of statistical tests for detecting differential expression using Affymetrix oligonucleotide microarrays.

    PubMed

    Vardhanabhuti, Saran; Blakemore, Steven J; Clark, Steven M; Ghosh, Sujoy; Stephens, Richard J; Rajagopalan, Dilip

    2006-01-01

    Signal quantification and detection of differential expression are critical steps in the analysis of Affymetrix microarray data. Many methods have been proposed in the literature for each of these steps. The goal of this paper is to evaluate several signal quantification methods (GCRMA, RSVD, VSN, MAS5, and Resolver) and statistical methods for differential expression (t test, Cyber-T, SAM, LPE, RankProducts, Resolver RatioBuild). Our particular focus is on the ability to detect differential expression via statistical tests. We have used two different datasets for our evaluation. First, we have used the HG-U133 Latin Square spike in dataset developed by Affymetrix. Second, we have used data from an in-house rat liver transcriptomics study following 30 different drug treatments generated using the Affymetrix RAE230A chip. Our overall recommendation based on this study is to use GCRMA for signal quantification. For detection of differential expression, GCRMA coupled with Cyber-T or SAM is the best approach, as measured by area under the receiver operating characteristic (ROC) curve. The integrated pipeline in Resolver RatioBuild combining signal quantification and detection of differential expression is an equally good alternative for detecting differentially expressed genes. For most of the differential expression algorithms we considered, the performance using MAS5 signal quantification was inferior to that of the other methods we evaluated. PMID:17233564

  2. Microintaglio Printing of In situ Synthesized Proteins Enables Rapid Printing of High-Density Protein Microarrays Directly from DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Biyani, Manish; Moriyasu, Junpei; Tanaka, Yoko; Sato, Shusuke; Ueno, Shingo; Ichiki, Takanori

    2013-08-01

    A simple and versatile approach to the simultaneous on-chip synthesis and printing of proteins has been studied for high-density protein microarray applications. The method used is based on the principle of intaglio printing using microengraved plates. Unlike conventional approaches that require multistep reactions for synthesizing proteins off the chip followed by printing using a robotic spotter, our approach demonstrates the following: (i) parallel and spotter-free printing of high-density protein microarrays directly from a type of DNA microarray and (ii) microcompartmentalization of cell-free coupled transcription/translation reaction and direct transferring of picoliter protein solution per spot to pattern microarrays of 25-100 µm features.

  3. Rapid Identification of Carbapenemase Genes in Gram-Negative Bacteria with an Oligonucleotide Microarray-Based Assay

    PubMed Central

    Braun, Sascha D.; Monecke, Stefan; Thürmer, Alexander; Ruppelt, Antje; Makarewicz, Oliwia; Pletz, Mathias; Reißig, Annett; Slickers, Peter; Ehricht, Ralf

    2014-01-01

    Rapid molecular identification of carbapenemase genes in Gram-negative bacteria is crucial for infection control and prevention, surveillance and for epidemiological purposes. Furthermore, it may have a significant impact upon determining the appropriate initial treatment and greatly benefit for critically ill patients. A novel oligonucleotide microarray-based assay was developed to simultaneously detect genes encoding clinically important carbapenemases as well as selected extended (ESBL) and narrow spectrum (NSBL) beta-lactamases directly from clonal culture material within few hours. Additionally, a panel of species specific markers was included to identify Escherichia coli, Pseudomonas aeruginosa, Citrobacter freundii/braakii, Klebsiella pneumoniae and Acinetobacter baumannii. The assay was tested using a panel of 117 isolates collected from urinary, blood and stool samples. For these isolates, phenotypic identifications and susceptibility tests were available. An independent detection of carbapenemase, ESBL and NSBL genes was carried out by various external reference laboratories using PCR methods. In direct comparison, the microarray correctly identified 98.2% of the covered carbapenemase genes. This included blaVIM (13 out of 13), blaGIM (2/2), blaKPC (27/27), blaNDM (5/5), blaIMP-2/4/7/8/13/14/15/16/31 (10/10), blaOXA-23 (12/13), blaOXA-40-group (7/7), blaOXA-48-group (32/33), blaOXA-51 (1/1) and blaOXA-58 (1/1). Furthermore, the test correctly identified additional beta-lactamases [blaOXA-1 (16/16), blaOXA-2 (4/4), blaOXA-9 (33/33), OXA-10 (3/3), blaOXA-51 (25/25), blaOXA-58 (2/2), CTX-M1/M15 (17/17) and blaVIM (1/1)]. In direct comparison to phenotypical identification obtained by VITEK or MALDI-TOF systems, 114 of 117 (97.4%) isolates, including Acinetobacter baumannii (28/28), Enterobacter spec. (5/5), Escherichia coli (4/4), Klebsiella pneumoniae (62/63), Klebsiella oxytoca (0/2), Pseudomonas aeruginosa (12/12), Citrobacter freundii (1/1) and

  4. Microarrays

    ERIC Educational Resources Information Center

    Plomin, Robert; Schalkwyk, Leonard C.

    2007-01-01

    Microarrays are revolutionizing genetics by making it possible to genotype hundreds of thousands of DNA markers and to assess the expression (RNA transcripts) of all of the genes in the genome. Microarrays are slides the size of a postage stamp that contain millions of DNA sequences to which single-stranded DNA or RNA can hybridize. This…

  5. Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

    PubMed

    Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

    2011-08-01

    The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research. PMID:21127932

  6. Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

    PubMed

    McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

    2015-07-01

    The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland. PMID:25631743

  7. An oligonucleotide microarray for transcriptome analysis of Schistosoma mansoni and its application/use to investigate gender-associated gene expression.

    PubMed

    Fitzpatrick, Jennifer M; Johnston, David A; Williams, Gary W; Williams, Debbie J; Freeman, Tom C; Dunne, David W; Hoffmann, Karl F

    2005-05-01

    Global profiling transcriptomes of parasitic helminths offers the potential to simultaneously identify co-ordinately expressed genes, novel genetic programs and uniquely utilized metabolic pathways, which together provide an extensive and new resource for vaccine and drug discovery. We have exploited this post-genomic approach to fabricate the first oligonucleotide DNA microarray for gene expression analysis of the parasitic trematode Schistosoma mansoni. A total of 17,329 S. mansoni DNA sequences were used to design a microarray consisting of 7335 parasite elements or approximately 50% of this parasite's transcriptome. Here, we describe the design of this new microarray resource and its evaluation by extending studies into gender-associated gene expression in adult schistosomes. We demonstrate a high degree of reproducibility in detecting transcriptional differences among biologically replicated experiments and the ability of the microarray to distinguish between the expression of closely related gene family members. Importantly, for issues related to sexual dimorphism, labour division, gamete production and drug target discovery, 197 transcripts demonstrated a gender-biased pattern of gene expression in the adult schistosome, greatly extending the number of sex-associated genes. These data demonstrate the power of this new resource to facilitate a greater understanding into the biological complexities of schistosome development and maturation useful for identifying novel intervention strategies. PMID:15811522

  8. Development of the first oligonucleotide microarray for global gene expression profiling in guinea pigs: defining the transcription signature of infectious diseases

    PubMed Central

    2012-01-01

    Background The Guinea pig (Cavia porcellus) is one of the most extensively used animal models to study infectious diseases. However, despite its tremendous contribution towards understanding the establishment, progression and control of a number of diseases in general and tuberculosis in particular, the lack of fully annotated guinea pig genome sequence as well as appropriate molecular reagents has severely hampered detailed genetic and immunological analysis in this animal model. Results By employing the cross-species hybridization technique, we have developed an oligonucleotide microarray with 44,000 features assembled from different mammalian species, which to the best of our knowledge is the first attempt to employ microarray to study the global gene expression profile in guinea pigs. To validate and demonstrate the merit of this microarray, we have studied, as an example, the expression profile of guinea pig lungs during the advanced phase of M. tuberculosis infection. A significant upregulation of 1344 genes and a marked down regulation of 1856 genes in the lungs identified a disease signature of pulmonary tuberculosis infection. Conclusion We report the development of first comprehensive microarray for studying the global gene expression profile in guinea pigs and validation of its usefulness with tuberculosis as a case study. An important gap in the area of infectious diseases has been addressed and a valuable molecular tool is provided to optimally harness the potential of guinea pig model to develop better vaccines and therapies against human diseases. PMID:23031549

  9. Exploration of high-density protein microarrays for antibody validation and autoimmunity profiling.

    PubMed

    Sjöberg, Ronald; Mattsson, Cecilia; Andersson, Eni; Hellström, Cecilia; Uhlen, Mathias; Schwenk, Jochen M; Ayoglu, Burcu; Nilsson, Peter

    2016-09-25

    High-density protein microarrays of recombinant human protein fragments, representing 12,412 unique Ensembl Gene IDs, have here been produced and explored. These protein microarrays were used to analyse antibody off-target interactions, as well as for profiling the human autoantibody repertoire in plasma against the antigens represented by the protein fragments. Affinity-purified polyclonal antibodies produced within the Human Protein Atlas (HPA) were analysed on microarrays of three different sizes, ranging from 384 antigens to 21,120 antigens, for evaluation of the antibody validation criteria in the HPA. Plasma samples from secondary progressive multiple sclerosis patients were also screened in order to explore the feasibility of these arrays for broad-scale profiling of autoantibody reactivity. Furthermore, analysis on these near proteome-wide microarrays was complemented with analysis on HuProt™ Human Proteome protein microarrays. The HPA recombinant protein microarray with 21,120 antigens and the HuProt™ Human Proteome protein microarray are currently the largest protein microarray platforms available to date. The results on these arrays show that the Human Protein Atlas antibodies have few off-target interactions if the antibody validation criteria are kept stringent and demonstrate that the HPA-produced high-density recombinant protein fragment microarrays allow for a high-throughput analysis of plasma for identification of possible autoantibody targets in the context of various autoimmune conditions. PMID:26417875

  10. Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis

    PubMed Central

    2011-01-01

    Background Cytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence in situ hybridization (FISH). Results Using a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23. Conclusions Our results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future. PMID:22087757

  11. Development of a High-density oligo-nucleotide microarray for rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Progress in aquaculture is hindered by a lack of knowledge about the biochemical processes affecting fish production efficiency and quality. Rainbow trout is the most cultivated cold freshwater fish in the US and one of the most intensively studied due to interest as a research model organism for ca...

  12. Reusable conductimetric array of interdigitated microelectrodes for the readout of low-density microarrays.

    PubMed

    Mallén, Maria; Díaz-González, María; Bonilla, Diana; Salvador, Juan P; Marco, María P; Baldi, Antoni; Fernández-Sánchez, César

    2014-06-17

    Low-density protein microarrays are emerging tools in diagnostics whose deployment could be primarily limited by the cost of fluorescence detection schemes. This paper describes an electrical readout system of microarrays comprising an array of gold interdigitated microelectrodes and an array of polydimethylsiloxane microwells, which enabled multiplexed detection of up to thirty six biological events on the same substrate. Similarly to fluorescent readout counterparts, the microarray can be developed on disposable glass slide substrates. However, unlike them, the presented approach is compact and requires a simple and inexpensive instrumentation. The system makes use of urease labeled affinity reagents for developing the microarrays and is based on detection of conductivity changes taking place when ionic species are generated in solution due to the catalytic hydrolysis of urea. The use of a polydimethylsiloxane microwell array facilitates the positioning of the measurement solution on every spot of the microarray. Also, it ensures the liquid tightness and isolation from the surrounding ones during the microarray readout process, thereby avoiding evaporation and chemical cross-talk effects that were shown to affect the sensitivity and reliability of the system. The performance of the system is demonstrated by carrying out the readout of a microarray for boldenone anabolic androgenic steroid hormone. Analytical results are comparable to those obtained by fluorescent scanner detection approaches. The estimated detection limit is 4.0 ng mL(-1), this being below the threshold value set by the World Anti-Doping Agency and the European Community. PMID:24890693

  13. Flow-pattern Guided Fabrication of High-density Barcode Antibody Microarray.

    PubMed

    Ramirez, Lisa S; Wang, Jun

    2016-01-01

    Antibody microarray as a well-developed technology is currently challenged by a few other established or emerging high-throughput technologies. In this report, we renovate the antibody microarray technology by using a novel approach for manufacturing and by introducing new features. The fabrication of our high-density antibody microarray is accomplished through perpendicularly oriented flow-patterning of single stranded DNAs and subsequent conversion mediated by DNA-antibody conjugates. This protocol outlines the critical steps in flow-patterning DNA, producing and purifying DNA-antibody conjugates, and assessing the quality of the fabricated microarray. The uniformity and sensitivity are comparable with conventional microarrays, while our microarray fabrication does not require the assistance of an array printer and can be performed in most research laboratories. The other major advantage is that the size of our microarray units is 10 times smaller than that of printed arrays, offering the unique capability of analyzing functional proteins from single cells when interfacing with generic microchip designs. This barcode technology can be widely employed in biomarker detection, cell signaling studies, tissue engineering, and a variety of clinical applications. PMID:26780370

  14. A microfluidic device for the automated electrical readout of low-density glass-slide microarrays.

    PubMed

    Díaz-González, María; Salvador, J Pablo; Bonilla, Diana; Marco, M Pilar; Fernández-Sánchez, César; Baldi, Antoni

    2015-12-15

    Microarrays are a powerful platform for rapid and multiplexed analysis in a wide range of research fields. Electrical readout systems have emerged as an alternative to conventional optical methods for microarray analysis thanks to its potential advantages like low-cost, low-power and easy miniaturization of the required instrumentation. In this work an automated electrical readout system for low-cost glass-slide microarrays is described. The system enables the simultaneous conductimetric detection of up to 36 biorecognition events by incorporating an array of interdigitated electrode transducers. A polydimethylsiloxane microfluidic structure has been designed that creates microwells over the transducers and incorporates the microfluidic channels required for filling and draining them with readout and cleaning solutions, thus making the readout process fully automated. Since the capture biomolecules are not immobilized on the transducer surface this readout system is reusable, in contrast to previously reported electrochemical microarrays. A low-density microarray based on a competitive enzymatic immunoassay for atrazine detection was used to test the performance of the readout system. The electrical assay shows a detection limit of 0.22±0.03 μg L(-1) similar to that obtained with fluorescent detection and allows the direct determination of the pesticide in polluted water samples. These results proved that an electrical readout system such as the one presented in this work is a reliable and cost-effective alternative to fluorescence scanners for the analysis of low-density microarrays. PMID:26210466

  15. Fast DNA serotyping and antimicrobial resistance gene determination of salmonella enterica with an oligonucleotide microarray-based assay.

    PubMed

    Braun, Sascha D; Ziegler, Albrecht; Methner, Ulrich; Slickers, Peter; Keiling, Silke; Monecke, Stefan; Ehricht, Ralf

    2012-01-01

    Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4-5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping. PMID:23056321

  16. Fast DNA Serotyping and Antimicrobial Resistance Gene Determination of Salmonella enterica with an Oligonucleotide Microarray-Based Assay

    PubMed Central

    Braun, Sascha D.; Ziegler, Albrecht; Methner, Ulrich; Slickers, Peter; Keiling, Silke; Monecke, Stefan; Ehricht, Ralf

    2012-01-01

    Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4–5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping. PMID:23056321

  17. Heterologous oligonucleotide microarrays for transcriptomics in a non-model species; a proof-of-concept study of drought stress in Musa

    PubMed Central

    Davey, Mark W; Graham, Neil S; Vanholme, Bartel; Swennen, Rony; May, Sean T; Keulemans, Johan

    2009-01-01

    Background 'Systems-wide' approaches such as microarray RNA-profiling are ideally suited to the study of the complex overlapping responses of plants to biotic and abiotic stresses. However, commercial microarrays are only available for a limited number of plant species and development costs are so substantial as to be prohibitive for most research groups. Here we evaluate the use of cross-hybridisation to Affymetrix oligonucleotide GeneChip® microarrays to profile the response of the banana (Musa spp.) leaf transcriptome to drought stress using a genomic DNA (gDNA)-based probe-selection strategy to improve the efficiency of detection of differentially expressed Musa transcripts. Results Following cross-hybridisation of Musa gDNA to the Rice GeneChip® Genome Array, ~33,700 gene-specific probe-sets had a sufficiently high degree of homology to be retained for transcriptomic analyses. In a proof-of-concept approach, pooled RNA representing a single biological replicate of control and drought stressed leaves of the Musa cultivar 'Cachaco' were hybridised to the Affymetrix Rice Genome Array. A total of 2,910 Musa gene homologues with a >2-fold difference in expression levels were subsequently identified. These drought-responsive transcripts included many functional classes associated with plant biotic and abiotic stress responses, as well as a range of regulatory genes known to be involved in coordinating abiotic stress responses. This latter group included members of the ERF, DREB, MYB, bZIP and bHLH transcription factor families. Fifty-two of these drought-sensitive Musa transcripts were homologous to genes underlying QTLs for drought and cold tolerance in rice, including in 2 instances QTLs associated with a single underlying gene. The list of drought-responsive transcripts also included genes identified in publicly-available comparative transcriptomics experiments. Conclusion Our results demonstrate that despite the general paucity of nucleotide sequence data in

  18. Use of Low-Density DNA Microarrays and Photopolymerization for Genotyping Foodborne-Associated Noroviruses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Human noroviruses cause up to 21 million cases of foodborne disease in the United States annually and are the most common cause of acute gastroenteritis in industrialized countries. To reduce the burden of foodborne disease associated with viruses, the use of low density DNA microarrays in conjunct...

  19. A Low Density Microarray Method for the Identification of Human Papillomavirus Type 18 Variants

    PubMed Central

    Meza-Menchaca, Thuluz; Williams, John; Rodríguez-Estrada, Rocío B.; García-Bravo, Aracely; Ramos-Ligonio, Ángel; López-Monteon, Aracely; Zepeda, Rossana C.

    2013-01-01

    We describe a novel microarray based-method for the screening of oncogenic human papillomavirus 18 (HPV-18) molecular variants. Due to the fact that sequencing methodology may underestimate samples containing more than one variant we designed a specific and sensitive stacking DNA hybridization assay. This technology can be used to discriminate between three possible phylogenetic branches of HPV-18. Probes were attached covalently on glass slides and hybridized with single-stranded DNA targets. Prior to hybridization with the probes, the target strands were pre-annealed with the three auxiliary contiguous oligonucleotides flanking the target sequences. Screening HPV-18 positive cell lines and cervical samples were used to evaluate the performance of this HPV DNA microarray. Our results demonstrate that the HPV-18's variants hybridized specifically to probes, with no detection of unspecific signals. Specific probes successfully reveal detectable point mutations in these variants. The present DNA oligoarray system can be used as a reliable, sensitive and specific method for HPV-18 variant screening. Furthermore, this simple assay allows the use of inexpensive equipment, making it accessible in resource-poor settings. PMID:24077317

  20. Microbial distributions detected by an oligonucleotide microarray across geochemical zones associated with methane in marine sediments from the Ulleung Basin

    SciTech Connect

    Briggs, Brandon R; Graw, Michael; Brodie, Eoin L; Bahk, Jang-Jun; Kim, Sung-Han; Hyun, Jung-Ho; Kim, Ji-Hoon; Torres, Marta; Colwell, Frederick S

    2013-11-01

    The biogeochemical processes that occur in marine sediments on continental margins are complex; however, from one perspective they can be considered with respect to three geochemical zones based on the presence and form of methane: sulfate–methane transition (SMTZ), gas hydrate stability zone (GHSZ), and free gas zone (FGZ). These geochemical zones may harbor distinct microbial communities that are important in biogeochemical carbon cycles. The objective of this study was to describe the microbial communities in sediments from the SMTZ, GHSZ, and FGZ using molecular ecology methods (i.e. PhyloChip microarray analysis and terminal restriction fragment length polymorphism (T-RFLP)) and examining the results in the context of non-biological parameters in the sediments. Non-metric multidimensional scaling and multi-response permutation procedures were used to determine whether microbial community compositions were significantly different in the three geochemical zones and to correlate samples with abiotic characteristics of the sediments. This analysis indicated that microbial communities from all three zones were distinct from one another and that variables such as sulfate concentration, hydrate saturation of the nearest gas hydrate layer, and depth (or unmeasured variables associated with depth e.g. temperature, pressure) were correlated to differences between the three zones. The archaeal anaerobic methanotrophs typically attributed to performing anaerobic oxidation of methane were not detected in the SMTZ; however, the marine benthic group-B, which is often found in SMTZ, was detected. Within the GHSZ, samples that were typically closer to layers that contained higher hydrate saturation had indicator sequences related to Vibrio-type taxa. These results suggest that the biogeographic patterns of microbial communities in marine sediments are distinct based on geochemical zones defined by methane.

  1. PCR amplfication on a microarray of gel-immobilized oligonucleotides : detection of bacterial toxin- and drug-resistent genes and their mutations.

    SciTech Connect

    Strizhkov, B. N.; Drobyshev, A. L.; Mikhailovich, V. M.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2000-10-01

    PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.

  2. A functional genomics tool for the Pacific bluefin tuna: Development of a 44K oligonucleotide microarray from whole-genome sequencing data for global transcriptome analysis.

    PubMed

    Yasuike, Motoshige; Fujiwara, Atushi; Nakamura, Yoji; Iwasaki, Yuki; Nishiki, Issei; Sugaya, Takuma; Shimizu, Akio; Sano, Motohiko; Kobayashi, Takanori; Ototake, Mitsuru

    2016-02-01

    Bluefin tunas are one of the most important fishery resources worldwide. Because of high market values, bluefin tuna farming has been rapidly growing during recent years. At present, the most common form of the tuna farming is based on the stocking of wild-caught fish. Therefore, concerns have been raised about the negative impact of the tuna farming on wild stocks. Recently, the Pacific bluefin tuna (PBT), Thunnus orientalis, has succeeded in completing the reproduction cycle under aquaculture conditions, but production bottlenecks remain to be solved because of very little biological information on bluefin tunas. Functional genomics approaches promise to rapidly increase our knowledge on biological processes in the bluefin tuna. Here, we describe the development of the first 44K PBT oligonucleotide microarray (oligo-array), based on whole-genome shotgun (WGS) sequencing and large-scale expressed sequence tags (ESTs) data. In addition, we also introduce an initial 44K PBT oligo-array experiment using in vitro grown peripheral blood leukocytes (PBLs) stimulated with immunostimulants such as lipopolysaccharide (LPS: a cell wall component of Gram-negative bacteria) or polyinosinic:polycytidylic acid (poly I:C: a synthetic mimic of viral infection). This pilot 44K PBT oligo-array analysis successfully addressed distinct immune processes between LPS- and poly I:C- stimulated PBLs. Thus, we expect that this oligo-array will provide an excellent opportunity to analyze global gene expression profiles for a better understanding of diseases and stress, as well as for reproduction, development and influence of nutrition on tuna aquaculture production. PMID:26477480

  3. Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions.

    PubMed

    Fasolo, Joseph; Im, Hogune; Snyder, Michael P

    2015-01-01

    High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis to determine a high confidence set of protein interactions for downstream validation in vivo. PMID:26274875

  4. An Undergraduate Laboratory Exercise to Study the Effect of Darkness on Plant Gene Expression Using DNA Microarray

    ERIC Educational Resources Information Center

    Chang, Ming-Mei; Briggs, George M.

    2007-01-01

    DNA microarrays are microscopic arrays on a solid surface, typically a glass slide, on which DNA oligonucleotides are deposited or synthesized in a high-density matrix with a predetermined spatial order. Several types of DNA microarrays have been developed and used for various biological studies. Here, we developed an undergraduate laboratory…

  5. Density variant glycan microarray for evaluating cross-linking of mucin-like glycoconjugates by lectins.

    PubMed

    Godula, Kamil; Bertozzi, Carolyn R

    2012-09-26

    Interactions of mucin glycoproteins with cognate receptors are dictated by the structures and spatial organization of glycans that decorate the mucin polypeptide backbone. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. In this work, we employed a microarray platform comprising synthetic glycopolymers that emulate natural mucins arrayed at different surface densities to evaluate how glycan valency and spatial separation affect the preferential binding mode of a particular lectin. We evaluated a panel of four lectins (Soybean agglutinin (SBA), Wisteria floribunda lectin (WFL), Vicia villosa-B-4 agglutinin (VVA), and Helix pomatia agglutin (HPA)) with specificity for α-N-acetylgalactosamine (α-GalNAc), an epitope displayed on mucins overexpressed in many adenocarcinomas. While these lectins possess the ability to agglutinate A(1)-blood cells carrying the α-GalNAc epitope and cross-link low valency glycoconjugates, only SBA showed a tendency to form intermolecular cross-links among the arrayed polyvalent mucin mimetics. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. Our findings indicate that glycan valency can set thresholds for cross-linking by lectins. More broadly, well-defined synthetic glycopolymers enable the integration of glycoconjugate structural and spatial diversity in a single microarray screening platform. PMID:22967056

  6. Microarrays with varying carbohydrate density reveal distinct subpopulations of serum antibodies.

    PubMed

    Oyelaran, Oyindasola; Li, Qian; Farnsworth, David; Gildersleeve, Jeffrey C

    2009-07-01

    Antigen arrays have become important tools for profiling complex mixtures of proteins such as serum antibodies. These arrays can be used to better understand immune responses, discover new biomarkers, and guide the development of vaccines. Nevertheless, they are not perfect and improved array designs would enhance the information derived from this technology. In this study, we describe and evaluate a strategy for varying antigen density on an array and then use the array to study binding of lectins, monoclonal antibodies, and serum antibodies. To vary density, neoglycoproteins containing differing amounts of carbohydrate were synthesized and used to make a carbohydrate microarray with variations in both structure and density. We demonstrate that this method provides variations in density on the array surface within a range that is relevant for biological recognition events. The array was used to evaluate density dependent binding properties of three lectins (Vicia villosa lectin B4, Helix pomatia agglutinin, and soybean agglutinin) and three monoclonal antibodies (HBTn-1, B1.1, and Bric111) that bind the tumor-associated Tn antigen. In addition, serum antibodies were profiled from 30 healthy donors. The results show that variations in antigen density are required to detect the full spectrum of antibodies that bind a particular antigen and can be used to reveal differences in antibody populations between individuals that are not detectable using a single antigen density. PMID:19366269

  7. Working with Oligonucleotide Arrays.

    PubMed

    Carvalho, Benilton S

    2016-01-01

    Preprocessing microarray data consists of a number of statistical procedures that convert the observed intensities into quantities that represent biological events of interest, like gene expression and allele-specific abundances. Here, we present a summary of the theory behind microarray data preprocessing for expression, whole transcriptome and SNP designs and focus on the computational protocol used to obtain processed data that will be used on downstream analyses. We describe the main features of the oligo Bioconductor package, an application designed to support oligonucleotide microarrays using the R statistical environment and the infrastructure provided by Bioconductor, allowing the researcher to handle probe-level data and interface with advanced statistical tools under a simplified framework. We demonstrate the use of the package by preprocessing data originated from three different designs. PMID:27008013

  8. Global Analysis of Human Nonreceptor Tyrosine Kinase Specificity Using High-Density Peptide Microarrays

    PubMed Central

    2015-01-01

    Protein kinases phosphorylate substrates in the context of specific phosphorylation site sequence motifs. The knowledge of the specific sequences that are recognized by kinases is useful for mapping sites of phosphorylation in protein substrates and facilitates the generation of model substrates to monitor kinase activity. Here, we have adapted a positional scanning peptide library method to a microarray format that is suitable for the rapid determination of phosphorylation site motifs for tyrosine kinases. Peptide mixtures were immobilized on glass slides through a layer of a tyrosine-free Y33F mutant avidin to facilitate the analysis of phosphorylation by radiolabel assay. A microarray analysis provided qualitatively similar results in comparison with the solution phase peptide library “macroarray” method. However, much smaller quantities of kinases were required to phosphorylate peptides on the microarrays, which thus enabled a proteome scale analysis of kinase specificity. We illustrated this capability by microarray profiling more than 80% of the human nonreceptor tyrosine kinases (NRTKs). Microarray results were used to generate a universal NRTK substrate set of 11 consensus peptides for in vitro kinase assays. Several substrates were highly specific for their cognate kinases, which should facilitate their incorporation into kinase-selective biosensors. PMID:25164267

  9. Quality Control Usage in High-Density Microarrays Reveals Differential Gene Expression Profiles in Ovarian Cancer.

    PubMed

    Villegas-Ruiz, Vanessa; Moreno, Jose; Jacome-Lopez, Karina; Zentella-Dehesa, Alejandro; Juarez-Mendez, Sergio

    2016-01-01

    There are several existing reports of microarray chip use for assessment of altered gene expression in different diseases. In fact, there have been over 1.5 million assays of this kind performed over the last twenty years, which have influenced clinical and translational research studies. The most commonly used DNA microarray platforms are Affymetrix GeneChip and Quality Control Software along with their GeneChip Probe Arrays. These chips are created using several quality controls to confirm the success of each assay, but their actual impact on gene expression profiles had not been previously analyzed until the appearance of several bioinformatics tools for this purpose. We here performed a data mining analysis, in this case specifically focused on ovarian cancer, as well as healthy ovarian tissue and ovarian cell lines, in order to confirm quality control results and associated variation in gene expression profiles. The microarray data used in our research were downloaded from ArrayExpress and Gene Expression Omnibus (GEO) and analyzed with Expression Console Software using RMA, MAS5 and Plier algorithms. The gene expression profiles were obtained using Partek Genomics Suite v6.6 and data were visualized using principal component analysis, heat map, and Venn diagrams. Microarray quality control analysis showed that roughly 40% of the microarray files were false negative, demonstrating over- and under-estimation of expressed genes. Additionally, we confirmed the results performing second analysis using independent samples. About 70% of the significant expressed genes were correlated in both analyses. These results demonstrate the importance of appropriate microarray processing to obtain a reliable gene expression profile. PMID:27268623

  10. Fiber optic chemical sensors: The evolution of high- density fiber-optic DNA microarrays

    NASA Astrophysics Data System (ADS)

    Ferguson, Jane A.

    2001-06-01

    Sensors were developed for multianalyte monitoring, fermentation monitoring, lactate analysis, remote oxygen detection for use in bioremediation monitoring and in a fuel spill clean-up project, heavy metal analysis, and high density DNA microarrays. The major focus of this thesis involved creating and improving high-density DNA gene arrays. Fiber optic sensors are created using fluorescent indicators, polymeric supports, and optical fiber substrates. The fluorescent indicator is entrapped in a polymer layer and attached to the tip of the optical fiber. The tip of the fiber bearing the sensing layer (the distal end) is placed in the sample of interest while the other end of the fiber (the proximal end) is connected to an analysis system. Any length of fiber can be used without compromising the integrity or sensitivity of the system. A fiber optic oxygen sensor was designed incorporating an oxygen sensitive fluorescent dye and a gas permeable polymer attached to an optical fiber. The construction simplicity and ruggedness of the sensor enabled its deployment for in situ chemical oxidation and bioremediation studies. Optical fibers were also used as the substrate to detect biomolecules in solution. To monitor bioprocesses, the production of the analyte of interest must be coupled with a species that is optically measurable. For example, oxygen is consumed in many metabolic functions. The fiber optic oxygen sensor is equipped with an additional sensing layer. Upon contact with a specific biochemical in the sample, a reaction occurs in the additional sensing layer that either consumes or produces oxygen. This dual layer system was used to monitor the presence of lactate, an important metabolite for clinical and bioprocess analysis. In many biological and environmental systems, the generation of one species occurs coincidentally with the generation or consumption of another species. A multianalyte sensor was prepared that can monitor the simultaneous activity of pH, CO2

  11. Dynamic of active microorganisms inhabiting a bioleaching industrial heap of low‐grade copper sulfide ore monitored by real‐time PCR and oligonucleotide prokaryotic acidophile microarray

    PubMed Central

    Remonsellez, Francisco; Galleguillos, Felipe; Moreno‐Paz, Mercedes; Parro, Víctor; Acosta, Mauricio; Demergasso, Cecilia

    2009-01-01

    Summary The bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commercial bioleaching operations. Here we report the first quantitative description of the dynamic of active communities in an industrial bioleaching heap. Acidithiobacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abundance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithiobacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium‐like bacteria reached their highest abundance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA‐based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium‐like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe3+/Fe2+ ion rate, are primary factors shaping the microbial dynamic in the heap. PMID:21255296

  12. Strategies in the preparation of DNA oligonucleotide arrays for diagnostic applications.

    PubMed

    Beaucage, S L

    2001-08-01

    This report emphasizes the interfacial chemistry that is required to ensure proper attachment of oligonucleotides onto the surface of microarrays. For example, strategies for the covalent attachment of pre-synthesized oligonucleotides to glass slides, gold films, polyacrylamide gel pads, polypyrrole films, and optical fibers are surveyed in an attempt to better define the parameters for optimal formation and detection of DNA hybrids. These parameters include among others, the nature and length of the linkers attaching oligonucleotides to the arrays, and the surface density of oligonucleotides required for unhindered hybridization with DNA targets. Sensitive detection methods such as the use of light-scattering techniques, molecular beacons, surface plasmon resonance, attenuated total internal reflection-FTIR, and the evanescent field excitation of fluorescence from surface-bound fluorophores have been developed to study the kinetics and specificity of hybridization events. Finally, the synthesis of oligonucleotides directly on glass surfaces and polypropylene sheets has been investigated to enable DNA sequencing by hybridization and achieve oligonucleotide densities of ca. 10(6) sequences per cm(2) on DNA chips. PMID:11472237

  13. Increased Expression of Matrix Extracellular Phosphoglycoprotein (MEPE) in Cortical Bone of the Rat Tibia after Mechanical Loading: Identification by Oligonucleotide Microarray

    PubMed Central

    Reijnders, Christianne M. A.; van Essen, Huib W.; van Rens, Birgitte T. T. M.; van Beek, Johannes H. G. M.; Ylstra, Bauke; Blankenstein, Marinus A.; Lips, Paul; Bravenboer, Nathalie

    2013-01-01

    Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG-TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form), fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up-regulation of MEPE and the down-regulation of creatine kinase (muscle form) and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation. PMID:24255709

  14. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manufacturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthesized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microarrays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

  15. The Current Status of DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Shi, Leming; Perkins, Roger G.; Tong, Weida

    DNA microarray technology that allows simultaneous assay of thousands of genes in a single experiment has steadily advanced to become a mainstream method used in research, and has reached a stage that envisions its use in medical applications and personalized medicine. Many different strategies have been developed for manufacturing DNA microarrays. In this chapter, we discuss the manu facturing characteristics of seven microarray platforms that were used in a recently completed large study by the MicroArray Quality Control (MAQC) consortium, which evaluated the concordance of results across these platforms. The platforms can be grouped into three categories: (1) in situ synthesis of oligonucleotide probes on microarrays (Affymetrix GeneChip® arrays based on photolithography synthesis and Agilent's arrays based on inkjet synthesis); (2) spotting of presynthe-sized oligonucleotide probes on microarrays (GE Healthcare's CodeLink system, Applied Biosystems' Genome Survey Microarrays, and the custom microarrays printed with Operon's oligonucleotide set); and (3) deposition of presynthesized oligonucleotide probes on bead-based microarrays (Illumina's BeadChip microar-rays). We conclude this chapter with our views on the challenges and opportunities toward acceptance of DNA microarray data in clinical and regulatory settings.

  16. Construction of High-Density Tissue Microarrays at Low Cost by Using Self-Made Manual Microarray Kits and Recipient Paraffin Blocks

    PubMed Central

    Choi, Chang Hwan; Kim, Kyu Ho; Song, Ju Young; Kim, Lucia; Park, In Suh; Han, Jee Young; Kim, Joon Mee; Chu, Young Chae

    2012-01-01

    Background Advances of tissue microarray (TMA) technology have enabled simultaneous in situ analysis of biomarker expression in a large number of archived pathology specimens. However, the relatively high cost of TMA construction may hamper many researchers from using this essential tool of modern pathology research. We discuss methods for making TMA kits and recipient blocks for manual construction of high-density TMAs at low cost. Methods Ordinary cannula piercing needles, hypodermic needles, bone marrow biopsy needles, metallic ink cartridges of ballpoint pens, and disposable skin biopsy punches were used to construct self-made manual TMA kits. The recipient blocks were manufactured by boring holes in the conventional bare paraffin blocks. A mini electric hand drill and a microcompound table assembled on a drill stand were used to maximize the capacity of the recipient blocks. Results By using TMA kits made from cannula piercing needles (16- and 18-gauge), it was possible to construct TMAs with 1 mm×140 cores, 0.6 mm×320 cores, 2 mm×70 cores, 3 mm×35 cores, and 5 mm×12 cores. The capacity of the recipient blocks could be dramatically increased by drilling holes. Conclusions Construction of TMAs using self-made TMA kits is an inexpensive alternative to construction of TMAs using commercial devices. PMID:23323107

  17. High-density universal 16S rRNA microarray analysis revealsbroader diversity than typical clone library when sampling theenvironment

    SciTech Connect

    DeSantis, Todd Z.; Brodie, Eoin L.; Moberg, Jordan P.; Zubieta,Ingrid X.; Piceno, Yvette M.; Andersen, Gary L.

    2006-06-15

    Molecular approaches aimed at detection of a broad-range ofprokaryotes in the environment routinely rely upon classifyingheterogeneous 16S rRNA genes amplified by PCR using primers with broadspecificity. The general method of sampling and categorizing DNA has beento clone then sequence the PCR products. However, the number of clonesrequired to adequately catalogue the majority of taxa in a sample isunwieldy. Alternatively, hybridizing target sequences to a universal 16SrRNA gene microarray may provide a more rapid and comprehensive view ofprokaryotic community composition. This study investigated the breadthand accuracy of a microarray in detecting diverse 16S rRNA gene sequencetypes compared to clone-and-sequencing using three environmental samples:urban aerosol, subsurface soil and subsurface water. PCR productsgenerated from universal 16S rRNA gene-targeted primers were classifiedusing either the clone-and-sequence method or by hybridization to a novelhigh-density microarray of 297,851 probes complementary to 842prokaryotic sub-families. The three clone libraries comprised 1,391high-quality sequences. Approximately 8 percent of the clones could notbe placed into a known sub-family and were considered novel. Themicroarray results confirmed the majority of clone-detected sub-familiesand additionally demonstrated greater amplicon diversity extending intophyla not observed by the cloning method. Sequences matching OTUs withinthe phyla Nitrospira, Planctomycetes, and TM7, which were uniquelydetected by the array, were verified with specific primers and subsequentamplicon sequencing. Sub-family richness detected by the arraycorresponded well with non-parametric richness predictions extrapolatedfrom clone libraries except in the water community where clone-basedrichness predictions were greatly exceeded. It was concluded thatalthough the microarray is unreliable inidentifying novel prokaryotictaxa, it reveals greater diversity in environmental samples thansequencing a

  18. Cytokines and therapeutic oligonucleotides.

    PubMed

    Hartmann, G; Bidlingmaier, M; Eigler, A; Hacker, U; Endres, S

    1997-12-01

    Therapeutic oligonucleotides - short strands of synthetic nucleic acids - encompass antisense and aptamer oligonucleotides. Antisense oligonucleotides are designed to bind to target RNA by complementary base pairing and to inhibit translation of the target protein. Antisense oligonucleotides enable specific inhibition of cytokine synthesis. In contrast, aptamer oligonucleotides are able to bind directly to specific proteins. This binding depends on the sequence of the oligonucleotide. Aptamer oligonucleotides with CpG motifs can exert strong immunostimulatory effects. Both kinds of therapeutic oligonucleotides - antisense and aptamer oligonucleotides - provide promising tools to modulate immunological functions. Recently, therapeutic oligonucleotides have moved towards clinical application. An antisense oligonucleotide directed against the proinflammatory intercellular adhesion molecule 1 (ICAM-1) is currently being tested in clinical trials for therapy of inflammatory disease. Immunostimulatory aptamer oligonucleotides are in preclinical development for immunotherapy. In the present review we summarize the application of therapeutic oligonucleotides to modulate immunological functions. We include technological aspects as well as current therapeutic concepts and clinical studies. PMID:9740353

  19. The role of group index engineering in series-connected photonic crystal microcavities for high density sensor microarrays

    SciTech Connect

    Zou, Yi Zhu, Liang; Chen, Ray T.; Chakravarty, Swapnajit

    2014-04-07

    We experimentally demonstrate an efficient and robust method for series connection of photonic crystal microcavities that are coupled to photonic crystal waveguides in the slow light transmission regime. We demonstrate that group index taper engineering provides excellent optical impedance matching between the input and output strip waveguides and the photonic crystal waveguide, a nearly flat transmission over the entire guided mode spectrum and clear multi-resonance peaks corresponding to individual microcavities that are connected in series. Series connected photonic crystal microcavities are further multiplexed in parallel using cascaded multimode interference power splitters to generate a high density silicon nanophotonic microarray comprising 64 photonic crystal microcavity sensors, all of which are interrogated simultaneously at the same instant of time.

  20. Development of a human mitochondrial oligonucleotide microarray (h-MitoArray) and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

    PubMed Central

    Čížková, Alena; Stránecký, Viktor; Ivánek, Robert; Hartmannová, Hana; Nosková, Lenka; Piherová, Lenka; Tesařová, Markéta; Hansíková, Hana; Honzík, Tomáš; Zeman, Jiří; Divina, Petr; Potocká, Andrea; Paul, Jan; Sperl, Wolfgang; Mayr, Johannes A; Seneca, Sara; Houštĕk, Josef; Kmoch, Stanislav

    2008-01-01

    Background To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray) allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. Results Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate disease causing genes for

  1. Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology

    PubMed Central

    Miller, Melissa B.; Tang, Yi-Wei

    2009-01-01

    Summary: The introduction of in vitro nucleic acid amplification techniques, led by real-time PCR, into the clinical microbiology laboratory has transformed the laboratory detection of viruses and select bacterial pathogens. However, the progression of the molecular diagnostic revolution currently relies on the ability to efficiently and accurately offer multiplex detection and characterization for a variety of infectious disease pathogens. Microarray analysis has the capability to offer robust multiplex detection but has just started to enter the diagnostic microbiology laboratory. Multiple microarray platforms exist, including printed double-stranded DNA and oligonucleotide arrays, in situ-synthesized arrays, high-density bead arrays, electronic microarrays, and suspension bead arrays. One aim of this paper is to review microarray technology, highlighting technical differences between them and each platform's advantages and disadvantages. Although the use of microarrays to generate gene expression data has become routine, applications pertinent to clinical microbiology continue to rapidly expand. This review highlights uses of microarray technology that impact diagnostic microbiology, including the detection and identification of pathogens, determination of antimicrobial resistance, epidemiological strain typing, and analysis of microbial infections using host genomic expression and polymorphism profiles. PMID:19822891

  2. A New Generation Microarray for the Simultaneous Detection and Identification of Yersinia pestis and Bacillus anthracis in Food

    PubMed Central

    Goji, Noriko; MacMillan, Trevor; Amoako, Kingsley Kwaku

    2012-01-01

    The use of microarrays as a multiple analytic system has generated increased interest and provided a powerful analytical tool for the simultaneous detection of pathogens in a single experiment. A wide array of applications for this technology has been reported. A low density oligonucleotide microarray was generated from the genetic sequences of Y. pestis and B. anthracis and used to fabricate a microarray chip. The new generation chip, consisting of 2,240 spots in 4 quadrants with the capability of stripping/rehybridization, was designated as “Y-PESTIS/B-ANTHRACIS 4x2K Array.” The chip was tested for specificity using DNA from a panel of bacteria that may be potentially present in food. In all, 37 unique Y. pestis-specific and 83 B. anthracis-specific probes were identified. The microarray assay distinguished Y. pestis and B. anthracis from the other bacterial species tested and correctly identified the Y. pestis-specific oligonucleotide probes using DNA extracted from experimentally inoculated milk samples. Using a whole genome amplification method, the assay was able to detect as low as 1 ng genomic DNA as the start sample. The results suggest that oligonucleotide microarray can specifically detect and identify Y. pestis and B. anthracis and may be a potentially useful diagnostic tool for detecting and confirming the organisms in food during a bioterrorism event. PMID:23125935

  3. Controlling microarray DNA hybridization efficiency by probe-surface distance and external surface electrostatics

    NASA Astrophysics Data System (ADS)

    Qamhieh, K.; Pettitt, B. Montgomery

    2015-03-01

    DNA microarrays are analytical devices designed to determine the composition of multicomponent solutions of nucleic acids, DNA or RNA. These devices are promising technology for diverse applications, including sensing, diagnostics, and drug/gene delivery. Here, we modify a hybridization adsorption isotherm to study the effects of probe-surface distance and the external electrostatic fields, on the oligonucleotide hybridization in microarray and how these effects are varies depending on surface probe density and target concentration. This study helps in our understanding on-surface hybridization mechanisms, and from it we can observe a significant effect of the probe-surface distance, and the external electrostatic fields, on the hybridization yield. In addition we present a simple new criteria to control the oligonucleotide hybridization efficiency by providing a chart illustrating the effects of all factors on the DNA-hybridization efficiency.

  4. Controlled Breast Cancer Microarrays for the Deconvolution of Cellular Multilayering and Density Effects upon Drug Responses

    PubMed Central

    Håkanson, Maria; Kobel, Stefan; Lutolf, Matthias P.; Textor, Marcus; Cukierman, Edna; Charnley, Mirren

    2012-01-01

    Background Increasing evidence shows that the cancer microenvironment affects both tumorigenesis and the response of cancer to drug treatment. Therefore in vitro models that selectively reflect characteristics of the in vivo environment are greatly needed. Current methods allow us to screen the effect of extrinsic parameters such as matrix composition and to model the complex and three-dimensional (3D) cancer environment. However, 3D models that reflect characteristics of the in vivo environment are typically too complex and do not allow the separation of discrete extrinsic parameters. Methodology/Principal Findings In this study we used a poly(ethylene glycol) (PEG) hydrogel-based microwell array to model breast cancer cell behavior in multilayer cell clusters that allows a rigorous control of the environment. The innovative array fabrication enables different matrix proteins to be integrated into the bottom surface of microwells. Thereby, extrinsic parameters including dimensionality, type of matrix coating and the extent of cell-cell adhesion could be independently studied. Our results suggest that cell to matrix interactions and increased cell-cell adhesion, at high cell density, induce independent effects on the response to Taxol in multilayer breast cancer cell clusters. In addition, comparing the levels of apoptosis and proliferation revealed that drug resistance mediated by cell-cell adhesion can be related to altered cell cycle regulation. Conversely, the matrix-dependent response to Taxol did not correlate with proliferation changes suggesting that cell death inhibition may be responsible for this effect. Conclusions/Significance The application of the PEG hydrogel platform provided novel insight into the independent role of extrinsic parameters controlling drug response. The presented platform may not only become a useful tool for basic research related to the role of the cancer microenvironment but could also serve as a complementary platform for in

  5. Disc-based microarrays: principles and analytical applications.

    PubMed

    Morais, Sergi; Puchades, Rosa; Maquieira, Ángel

    2016-07-01

    The idea of using disk drives to monitor molecular biorecognition events on regular optical discs has received considerable attention during the last decade. CDs, DVDs, Blu-ray discs and other new optical discs are universal and versatile supports with the potential for development of protein and DNA microarrays. Besides, standard disk drives incorporated in personal computers can be used as compact and affordable optical reading devices. Consequently, a CD technology, resulting from the audio-video industry, has been used to develop analytical applications in health care, environmental monitoring, food safety and quality assurance. The review presents and critically evaluates the current state of the art of disc-based microarrays with illustrative examples, including past, current and future developments. Special mention is made of the analytical developments that use either chemically activated or raw standard CDs where proteins, oligonucleotides, peptides, haptens or other biological probes are immobilized. The discs are also used to perform the assays and must maintain their readability with standard optical drives. The concept and principle of evolving disc-based microarrays and the evolution of disk drives as optical detectors are also described. The review concludes with the most relevant uses ordered chronologically to provide an overview of the progress of CD technology applications in the life sciences. Also, it provides a selection of important references to the current literature. Graphical Abstract High density disc-based microarrays. PMID:26922341

  6. Cross-species hybridisation of human and bovine orthologous genes on high density cDNA microarrays

    PubMed Central

    Adjaye, James; Herwig, Ralf; Herrmann, Doris; Wruck, Wasco; BenKahla, Alia; Brink, Thore C; Nowak, Monika; Carnwath, Joseph W; Hultschig, Claus; Niemann, Heiner; Lehrach, Hans

    2004-01-01

    Background Cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The usefulness of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. The objective of this study was to investigate the feasibility and reproducibility of cross-species analysis employing a human cDNA microarray as probe. Results As a proof of principle, total RNA derived from human and bovine fetal brains was used as a source of labelled targets for hybridisation onto a human cDNA microarray composed of 349 characterised genes. Each gene was spotted 20 times representing 6,980 data points thus enabling highly reproducible spot quantification. Employing high stringency hybridisation and washing conditions, followed by data analysis, revealed slight differences in the expression levels and reproducibility of the signals between the two species. We also assigned each of the genes into three expression level categories- i.e. high, medium and low. The correlation co-efficient of cross hybridisation between the orthologous genes was 0.94. Verification of the array data by semi-quantitative RT-PCR using common primer sequences enabled co-amplification of both human and bovine transcripts. Finally, we were able to assign gene names to previously uncharacterised bovine ESTs. Conclusions Results of our study demonstrate the harnessing and utilisation power of comparative genomics and prove the feasibility of using human microarrays to facilitate the identification of co-expressed orthologous genes in common tissues derived from different

  7. Supplementing High-Density SNP Microarrays for Additional Coverage of Disease-Related Genes: Addiction as a Paradigm

    SciTech Connect

    SacconePhD, Scott F; Chesler, Elissa J; Bierut, Laura J; Kalivas, Peter J; Lerman, Caryn; Saccone, Nancy L; Uhl, George R; Li, Chuan-Yun; Philip, Vivek M; Edenberg, Howard; Sherry, Steven; Feolo, Michael; Moyzis, Robert K; Rutter, Joni L

    2009-01-01

    Commercial SNP microarrays now provide comprehensive and affordable coverage of the human genome. However, some diseases have biologically relevant genomic regions that may require additional coverage. Addiction, for example, is thought to be influenced by complex interactions among many relevant genes and pathways. We have assembled a list of 486 biologically relevant genes nominated by a panel of experts on addiction. We then added 424 genes that showed evidence of association with addiction phenotypes through mouse QTL mappings and gene co-expression analysis. We demonstrate that there are a substantial number of SNPs in these genes that are not well represented by commercial SNP platforms. We address this problem by introducing a publicly available SNP database for addiction. The database is annotated using numeric prioritization scores indicating the extent of biological relevance. The scores incorporate a number of factors such as SNP/gene functional properties (including synonymy and promoter regions), data from mouse systems genetics and measures of human/mouse evolutionary conservation. We then used HapMap genotyping data to determine if a SNP is tagged by a commercial microarray through linkage disequilibrium. This combination of biological prioritization scores and LD tagging annotation will enable addiction researchers to supplement commercial SNP microarrays to ensure comprehensive coverage of biologically relevant regions.

  8. Supplementing High-Density SNP Microarrays for Additional Coverage of Disease-Related Genes: Addiction as a Paradigm

    PubMed Central

    Saccone, Scott F.; Bierut, Laura J.; Chesler, Elissa J.; Kalivas, Peter W.; Lerman, Caryn; Saccone, Nancy L.; Uhl, George R.; Li, Chuan-Yun; Philip, Vivek M.; Edenberg, Howard J.; Sherry, Stephen T.; Feolo, Michael; Moyzis, Robert K.; Rutter, Joni L.

    2009-01-01

    Commercial SNP microarrays now provide comprehensive and affordable coverage of the human genome. However, some diseases have biologically relevant genomic regions that may require additional coverage. Addiction, for example, is thought to be influenced by complex interactions among many relevant genes and pathways. We have assembled a list of 486 biologically relevant genes nominated by a panel of experts on addiction. We then added 424 genes that showed evidence of association with addiction phenotypes through mouse QTL mappings and gene co-expression analysis. We demonstrate that there are a substantial number of SNPs in these genes that are not well represented by commercial SNP platforms. We address this problem by introducing a publicly available SNP database for addiction. The database is annotated using numeric prioritization scores indicating the extent of biological relevance. The scores incorporate a number of factors such as SNP/gene functional properties (including synonymy and promoter regions), data from mouse systems genetics and measures of human/mouse evolutionary conservation. We then used HapMap genotyping data to determine if a SNP is tagged by a commercial microarray through linkage disequilibrium. This combination of biological prioritization scores and LD tagging annotation will enable addiction researchers to supplement commercial SNP microarrays to ensure comprehensive coverage of biologically relevant regions. PMID:19381300

  9. Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

    DOEpatents

    Golova, Julia B.; Chernov, Boris K.

    2010-04-27

    New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

  10. Use of a low-density microarray for studying gene expression patterns induced by hepatotoxicants on primary cultures of rat hepatocytes.

    PubMed

    de Longueville, Francoise; Atienzar, Franck A; Marcq, Laurence; Dufrane, Simon; Evrard, Stéphanie; Wouters, Lydia; Leroux, Florence; Bertholet, Vincent; Gerin, Brigitte; Whomsley, Rhys; Arnould, Thierry; Remacle, José; Canning, Mickael

    2003-10-01

    In the field of gene expression analysis, DNA microarray technology is having a major impact on many different areas including toxicology. For instance, a number of studies have shown that transcription profiling can generate the information needed to assign a compound to a mode-of-action class. In this study, we investigated whether compounds inducing similar toxicological endpoints produce similar changes in gene expression. In vitro primary rat hepatocytes were exposed to 11 different hepatotoxicants: acetaminophen, amiodarone, clofibrate, erythromycin estolate, isoniazid, alpha-naphtylylisothiocyanate, beta-naphtoflavone, 4-pentenoic acid, phenobarbital, tetracycline, and zileuton. These molecules were selected on the basis of their variety of hepatocellular effects observed such as necrosis, cholestasis, steatosis, and induction of CYP P450 enzymes. We used a low-density DNA microarray containing 59 genes chosen as relevant toxic and metabolic markers. The in vitro gene expression data generated in this study were generally in good agreement with the literature, which mainly concerns in vivo data. Furthermore, gene expression profiles observed in this study have been confirmed for several genes by real-time PCR assays. All the tested drugs generated a specific gene expression profile. Our results show that even with a relatively limited gene set, gene expression profiling allows a certain degree of classification of compounds with similar hepatocellular toxicities such as cholestasis, necrosis. The clustering analysis revealed that the compounds known to cause steatosis were linked, suggesting that they functionally regulate similar genes and possibly act through the same mechanisms of action. On the other hand, the drugs inducing necrosis and cholestasis were pooled in the same cluster. The drugs arbitrarily classified as the CYP450 inducers formed individual clusters. In conclusion, this study suggests that low-density microarrays could be useful in

  11. Efficiency, error and yield in light-directed maskless synthesis of DNA microarrays

    PubMed Central

    2011-01-01

    Background Light-directed in situ synthesis of DNA microarrays using computer-controlled projection from a digital micromirror device--maskless array synthesis (MAS)--has proved to be successful at both commercial and laboratory scales. The chemical synthetic cycle in MAS is quite similar to that of conventional solid-phase synthesis of oligonucleotides, but the complexity of microarrays and unique synthesis kinetics on the glass substrate require a careful tuning of parameters and unique modifications to the synthesis cycle to obtain optimal deprotection and phosphoramidite coupling. In addition, unintended deprotection due to scattering and diffraction introduce insertion errors that contribute significantly to the overall error rate. Results Stepwise phosphoramidite coupling yields have been greatly improved and are now comparable to those obtained in solid phase synthesis of oligonucleotides. Extended chemical exposure in the synthesis of complex, long oligonucleotide arrays result in lower--but still high--final average yields which approach 99%. The new synthesis chemistry includes elimination of the standard oxidation until the final step, and improved coupling and light deprotection. Coupling Insertions due to stray light are the limiting factor in sequence quality for oligonucleotide synthesis for gene assembly. Diffraction and local flare are by far the largest contributors to loss of optical contrast. Conclusions Maskless array synthesis is an efficient and versatile method for synthesizing high density arrays of long oligonucleotides for hybridization- and other molecular binding-based experiments. For applications requiring high sequence purity, such as gene assembly, diffraction and flare remain significant obstacles, but can be significantly reduced with straightforward experimental strategies. PMID:22152062

  12. Gene Expression Profiling of Microdissected Pancreatic Ductal Carcinomas Using High-Density DNA Microarrays1,3

    PubMed Central

    Grützmann, Robert; Pilarsky, Christian; Ammerpohl, Ole; Lüttges, Jutta; Böhme, Armin; Sipos, Bence; Foerder, Melanie; Alldinger, Ingo; Jahnke, Beatrix; Schackert, Hans Konrad; Kalthoff, Holger; Kremer, Bernd; Klöppel, Günter; Saeger, Hans Detlev

    2004-01-01

    Abstract Pancreatic ductal adenocarcinoma (PDAC) remains an important cause of malignancy-related death and is the eighth most common cancer with the lowest overall 5-year relative survival rate. To identify new molecular markers and candidates for new therapeutic regimens, we investigated the gene expression profile of microdissected cells from 11 normal pancreatic ducts, 14 samples of PDAC, and 4 well-characterized pancreatic cancer cell lines using the Affymetrix U133 GeneChip set. RNA was extracted from microdissected samples and cell lines, amplified, and labeled using a repetitive in vitro transcription protocol. Differentially expressed genes were identified using the significance analysis of microarrays program. We found 616 differentially expressed genes. Within these, 140 were also identified in PDAC by others, such as Galectin-1, Galectin-3, and MT-SP2. We validated the differential expression of several genes (e.g., CENPF, MCM2, MCM7, RAMP, IRAK1, and PTTG1) in PDAC by immunohistochemistry and reverse transcription polymerase chain reaction. We present a whole genome expression study of microdissected tissues from PDAC, from microdissected normal ductal pancreatic cells and pancreatic cancer cell lines using highdensity microarrays. Within the panel of genes, we identified novel differentially expressed genes, which have not been associated with the pathogenesis of PDAC before. PMID:15548371

  13. Discovery of Genomic DNA Polymorphisms using oligonucleotide arrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are applying oligonucleotide microarray technology as a means of rapidly discovering DNA-based markers for genetic mapping in finger millet and rice. A test genome scan was conducted using the full genome sequences of rice varieties 93-11 (indica) and Nipponbare (japonica) compared with the Affy...

  14. Gene Assembly from Chip-Synthesized Oligonucleotides

    PubMed Central

    Eroshenko, Nikolai; Kosuri, Sriram; Marblestone, Adam H; Conway, Nicholas; Church, George M.

    2012-01-01

    De novo synthesis of long double-stranded DNA constructs has a myriad of applications in biology and biological engineering. However, its widespread adoption has been hindered by high costs. Cost can be significantly reduced by using oligonucleotides synthesized on high-density DNA chips. However, most methods for using off-chip DNA for gene synthesis have failed to scale due to the high error rates, low yields, and high chemical complexity of the chip-synthesized oligonucleotides. We have recently demonstrated that some commercial DNA chip manufacturers have improved error rates, and that the issues of chemical complexity and low yields can be solved by using barcoded primers to accurately and efficiently amplify subpools of oligonucleotides. This article includes protocols for computationally designing the DNA chip, amplifying the oligonucleotide subpools, and assembling 500-800 basepair (bp) constructs. PMID:25077042

  15. Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach

    PubMed Central

    Lessa-Aquino, Carolina; Borges Rodrigues, Camila; Pablo, Jozelyn; Sasaki, Rie; Jasinskas, Algis; Liang, Li; Wunder, Elsio A.; Ribeiro, Guilherme S.; Vigil, Adam; Galler, Ricardo; Molina, Douglas; Liang, Xiaowu; Reis, Mitermayer G.; Ko, Albert I.; Medeiros, Marco Alberto; Felgner, Philip L.

    2013-01-01

    Background Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease. Methodology In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results. Principal findings We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients. Conclusions Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for

  16. High-Density 16S Microarray and Clone Library-Based Microbial Community Composition of the Phoenix Spacecraft Assembly Clean Room

    NASA Astrophysics Data System (ADS)

    Vaishampayan, Parag; Osman, Shariff; Andersen, Gary; Venkateswaran, Kasthuri

    2010-06-01

    The bacterial diversity and comparative community structure of a clean room used for assembling the Phoenix spacecraft was characterized throughout the spacecraft assembly process by using 16S rRNA gene cloning/sequencing and DNA microarray (PhyloChip) technologies. Samples were collected from several locations of the clean room at three time points: before Phoenix's arrival (PHX-B), during hardware assembly (PHX-D), and after the spacecraft was removed for launch (PHX-A). Bacterial diversity comprised of all major bacterial phyla of PHX-B was found to be statistically different from PHX-D and PHX-A samples. Due to stringent cleaning and decontamination protocols during assembly, PHX-D bacterial diversity was dramatically reduced when compared to PHX-B and PHX-A samples. Comparative community analysis based on PhyloChip results revealed similar overall trends as were seen in clone libraries, but the high-density phylogenetic microarray detected larger diversity in all sampling events. The decrease in community complexity in PHX-D compared to PHX-B, and the subsequent recurrence of these organisms in PHX-A, speaks to the effectiveness of NASA cleaning protocols. However, the persistence of a subset of bacterial signatures throughout all spacecraft assembly phases underscores the need for continued refinement of sterilization technologies and the implementation of safeguards that monitor and inventory microbial contaminants.

  17. In-house Manual Construction of High-Density and High-Quality Tissue Microarrays by Using Homemade Recipient Agarose-Paraffin Blocks

    PubMed Central

    Kim, Kyu Ho; Choi, Yeon Il; Kim, Lucia; Park, In Suh; Han, Jee Young; Kim, Joon Mee; Chu, Young Chae

    2013-01-01

    Background Self-made tissue punches can be effectively used to punch holes in blank recipient paraffin blocks and extract tissue cores from the donor paraffin blocks for the low-cost construction of tissue microarrays (TMAs). However, variable degrees of section distortion and loss of the tissue cores can occurs during cutting of the TMAs, posing technical problems for in-house manual construction of high-density TMAs. We aimed to update the method for in-house manual TMA construction to improve the quality of high-density TMAs. Methods Blocks of agarose gel were subjected to the standard tissue processing and embedding procedure to prepare recipient agarose-paraffin blocks. The self-made tissue punches and recipient agarose-paraffin blocks were used to construct TMAs, which were completely melted and re-embedded in paraffin to make finished TMA blocks. Results The donor tissue cores were completely integrated into the surrounding paraffin of the recipient blocks. This method enabled us to construct high-density TMAs with significantly less section distortion or loss of tissue cores during microtomy. Conclusions Simple and inexpensive construction of high-density and high-quality TMAs can be warranted by using paraffinized agarose gels as recipient blocks. PMID:23837016

  18. Comparing Bacterial DNA Microarray Fingerprints

    SciTech Connect

    Willse, Alan R.; Chandler, Darrell P.; White, Amanda M.; Protic, Miroslava; Daly, Don S.; Wunschel, Sharon C.

    2005-08-15

    Detecting subtle genetic differences between microorganisms is an important problem in molecular epidemiology and microbial forensics. In a typical investigation, gel electrophoresis is used to compare randomly amplified DNA fragments between microbial strains, where the patterns of DNA fragment sizes are proxies for a microbe's genotype. The limited genomic sample captured on a gel is often insufficient to discriminate nearly identical strains. This paper examines the application of microarray technology to DNA fingerprinting as a high-resolution alternative to gel-based methods. The so-called universal microarray, which uses short oligonucleotide probes that do not target specific genes or species, is intended to be applicable to all microorganisms because it does not require prior knowledge of genomic sequence. In principle, closely related strains can be distinguished if the number of probes on the microarray is sufficiently large, i.e., if the genome is sufficiently sampled. In practice, we confront noisy data, imperfectly matched hybridizations, and a high-dimensional inference problem. We describe the statistical problems of microarray fingerprinting, outline similarities with and differences from more conventional microarray applications, and illustrate the statistical fingerprinting problem for 10 closely related strains from three Bacillus species, and 3 strains from non-Bacillus species.

  19. Photonic crystal microcavity engineering and high-density bio-patterning for chip-integrated microarray applications

    NASA Astrophysics Data System (ADS)

    Chakravarty, Swapnajit; Lai, Wei-Cheng; Zou, Yi; Chen, Ray T.

    2012-01-01

    While Q ~ 1million has been demonstrated in freely suspended photonic crystal (PC) membranes, the reduced refractive index contrast when PC microcavities are immersed in phosphate buffered saline (PBS), a typical ambient for biomolecules, reduces Q by more than 2 orders of magnitude. We experimentally demonstrate photonic crystal microcavity based resonant sensors coupled to photonic crystal waveguides in silicon on insulator for chemical and bio-sensing. Linear L-type microcavities are considered. In contrast to cavities with small modes volumes but low quality factors for bio-sensing, we show that increasing the length of the microcavity enhances the quality factor of the resonance by an order of magnitude and also increases the resonance wavelength shift while still retaining compact device characteristics. Q~26,760 and sensitivity down to 7.5ng/ml and ~9pg/mm2 in bio-sensing was experimentally demonstrated in SOI devices for goat anti-rabbit IgG antibodies with Kd~10-6M. The increase in cavity length follows from fundamental engineering limitations in ink-jet printing or microfluidic channels when unique receptor biomolecules are coated on separate adjacent sensors in a microarray.

  20. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

  1. Analysis of dust samples from the Middle East using high-density resequencing micro-array RPM-TEI

    NASA Astrophysics Data System (ADS)

    Leski, T. A.; Gregory, M. J.; Malanoski, A. P.; Smith, J. P.; Glaven, R. H.; Wang, Z.; Stenger, D. A.; Lin, B.

    2010-04-01

    A previously developed resequencing microarray, "Tropical and Emerging Infections (RPM-TEI v.1.0 chip)", designed to identify and discriminate between tropical diseases and other potential biothreat agents, their near-neighbor species, and/or potential confounders, was used to characterize the microbes present in the silt/clay fraction of surface soils and airborne dust collected from the Middle East. Local populations and U.S. military personnel deployed to the Middle East are regularly subjected to high levels of airborne desert dust containing a significant fraction of inhalable particles and some portion require clinical aid. Not all of the clinical symptoms can be directly attributed to the physical action of material in the human respiratory tract. To better understand the potential health effects of the airborne dust, the composition of the microbial communities associated with surface soil and/or airborne dust (air filter) samples from 19 different sites in Iraq and Kuwait was identified using RPM-TEI v.1.0. Results indicated that several microorganisms including a class of rapidly growing Mycobacterium, Bacillus, Brucella, Clostridium and Coxiella burnetti, were present in the samples. The presence of these organisms in the surface soils and the inhalable fraction of airborne dust analyzed may pose a human health risk and warrants further investigation. Better understanding of the factors influencing the composition of these microbial communities is important to address questions related to human health and is critical to achieving Force Health Protection for the Warfighter operating in the Middle East, Afghanistan, North Africa and other arid regions.

  2. Assessment of a direct hybridization microarray strategy for comprehensive monitoring of genetically modified organisms (GMOs).

    PubMed

    Turkec, Aydin; Lucas, Stuart J; Karacanli, Burçin; Baykut, Aykut; Yuksel, Hakki

    2016-03-01

    Detection of GMO material in crop and food samples is the primary step in GMO monitoring and regulation, with the increasing number of GM events in the world market requiring detection solutions with high multiplexing capacity. In this study, we test the suitability of a high-density oligonucleotide microarray platform for direct, quantitative detection of GMOs found in the Turkish feed market. We tested 1830 different 60nt probes designed to cover the GM cassettes from 12 different GM cultivars (3 soya, 9 maize), as well as plant species-specific and contamination controls, and developed a data analysis method aiming to provide maximum throughput and sensitivity. The system was able specifically to identify each cultivar, and in 10/12 cases was sensitive enough to detect GMO DNA at concentrations of ⩽1%. These GMOs could also be quantified using the microarray, as their fluorescence signals increased linearly with GMO concentration. PMID:26471572

  3. Time-resolved Förster-resonance-energy-transfer DNA assay on an active CMOS microarray

    PubMed Central

    Schwartz, David Eric; Gong, Ping; Shepard, Kenneth L.

    2008-01-01

    We present an active oligonucleotide microarray platform for time-resolved Förster resonance energy transfer (TR-FRET) assays. In these assays, immobilized probe is labeled with a donor fluorophore and analyte target is labeled with a fluorescence quencher. Changes in the fluorescence decay lifetime of the donor are measured to determine the extent of hybridization. In this work, we demonstrate that TR-FRET assays have reduced sensitivity to variances in probe surface density compared with standard fluorescence-based microarray assays. Use of an active array substrate, fabricated in a standard complementary metal-oxide-semiconductor (CMOS) process, provides the additional benefits of reduced system complexity and cost. The array consists of 4096 independent single-photon avalanche diode (SPAD) pixel sites and features on-chip time-to-digital conversion. We demonstrate the functionality of our system by measuring a DNA target concentration series using TR-FRET with semiconductor quantum dot donors. PMID:18515059

  4. Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ricard-Blum, S.

    Proteins are key actors in the life of the cell, involved in many physiological and pathological processes. Since variations in the expression of messenger RNA are not systematically correlated with variations in the protein levels, the latter better reflect the way a cell functions. Protein microarrays thus supply complementary information to DNA chips. They are used in particular to analyse protein expression profiles, to detect proteins within complex biological media, and to study protein-protein interactions, which give information about the functions of those proteins [3-9]. They have the same advantages as DNA microarrays for high-throughput analysis, miniaturisation, and the possibility of automation. Section 18.1 gives a brief overview of proteins. Following this, Sect. 18.2 describes how protein microarrays can be made on flat supports, explaining how proteins can be produced and immobilised on a solid support, and discussing the different kinds of substrate and detection method. Section 18.3 discusses the particular format of protein microarrays in suspension. The diversity of protein microarrays and their applications are then reported in Sect. 18.4, with applications to therapeutics (protein-drug interactions) and diagnostics. The prospects for future developments of protein microarrays are then outlined in the conclusion. The bibliography provides an extensive list of reviews and detailed references for those readers who wish to go further in this area. Indeed, the aim of the present chapter is not to give an exhaustive or detailed analysis of the state of the art, but rather to provide the reader with the basic elements needed to understand how proteins are designed and used.

  5. High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients.

    PubMed

    Hecker, Michael; Fitzner, Brit; Wendt, Matthias; Lorenz, Peter; Flechtner, Kristin; Steinbeck, Felix; Schröder, Ina; Thiesen, Hans-Jürgen; Zettl, Uwe Klaus

    2016-04-01

    Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g.MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (pvalues <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392-411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS. PMID:26831522

  6. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Reck, Folkert; Kudick, Rene; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert; Wippo, Harald

    2001-01-01

    To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an informational Watson-Crick base-pairing system are unique to the pentopyranosyl-(4 + 2)-oligonucleotide isomer derived from the RNA-building block D-ribose, studies on the entire family of diastereoisomeric pyranosyL(4 - Z)-oligonucleotide systems deriving from D-ribose. L-lyxose. D-xylose, and L-arabinose were carried out. The result of these extended studies is unambiguous: not only pyranosyl-RNA, but all members of the pentopyranosyl(4 + 2)-oligonucleotide family are highly efficient Watson-Crick base-pairing systems. Their synthesis and pairing properties will be described in a series of publications in this journal.

  7. Uses of Dendrimers for DNA Microarrays

    PubMed Central

    Caminade, Anne-Marie; Padié, Clément; Laurent, Régis; Maraval, Alexandrine; Majoral, Jean-Pierre

    2006-01-01

    Biosensors such as DNA microarrays and microchips are gaining an increasing importance in medicinal, forensic, and environmental analyses. Such devices are based on the detection of supramolecular interactions called hybridizations that occur between complementary oligonucleotides, one linked to a solid surface (the probe), and the other one to be analyzed (the target). This paper focuses on the improvements that hyperbranched and perfectly defined nanomolecules called dendrimers can provide to this methodology. Two main uses of dendrimers for such purpose have been described up to now; either the dendrimer is used as linker between the solid surface and the probe oligonucleotide, or the dendrimer is used as a multilabeled entity linked to the target oligonucleotide. In the first case the dendrimer generally induces a higher loading of probes and an easier hybridization, due to moving away the solid phase. In the second case the high number of localized labels (generally fluorescent) induces an increased sensitivity, allowing the detection of small quantities of biological entities.

  8. Poly(oligonucleotide)

    PubMed Central

    2015-01-01

    Here we report the preparation of poly(oligonucleotide) brush polymers and amphiphilic brush copolymers from nucleic acid monomers via graft-through polymerization. We describe the polymerization of PNA-norbornyl monomers to yield poly-PNA (poly(peptide nucleic acid)) via ring-opening metathesis polymerization (ROMP) with the initiator, (IMesH2)(C5H5N)2(Cl)2RuCHPh.1 In addition, we present the preparation of poly-PNA nanoparticles from amphiphilic block copolymers and describe their hybridization to a complementary single-stranded DNA (ssDNA) oligonucleotide. PMID:25077676

  9. The delivery of therapeutic oligonucleotides.

    PubMed

    Juliano, Rudolph L

    2016-08-19

    The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. PMID:27084936

  10. Optimization of an oligonucleotide microchip for microbial identification studies: a non-equilibrium dissociation approach

    NASA Technical Reports Server (NTRS)

    Liu, W. T.; Mirzabekov, A. D.; Stahl, D. A.

    2001-01-01

    The utility of a high-density oligonucleotide microarray (microchip) for identifying strains of five closely related bacilli (Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus medusa and Bacillus subtilis) was demonstrated using an approach that compares the non-equilibrium dissociation rates ('melting curves') of all probe-target duplexes simultaneously. For this study, a hierarchical set of 30 oligonucleotide probes targeting the 16S ribosomal RNA of these bacilli at multiple levels of specificity (approximate taxonomic ranks of domain, kingdom, order, genus and species) was designed and immobilized in a high-density matrix of gel pads on a glass slide. Reproducible melting curves for probes with different levels of specificity were obtained using an optimized salt concentration. Clear discrimination between perfect match (PM) and mismatch (MM) duplexes was achieved. By normalizing the signals to an internal standard (a universal probe), a more than twofold discrimination (> 2.4x) was achieved between PM and 1-MM duplexes at the dissociation temperature at which 50% of the probe-target duplexes remained intact. This provided excellent differentiation among representatives of different Bacillus species, both individually and in mixtures of two or three. The overall pattern of hybridization derived from this hierarchical probe set also provided a clear 'chip fingerprint' for each of these closely related Bacillus species.

  11. Chromosome Microarray.

    PubMed

    Anderson, Sharon

    2016-01-01

    Over the last half century, knowledge about genetics, genetic testing, and its complexity has flourished. Completion of the Human Genome Project provided a foundation upon which the accuracy of genetics, genomics, and integration of bioinformatics knowledge and testing has grown exponentially. What is lagging, however, are efforts to reach and engage nurses about this rapidly changing field. The purpose of this article is to familiarize nurses with several frequently ordered genetic tests including chromosomes and fluorescence in situ hybridization followed by a comprehensive review of chromosome microarray. It shares the complexity of microarray including how testing is performed and results analyzed. A case report demonstrates how this technology is applied in clinical practice and reveals benefits and limitations of this scientific and bioinformatics genetic technology. Clinical implications for maternal-child nurses across practice levels are discussed. PMID:27276104

  12. Quantitative Determination and Subcellular Imaging of Cu in Single Cells via Laser Ablation-ICP-Mass Spectrometry Using High-Density Microarray Gelatin Standards.

    PubMed

    Van Malderen, Stijn J M; Vergucht, Eva; De Rijcke, Maarten; Janssen, Colin; Vincze, Laszlo; Vanhaecke, Frank

    2016-06-01

    This manuscript describes the development and characterization of a high-density microarray calibration standard, manufactured in-house and designed to overcome the limitations in precision, accuracy, and throughput of current calibration approaches for the quantification of elemental concentrations on the cellular level using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS). As a case study, the accumulation of Cu in the model organism Scrippsiella trochoidea resulting from transition metal exposure (ranging from 0.5 to 100 μg/L) was evaluated. After the Cu exposure, cells of this photosynthetic dinoflagellate were treated with a critical point drying protocol, transferred to a carbon stub, and sputter-coated with a Au layer for scanning electron microscopy (SEM) analysis. In subsequent LA-ICPMS analysis, approximately 100 cells of each population were individually ablated. This approach permitted the evaluation of the mean concentration of Cu in the cell population across different exposure levels and also allowed the examination of the cellular distribution of Cu within the populations. In a cross-validation exercise, subcellular LA-ICPMS imaging was demonstrated to corroborate synchrotron radiation confocal X-ray fluorescence (SR-XRF) microimaging of single cells investigated under in vivo conditions. PMID:27149342

  13. Compact, universal DNA microarrays to comprehensively determine transcription-factor binding site specificities

    PubMed Central

    Berger, Michael F.; Philippakis, Anthony A.; Qureshi, Aaron M.; He, Fangxue S.; Estep, Preston W.; Bulyk, Martha L.

    2015-01-01

    Transcription factors (TFs) regulate the expression of genes involved in myriad cellular processes through sequence-specific interactions with DNA. In order to predict DNA regulatory elements and the TFs targeting them with greater accuracy, detailed knowledge of the binding preferences of TFs is needed. Protein binding microarray (PBM) technology permits rapid, high-throughput characterization of the in vitro DNA binding specificities of proteins1. Here, we present a novel, maximally compact, synthetic DNA sequence design that represents all possible DNA sequence variants of a given length k (i.e., all “k-mers”) on a single, universal microarray. We constructed such all k-mer microarrays covering all 10 base pair (bp) binding sites by converting high-density single-stranded oligonucleotide arrays to double-stranded DNA arrays. Using these microarrays, we comprehensively determined the binding specificities over a full range of affinities for five TFs of diverse structural classes from yeast, worm, mouse, and human. Importantly, the unbiased coverage of all k-mers permits an interrogation of binding site preferences, including nucleotide interdependencies, at unprecedented resolution. PMID:16998473

  14. Use of a High-Density Protein Microarray to Identify Autoantibodies in Subjects with Type 2 Diabetes Mellitus and an HLA Background Associated with Reduced Insulin Secretion.

    PubMed

    Chang, Douglas C; Piaggi, Paolo; Hanson, Robert L; Knowler, William C; Bucci, John; Thio, Guene; Hohenadel, Maximilian G; Bogardus, Clifton; Krakoff, Jonathan

    2015-01-01

    New biomarkers for type 2 diabetes mellitus (T2DM) may aid diagnosis, drug development or clinical treatment. Evidence is increasing for the adaptive immune system's role in T2DM and suggests the presence of unidentified autoantibodies. While high-density protein microarrays have emerged as a useful technology to identify possible novel autoantigens in autoimmune diseases, its application in T2DM has lagged. In Pima Indians, the HLA haplotype (HLA-DRB1*02) is protective against T2DM and, when studied when they have normal glucose tolerance, subjects with this HLA haplotype have higher insulin secretion compared to those without the protective haplotype. Possible autoantibody biomarkers were identified using microarrays containing 9480 proteins in plasma from Pima Indians with T2DM without the protective haplotype (n = 7) compared with those with normal glucose regulation (NGR) with the protective haplotype (n = 11). A subsequent validation phase involving 45 cases and 45 controls, matched by age, sex and specimen storage time, evaluated 77 proteins. Eleven autoantigens had higher antibody signals among T2DM subjects with the lower insulin-secretion HLA background compared with NGR subjects with the higher insulin-secretion HLA background (p<0.05, adjusted for multiple comparisons). PPARG2 and UBE2M had lowest p-values (adjusted p = 0.023) while PPARG2 and RGS17 had highest case-to-control antibody signal ratios (1.7). A multi-protein classifier involving the 11 autoantigens had sensitivity, specificity, and area under the receiver operating characteristics curve of 0.73, 0.80, and 0.83 (95% CI 0.74-0.91, p = 3.4x10-8), respectively. This study identified 11 novel autoantigens which were associated with T2DM and an HLA background associated with reduced insulin secretion. While further studies are needed to distinguish whether these antibodies are associated with insulin secretion via the HLA background, T2DM more broadly, or a combination of the two, this study may

  15. DEVELOPMENT AND VALIDATION OF A 2,000 GENE MICROARRAY FOR THE FATHEAD MINNOW, PIMEPHALES PROMELAS

    EPA Science Inventory

    The development of the gene microarray has provided the field of ecotoxicology a new tool to identify modes of action (MOA) of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000 gene oligonucleotide microarray for the fathead minnow (P...

  16. High-density fiber optic biosensor arrays

    NASA Astrophysics Data System (ADS)

    Epstein, Jason R.; Walt, David R.

    2002-02-01

    Novel approaches are required to coordinate the immense amounts of information derived from diverse genomes. This concept has influenced the expanded role of high-throughput DNA detection and analysis in the biological sciences. A high-density fiber optic DNA biosensor was developed consisting of oligonucleotide-functionalized, 3.1 mm diameter microspheres deposited into the etched wells on the distal face of a 500 micrometers imaging fiber bundle. Imaging fiber bundles containing thousands of optical fibers, each associated with a unique oligonucleotide probe sequence, were the foundation for an optically connected, individually addressable DNA detection platform. Different oligonucleotide-functionalized microspheres were combined in a stock solution, and randomly dispersed into the etched wells. Microsphere positions were registered from optical dyes incorporated onto the microspheres. The distribution process provided an inherent redundancy that increases the signal-to-noise ratio as the square root of the number of sensors examined. The representative amount of each probe-type in the array was dependent on their initial stock solution concentration, and as other sequences of interest arise, new microsphere elements can be added to arrays without altering the existing detection capabilities. The oligonucleotide probe sequences hybridize to fluorescently-labeled, complementary DNA target solutions. Fiber optic DNA microarray research has included DNA-protein interaction profiles, microbial strain differentiation, non-labeled target interrogation with molecular beacons, and single cell-based assays. This biosensor array is proficient in DNA detection linked to specific disease states, single nucleotide polymorphism (SNP's) discrimination, and gene expression analysis. This array platform permits multiple detection formats, provides smaller feature sizes, and enables sensor design flexibility. High-density fiber optic microarray biosensors provide a fast

  17. A microarray for assessing transcription from pelagic marine microbial taxa

    PubMed Central

    Shilova, Irina N; Robidart, Julie C; James Tripp, H; Turk-Kubo, Kendra; Wawrik, Boris; Post, Anton F; Thompson, Anne W; Ward, Bess; Hollibaugh, James T; Millard, Andy; Ostrowski, Martin; J Scanlan, David; Paerl, Ryan W; Stuart, Rhona; Zehr, Jonathan P

    2014-01-01

    Metagenomic approaches have revealed unprecedented genetic diversity within microbial communities across vast expanses of the world's oceans. Linking this genetic diversity with key metabolic and cellular activities of microbial assemblages is a fundamental challenge. Here we report on a collaborative effort to design MicroTOOLs (Microbiological Targets for Ocean Observing Laboratories), a high-density oligonucleotide microarray that targets functional genes of diverse taxa in pelagic and coastal marine microbial communities. MicroTOOLs integrates nucleotide sequence information from disparate data types: genomes, PCR-amplicons, metagenomes, and metatranscriptomes. It targets 19 400 unique sequences over 145 different genes that are relevant to stress responses and microbial metabolism across the three domains of life and viruses. MicroTOOLs was used in a proof-of-concept experiment that compared the functional responses of microbial communities following Fe and P enrichments of surface water samples from the North Pacific Subtropical Gyre. We detected transcription of 68% of the gene targets across major taxonomic groups, and the pattern of transcription indicated relief from Fe limitation and transition to N limitation in some taxa. Prochlorococcus (eHLI), Synechococcus (sub-cluster 5.3) and Alphaproteobacteria SAR11 clade (HIMB59) showed the strongest responses to the Fe enrichment. In addition, members of uncharacterized lineages also responded. The MicroTOOLs microarray provides a robust tool for comprehensive characterization of major functional groups of microbes in the open ocean, and the design can be easily amended for specific environments and research questions. PMID:24477198

  18. Fingerprinting Closely Related Xanthomonas Pathovars with Random Nonamer Oligonucleotide Microarrays

    SciTech Connect

    Kingsley, Mark T.; Straub, Tim M.; Call, Douglas R.; Daly, Don S.; Wunschel, Sharon C.; Chandler, Darrell P.

    2002-12-01

    Current bacterial DNA typing methods are typically based upon gel-based fingerprinting methods. As such, they access a limited complement of genetic information and many independent restriction enzymes or probes is required to achieve statistical rigor and confidence in the resulting pattern of DNA fragments.

  19. An oligonucleotide microarray to characterize multidrug resistant plasmids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Bacteria plasmids are fragments of extra-chromosomal double stranded deoxyribonucleic acid (DNA) that can contain a variety of genes beneficial to the host organism like antibiotic drug resistance. Many of the Enterobacteriaceae carry multiple drug resistance (MDR) genes on large plasmids of replic...

  20. Using The Affymetrix Wheat Microarray As An Oat Expression Platform

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recent advances in sequencing have resulted in the sequence of a large number of plant expressed sequence tags (ESTs) to entire plant genomes. Using these EST sequences, oligonucleotide microarray chips have been developed for several species including barley (Hordeum vulgare), maize (Zea mays), ric...

  1. DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  2. Fluorescent oligonucleotides can serve as suitable alternatives to radiolabeled oligonucleotides.

    PubMed

    Ballal, Rahul; Cheema, Amrita; Ahmad, Waaqar; Rosen, Eliot M; Saha, Tapas

    2009-09-01

    Prolonged exposure to radiation from radionuclei used in medical research can cause DNA damage and mutation, which lead to several diseases including cancer. Radioactivity-based experiments are expensive and associated with specialized training, dedication of instruments, approvals, and cleanup with potential hazardous waste. The objective of this study was to find an alternative to the use of radioactivity in medical research using nucleic acid chemistry. FITC-labeled oligonucleotides that contain wild-type (wt) and modified base (8-oxo-G) at the same position and their complementary unlabeled strand were synthesized. Purified DNA repair enzyme, OGG1, and nuclear lysates from MCF-7 breast cancer cells were incubated with double-stranded FITC-labeled wt and 8-oxo-G oligonucleotide to demonstrate the OGG1 incision assay. We found that FITC-coupled oligonucleotides do not impose a steric hindrance during duplex formation, and the fluorescence intensity of the oligonucleotide is comparable with the intensity of the radioactive oligonucleotide. Moreover, we have seen that the OGG1 incision assay can be performed using these fluorescence oligonucleotides, replacing conventional use of radiolabeled oligonucleotides in the assay. Although the use of fluorescent-labeled oligonucleotides was described in detail for incision assays, the technique can be applied to replace a broad range of experiments, where radioactive oligonucleotides are used, eliminating the hazardous consequences of radiation. PMID:19721820

  3. Tissue microarrays: applications in genomic research.

    PubMed

    Watanabe, Aprill; Cornelison, Robert; Hostetter, Galen

    2005-03-01

    The widespread application of tissue microarrays in cancer research and the clinical pathology laboratory demonstrates a versatile and portable technology. The rapid integration of tissue microarrays into biomarker discovery and validation processes reflects the forward thinking of researchers who have pioneered the high-density tissue microarray. The precise arrangement of hundreds of archival clinical tissue samples into a composite tissue microarray block is now a proven method for the efficient and standardized analysis of molecular markers. With applications in cancer research, tissue microarrays are a valuable tool in validating candidate markers discovered in highly sensitive genome-wide microarray experiments. With applications in clinical pathology, tissue microarrays are used widely in immunohistochemistry quality control and quality assurance. The timeline of a biomarker implicated in prostate neoplasia, which was identified by complementary DNA expression profiling, validated by tissue microarrays and is now used as a prognostic immunohistochemistry marker, is reviewed. The tissue microarray format provides opportunities for digital imaging acquisition, image processing and database integration. Advances in digital imaging help to alleviate previous bottlenecks in the research pipeline, permit computer image scoring and convey telepathology opportunities for remote image analysis. The tissue microarray industry now includes public and private sectors with varying degrees of research utility and offers a range of potential tissue microarray applications in basic research, prognostic oncology and drug discovery. PMID:15833047

  4. Aptamer Microarrays

    SciTech Connect

    Angel-Syrett, Heather; Collett, Jim; Ellington, Andrew D.

    2009-01-02

    In vitro selection can yield specific, high-affinity aptamers. We and others have devised methods for the automated selection of aptamers, and have begun to use these reagents for the construction of arrays. Arrayed aptamers have proven to be almost as sensitive as their solution phase counterparts, and when ganged together can provide both specific and general diagnostic signals for proteins and other analytes. We describe here technical details regarding the production and processing of aptamer microarrays, including blocking, washing, drying, and scanning. We will also discuss the challenges involved in developing standardized and reproducible methods for binding and quantitating protein targets. While signals from fluorescent analytes or sandwiches are typically captured, it has proven possible for immobilized aptamers to be uniquely coupled to amplification methods not available to protein reagents, thus allowing for protein-binding signals to be greatly amplified. Into the future, many of the biosensor methods described in this book can potentially be adapted to array formats, thus further expanding the utility of and applications for aptamer arrays.

  5. DNA Microarrays for Identifying Fishes

    PubMed Central

    Nölte, M.; Weber, H.; Silkenbeumer, N.; Hjörleifsdottir, S.; Hreggvidsson, G. O.; Marteinsson, V.; Kappel, K.; Planes, S.; Tinti, F.; Magoulas, A.; Garcia Vazquez, E.; Turan, C.; Hervet, C.; Campo Falgueras, D.; Antoniou, A.; Landi, M.; Blohm, D.

    2008-01-01

    In many cases marine organisms and especially their diverse developmental stages are difficult to identify by morphological characters. DNA-based identification methods offer an analytically powerful addition or even an alternative. In this study, a DNA microarray has been developed to be able to investigate its potential as a tool for the identification of fish species from European seas based on mitochondrial 16S rDNA sequences. Eleven commercially important fish species were selected for a first prototype. Oligonucleotide probes were designed based on the 16S rDNA sequences obtained from 230 individuals of 27 fish species. In addition, more than 1200 sequences of 380 species served as sequence background against which the specificity of the probes was tested in silico. Single target hybridisations with Cy5-labelled, PCR-amplified 16S rDNA fragments from each of the 11 species on microarrays containing the complete set of probes confirmed their suitability. True-positive, fluorescence signals obtained were at least one order of magnitude stronger than false-positive cross-hybridisations. Single nontarget hybridisations resulted in cross-hybridisation signals at approximately 27% of the cases tested, but all of them were at least one order of magnitude lower than true-positive signals. This study demonstrates that the 16S rDNA gene is suitable for designing oligonucleotide probes, which can be used to differentiate 11 fish species. These data are a solid basis for the second step to create a “Fish Chip” for approximately 50 fish species relevant in marine environmental and fisheries research, as well as control of fisheries products. PMID:18270778

  6. Oligonucleotide Immobilization and Hybridization on Aldehyde-Functionalized Poly(2-hydroxyethyl methacrylate) Brushes.

    PubMed

    Bilgic, Tugba; Klok, Harm-Anton

    2015-11-01

    DNA biosensing requires high oligonucleotide binding capacity interface chemistries that can be tuned to maximize probe presentation as well as hybridization efficiency. This contribution investigates the feasibility of aldehyde-functionalized poly(2-hydroxyethyl methacrylate) (PHEMA) brush-based interfaces for oligonucleotide binding and hybridization. These polymer brushes, which allow covalent immobilization of oligonucleotides, are prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of HEMA followed by a postpolymerization oxidation step to generate side chain aldehyde groups. A series of polymer brushes covering a range of film thicknesses and grafting densities was investigated with regard to their oligonucleotide binding capacity as well as their ability to support oligonucleotide hybridization. Densely grafted brushes were found to have probe oligonucleotide binding capacities of up to ∼30 pmol/cm(2). Increasing the thickness of these densely grafted brush films, however, resulted in a decrease in the oligonucleotide binding capacity. Less densely grafted brushes possess binding capacities of ∼10 pmol/cm(2), which did not significantly depend on film thickness. The oligonucleotide hybridization efficiencies, however, were highest (93%) on those brushes that present the lowest surface concentration of the probe oligonucleotide. These results highlight the importance of optimizing the probe oligonucleotide surface concentration and binding interface chemistry. The versatility and tunability of the PHEMA-based brushes presented herein makes these films a very attractive platform for the immobilization and hybridization of oligonucleotides. PMID:26441148

  7. Configurable low-cost plotter device for fabrication of multi-color sub-cellular scale microarrays.

    PubMed

    Arrabito, Giuseppe; Schroeder, Hendrik; Schröder, Kathrin; Filips, Christian; Marggraf, Ulrich; Dopp, Christian; Venkatachalapathy, Muthukumaran; Dehmelt, Leif; Bastiaens, Philippe I H; Neyer, Andreas; Niemeyer, Christof M

    2014-07-23

    The construction and operation of a low-cost plotter for fabrication of microarrays for multiplexed single-cell analyses is reported. The printing head consists of polymeric pyramidal pens mounted on a rotation stage installed on an aluminium frame. This construction enables printing of microarrays onto glass substrates mounted on a tilt stage, controlled by a Lab-View operated user interface. The plotter can be assembled by typical academic workshops from components of less than 15,000 Euro. The functionality of the instrument is demonstrated by printing DNA microarrays on the area of 0.5 cm2 using up to three different oligonucleotides. Typical feature sizes are 5 μm diameter with a pitch of 15 μm, leading to densities of up to 10(4)-10(5) spots/mm2. The fabricated DNA microarrays are used to produce sub-cellular scale arrays of bioactive epidermal growth factor peptides by means of DNA-directed immobilization. The suitability of these biochips for cell biological studies is demonstrated by specific recruitment, concentration, and activation of EGF receptors within the plasma membrane of adherent living cells. This work illustrates that the presented plotter gives access to bio-functionalized arrays usable for fundamental research in cell biology, such as the manipulation of signal pathways in living cells at subcellular resolution. PMID:24678019

  8. A highly oriented hybrid microarray modified electrode fabricated by a template-free method for ultrasensitive electrochemical DNA recognition

    NASA Astrophysics Data System (ADS)

    Shi, Lei; Chu, Zhenyu; Dong, Xueliang; Jin, Wanqin; Dempsey, Eithne

    2013-10-01

    Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors.Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and

  9. The use of microarrays in microbial ecology

    SciTech Connect

    Andersen, G.L.; He, Z.; DeSantis, T.Z.; Brodie, E.L.; Zhou, J.

    2009-09-15

    Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogenetic microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer

  10. Fluid dynamical analysis of the distribution of ink jet printed biomolecules in microarray substrates for genotyping applications

    PubMed Central

    Dijksman, J. Frits; Pierik, Anke

    2008-01-01

    Oligonucleotide microarrays are tools used to analyze samples for the presence of specific DNA sequences. In the system as presented here, specific DNA sequences are first amplified by a polymerase chain reaction (PCR) during which process they are labeled with fluorophores. The amplicons are subsequently hybridized onto an oligonucleotide microarray, which in our case is a porous nylon membrane with microscopic spots. Each spot on the membrane contains oligonucleotides with a sequence complementary to part of one specific target sequence. The solution containing the amplicons flows by external agitation many times up and down through the porous substrate, thereby reducing the time delaying effect of diffusion. By excitation of the fluorophores the emitted pattern of fluorophores can be detected by a charge-coupled device camera. The recorded pattern is a characteristic of the composition of the sample. The oligonucleotide capture probes have been deposited on the substrate by using noncontact piezo ink jet printing, which is the focus of our study. The objective of this study is to understand the mechanisms that determine the distribution of the ink jet printed capture probes inside the membrane. The membrane is a porous medium: the droplets placed on the membrane penetrate in the microstructure of it. The three-dimensional (3D) distribution of the capture probes inside the membrane determines the distribution of the hybridized fluorescent PCR products inside the membrane and thus the emission of light when exposed to the light source. As the 3D distribution of the capture probes inside the membrane eventually determines the detection efficiency, this parameter can be controlled for optimization of the sensitivity of the assay. The main issues addressed here are how are the capture probes distributed inside the membrane and how does this distribution depend on the printing parameters. We will use two model systems to study the influences of different parameters: a

  11. The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

    PubMed Central

    2013-01-01

    Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

  12. Microarrays, Integrated Analytical Systems

    NASA Astrophysics Data System (ADS)

    Combinatorial chemistry is used to find materials that form sensor microarrays. This book discusses the fundamentals, and then proceeds to the many applications of microarrays, from measuring gene expression (DNA microarrays) to protein-protein interactions, peptide chemistry, carbodhydrate chemistry, electrochemical detection, and microfluidics.

  13. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries

    PubMed Central

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2016-01-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection–based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization. PMID:26054766

  14. Construction of Whole Genome Microarrays, and Expression Analysis of Desulfovibrio vulgaris cells in Metal-Reducing Conditions (Uranium and Chromium)

    SciTech Connect

    Fields, Matthew W.

    2005-06-01

    One of the major goals of the project is to construct whole-genome microarrays for Desulfovibrio vulgaris. Previous whole-genome microarrays constructed at ORNL have been PCR-amplimer based, and we wanted to re-evaluate the type of microarrays being built because oligonucleotide probes have several advantages. Microarrays have been generally constructed with two types of probes, PCR-generated probes that typically range in size between 200 and 2000 bp, and oligonucleotide probes with typical size of 20-70 nt. Producing PCR product-based DNA arrays can be a time-consuming procedure that includes PCR primer design, amplification, size verification, product purification, and product quantification. Also, some ORFs are difficult to amplify and thus the construction of comprehensive arrays can be a challenge. Recently, to alleviate some of the problems associated with PCR product-based microarrays, oligonucleotide microarrays that contain probes longer than 40 nt have been evaluated and used for whole genome expression studies. These microarrays should have higher specificity and are easy to construct, and can thus provide an important alternative approach to monitor gene expression. However, due to the smaller probe size, it is expected that the detection sensitivity of oligonucleotide arrays will be lower than PCR product-based probes.

  15. Living-Cell Microarrays

    PubMed Central

    Yarmush, Martin L.; King, Kevin R.

    2011-01-01

    Living cells are remarkably complex. To unravel this complexity, living-cell assays have been developed that allow delivery of experimental stimuli and measurement of the resulting cellular responses. High-throughput adaptations of these assays, known as living-cell microarrays, which are based on microtiter plates, high-density spotting, microfabrication, and microfluidics technologies, are being developed for two general applications: (a) to screen large-scale chemical and genomic libraries and (b) to systematically investigate the local cellular microenvironment. These emerging experimental platforms offer exciting opportunities to rapidly identify genetic determinants of disease, to discover modulators of cellular function, and to probe the complex and dynamic relationships between cells and their local environment. PMID:19413510

  16. Evaluation of 50-mer oligonucleotide arrays for detectingmicrobial populations in environmental samples.

    SciTech Connect

    Tiquia, S.M.; Wu, L.; Chong, S.C.; Passovets, S.; Xu, D.; Xu, Y.; Zhou, J.

    2004-03-30

    Microarrays fabricated with oligonucleotides longer than 40bp have been introduced for monitoring whole genome expression but havenot been evaluated with environmental samples. To determine the potentialof this type of microarray for environmental studies, a 50-meroligonucleotide microarray was constructed using 763 genes involved innitrogen cycling: nitrite reductase (nirS and nirK), ammoniamonooxygenase (amoA), nitrogenase (nifH), methane monooxygenase (pmoA),and sulfite reductase (dsrAB) from public databases and our own sequencecollections. The comparison of the sequences from pure cultures indicatedthat the developed microarrays could provide species-level resolution foranalyzing microorganisms involved in nitrification, denitrification,nitrogen fixation, methane oxidation, and sulfite reduction. Sensitivitytests suggested that the 50-mer oligonucleotide arrays could detectdominant populations in the environments, although sensitivity stillneeds to be improved. A significant quantitative relationship was alsoobtained with a mixture of DNAs from eight different bacteria. Theseresults suggest that the 50-mer oligonucleotide array can be used as aspecific and quantitative parallel tool for the detection of microbialpopulations in environmental samples.

  17. A highly oriented hybrid microarray modified electrode fabricated by a template-free method for ultrasensitive electrochemical DNA recognition.

    PubMed

    Shi, Lei; Chu, Zhenyu; Dong, Xueliang; Jin, Wanqin; Dempsey, Eithne

    2013-11-01

    Highly oriented growth of a hybrid microarray was realized by a facile template-free method on gold substrates for the first time. The proposed formation mechanism involves an interfacial structure-directing force arising from self-assembled monolayers (SAMs) between gold substrates and hybrid crystals. Different SAMs and variable surface coverage of the assembled molecules play a critical role in the interfacial directing forces and influence the morphologies of hybrid films. A highly oriented hybrid microarray was formed on the highly aligned and vertical SAMs of 1,4-benzenedithiol molecules with rigid backbones, which afforded an intense structure-directing power for the oriented growth of hybrid crystals. Additionally, the density of the microarray could be adjusted by controlling the surface coverage of assembled molecules. Based on the hybrid microarray modified electrode with a large specific area (ca. 10 times its geometrical area), a label-free electrochemical DNA biosensor was constructed for the detection of an oligonucleotide fragment of the avian flu virus H5N1. The DNA biosensor displayed a significantly low detection limit of 5 pM (S/N = 3), a wide linear response from 10 pM to 10 nM, as well as excellent selectivity, good regeneration and high stability. We expect that the proposed template-free method can provide a new reference for the fabrication of a highly oriented hybrid array and the as-prepared microarray modified electrode will be a promising paradigm in constructing highly sensitive and selective biosensors. PMID:24061929

  18. Modeling Formamide Denaturation of Probe-Target Hybrids for Improved Microarray Probe Design in Microbial Diagnostics

    PubMed Central

    Yilmaz, L. Safak; Loy, Alexander; Wright, Erik S.; Wagner, Michael; Noguera, Daniel R.

    2012-01-01

    Application of high-density microarrays to the diagnostic analysis of microbial communities is challenged by the optimization of oligonucleotide probe sensitivity and specificity, as it is generally unfeasible to experimentally test thousands of probes. This study investigated the adjustment of hybridization stringency using formamide with the idea that sensitivity and specificity can be optimized during probe design if the hybridization efficiency of oligonucleotides with target and non-target molecules can be predicted as a function of formamide concentration. Sigmoidal denaturation profiles were obtained using fluorescently labeled and fragmented 16S rRNA gene amplicon of Escherichia coli as the target with increasing concentrations of formamide in the hybridization buffer. A linear free energy model (LFEM) was developed and microarray-specific nearest neighbor rules were derived. The model simulated formamide melting with a denaturant m-value that increased hybridization free energy (ΔG°) by 0.173 kcal/mol per percent of formamide added (v/v). Using the LFEM and specific probe sets, free energy rules were systematically established to predict the stability of single and double mismatches, including bulged and tandem mismatches. The absolute error in predicting the position of experimental denaturation profiles was less than 5% formamide for more than 90 percent of probes, enabling a practical level of accuracy in probe design. The potential of the modeling approach for probe design and optimization is demonstrated using a dataset including the 16S rRNA gene of Rhodobacter sphaeroides as an additional target molecule. The LFEM and thermodynamic databases were incorporated into a computational tool (ProbeMelt) that is freely available at http://DECIPHER.cee.wisc.edu. PMID:22952791

  19. Towards MRI microarrays.

    PubMed

    Hall, Andrew; Mundell, Victoria J; Blanco-Andujar, Cristina; Bencsik, Martin; McHale, Glen; Newton, Michael I; Cave, Gareth W V

    2010-04-14

    Superparamagnetic iron oxide nanometre scale particles have been utilised as contrast agents to image staked target binding oligonucleotide arrays using MRI to correlate the signal intensity and T(2)* relaxation times in different NMR fluids. PMID:20379545

  20. Oligonucleotide conjugates for therapeutic applications

    PubMed Central

    Winkler, Johannes

    2013-01-01

    Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake machanisms and pharmacokinetic properties. PMID:23883124

  1. Detection and Genotyping of Arcobacter and Campylobacter Isolates from Retail Chicken Samples by Use of DNA Oligonucleotide Arrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To explore the use of DNA microarrays for pathogen detection in food, we have produced DNA oligonucleotide arrays to identify the presence of Arcobacter and Campylobacter in retail chicken. Probes were selected that target housekeeping and virulence-associated genes in both Arcobacter butzleri and ...

  2. Identifying Fishes through DNA Barcodes and Microarrays

    PubMed Central

    Kochzius, Marc; Seidel, Christian; Antoniou, Aglaia; Botla, Sandeep Kumar; Campo, Daniel; Cariani, Alessia; Vazquez, Eva Garcia; Hauschild, Janet; Hervet, Caroline; Hjörleifsdottir, Sigridur; Hreggvidsson, Gudmundur; Kappel, Kristina; Landi, Monica; Magoulas, Antonios; Marteinsson, Viggo; Nölte, Manfred; Planes, Serge; Tinti, Fausto; Turan, Cemal; Venugopal, Moleyur N.; Weber, Hannes; Blohm, Dietmar

    2010-01-01

    Background International fish trade reached an import value of 62.8 billion Euro in 2006, of which 44.6% are covered by the European Union. Species identification is a key problem throughout the life cycle of fishes: from eggs and larvae to adults in fisheries research and control, as well as processed fish products in consumer protection. Methodology/Principal Findings This study aims to evaluate the applicability of the three mitochondrial genes 16S rRNA (16S), cytochrome b (cyt b), and cytochrome oxidase subunit I (COI) for the identification of 50 European marine fish species by combining techniques of “DNA barcoding” and microarrays. In a DNA barcoding approach, neighbour Joining (NJ) phylogenetic trees of 369 16S, 212 cyt b, and 447 COI sequences indicated that cyt b and COI are suitable for unambiguous identification, whereas 16S failed to discriminate closely related flatfish and gurnard species. In course of probe design for DNA microarray development, each of the markers yielded a high number of potentially species-specific probes in silico, although many of them were rejected based on microarray hybridisation experiments. None of the markers provided probes to discriminate the sibling flatfish and gurnard species. However, since 16S-probes were less negatively influenced by the “position of label” effect and showed the lowest rejection rate and the highest mean signal intensity, 16S is more suitable for DNA microarray probe design than cty b and COI. The large portion of rejected COI-probes after hybridisation experiments (>90%) renders the DNA barcoding marker as rather unsuitable for this high-throughput technology. Conclusions/Significance Based on these data, a DNA microarray containing 64 functional oligonucleotide probes for the identification of 30 out of the 50 fish species investigated was developed. It represents the next step towards an automated and easy-to-handle method to identify fish, ichthyoplankton, and fish products. PMID

  3. Microarrays in hematology.

    PubMed

    Walker, Josef; Flower, Darren; Rigley, Kevin

    2002-01-01

    Microarrays are fast becoming routine tools for the high-throughput analysis of gene expression in a wide range of biologic systems, including hematology. Although a number of approaches can be taken when implementing microarray-based studies, all are capable of providing important insights into biologic function. Although some technical issues have not been resolved, microarrays will continue to make a significant impact on hematologically important research. PMID:11753074

  4. Production of complex nucleic acid libraries using highly parallel in situ oligonucleotide synthesis.

    PubMed

    Cleary, Michele A; Kilian, Kristopher; Wang, Yanqun; Bradshaw, Jeff; Cavet, Guy; Ge, Wei; Kulkarni, Amit; Paddison, Patrick J; Chang, Kenneth; Sheth, Nihar; Leproust, Eric; Coffey, Ernest M; Burchard, Julja; McCombie, W Richard; Linsley, Peter; Hannon, Gregory J

    2004-12-01

    Generation of complex libraries of defined nucleic acid sequences can greatly aid the functional analysis of protein and gene function. Previously, such studies relied either on individually synthesized oligonucleotides or on cellular nucleic acids as the starting material. As each method has disadvantages, we have developed a rapid and cost-effective alternative for construction of small-fragment DNA libraries of defined sequences. This approach uses in situ microarray DNA synthesis for generation of complex oligonucleotide populations. These populations can be recovered and either used directly or immortalized by cloning. From a single microarray, a library containing thousands of unique sequences can be generated. As an example of the potential applications of this technology, we have tested the approach for the production of plasmids encoding short hairpin RNAs (shRNAs) targeting numerous human and mouse genes. We achieved high-fidelity clone retrieval with a uniform representation of intended library sequences. PMID:15782200

  5. Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

    PubMed Central

    Narihiro, Takashi; Sekiguchi, Yuji

    2011-01-01

    Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

  6. Immobilization of DNA in polyacrylamide gel for the manufacture of DNA and DNA-oligonucleotide microchips.

    SciTech Connect

    Proudnikov, D.; Timofeev, E.; Mirzabekov, A.; Center for Mechanistic Biology and Biotechnology; Engelhardt Inst. of Molecular Biology

    1998-05-15

    Activated DNA was immobilized in aldehyde-containing polyacrylamide gel for use in manufacturing the MAGIChip (microarrays of gel-immobilized compounds on a chip). First, abasic sites were generated in DNA by partial acidic depurination. Amino groups were then introduced into the abasic sites by reaction with ethylenediamine and reduction of the aldimine bonds formed. It was found that DNA could be fragmented at the site of amino group incorporation or preserved mostly unfragmented. In similar reactions, both amino-DNA and amino-oligonucleotides were attached through their amines to polyacrylamide gel derivatized with aldehyde groups. Single- and double-stranded DNA of 40 to 972 nucleotides or base pairs were immobilized on the gel pads to manufacture a DNA microchip. The microchip was hybridized with fluorescently labeled DNA-specific oligonucleotide probes. This procedure for immobilization of amino compounds was used to manufacture MAGIChips containing both DNA and oligonucleotides.

  7. Thermodynamics of Oligonucleotide Duplex Melting

    ERIC Educational Resources Information Center

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  8. Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports.

    PubMed Central

    Guo, Z; Guilfoyle, R A; Thiel, A J; Wang, R; Smith, L M

    1994-01-01

    A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene. Images PMID:7816638

  9. Detection of transcriptional difference of porcine imprinted genes using different microarray platforms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Presently, multiple options exist for conducting gene expression profiling studies in swine. In order to determine the performance of some of the existing platforms, Affymetrix Porcine, Affymetrix Human U133+2.0, and the U.S. Pig Genome Coordination Program spotted glass oligonucleotide microarray p...

  10. Application of Equilibrium Models of Solution Hybridization to Microarray Design and Analysis

    PubMed Central

    Gharaibeh, Raad Z.; Newton, Joshua M.; Weller, Jennifer W.; Gibas, Cynthia J.

    2010-01-01

    Background The probe percent bound value, calculated using multi-state equilibrium models of solution hybridization, is shown to be useful in understanding the hybridization behavior of microarray probes having 50 nucleotides, with and without mismatches. These longer oligonucleotides are in widespread use on microarrays, but there are few controlled studies of their interactions with mismatched targets compared to 25-mer based platforms. Principal Findings 50-mer oligonucleotides with centrally placed single, double and triple mismatches were spotted on an array. Over a range of target concentrations it was possible to discriminate binding to perfect matches and mismatches, and the type of mismatch could be predicted accurately in the concentration midrange (100 pM to 200 pM) using solution hybridization modeling methods. These results have implications for microarray design, optimization and analysis methods. Conclusions Our results highlight the importance of incorporating biophysical factors in both the design and the analysis of microarrays. Use of the probe “percent bound” value predicted by equilibrium models of hybridization is confirmed to be important for predicting and interpreting the behavior of long oligonucleotide arrays, as has been shown for short oligonucleotide arrays. PMID:20548788

  11. Gene expression profiles of corn developing kernels of Tex6 using maize oligo-microarray

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Maize oligonuleotide microarray was used to analyze the temporal patterns of gene expression in late developmental maize kernels of Tex6 after 25 days after pollination (DAP). There was a total of 57,452 70-mer oligonucleotides on a set of two array-slides. Because of the resistant traits of Tex6, w...

  12. Microarray analysis of gene expression and diapause in Glassy-winged Sharpshooter (Homalodisca vitripennis: Hemiptera: Cicadellidae)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The condition of diapause in the glassy-winged sharpshooter, GWSS, Homalodisca vitripennis, is poorly understood. Diapause is better known from other, non hemipteran insects. We used oligonucleotide microarrays to address the specificities of transcriptional responses of adult female GWSS, which wer...

  13. Microarray Analysis of Antimicrobial Resistance Genes in Salmonella enterica from Preharvest Poultry Environment

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid detection of drug resistance profiles in Salmonella can be critical in treatment of salmonellosis. A 70-mer oligonucleotide microarray chip with 775 gene probes was used to detect antimicrobial resistance genes in 34 Salmonella isolates from a turkey production facility. The phenotypic antim...

  14. Microarray analysis identified Puccinia striiformis f. sp. tritici genes involved in infection and sporulation.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, one of the most important diseases of wheat worldwide. To identify Pst genes involved in infection and sporulation, a custom oligonucleotide Genechip was made using sequences of 442 genes selected from Pst cDNA libraries. Microarray analy...

  15. Phenotypic MicroRNA Microarrays

    PubMed Central

    Kwon, Yong-Jun; Heo, Jin Yeong; Kim, Hi Chul; Kim, Jin Yeop; Liuzzi, Michel; Soloveva, Veronica

    2013-01-01

    Microarray technology has become a very popular approach in cases where multiple experiments need to be conducted repeatedly or done with a variety of samples. In our lab, we are applying our high density spots microarray approach to microscopy visualization of the effects of transiently introduced siRNA or cDNA on cellular morphology or phenotype. In this publication, we are discussing the possibility of using this micro-scale high throughput process to study the role of microRNAs in the biology of selected cellular models. After reverse-transfection of microRNAs and siRNA, the cellular phenotype generated by microRNAs regulated NF-κB expression comparably to the siRNA. The ability to print microRNA molecules for reverse transfection into cells is opening up the wide horizon for the phenotypic high content screening of microRNA libraries using cellular disease models.

  16. Microarrays: an overview.

    PubMed

    Lee, Norman H; Saeed, Alexander I

    2007-01-01

    Gene expression microarrays are being used widely to address a myriad of complex biological questions. To gather meaningful expression data, it is crucial to have a firm understanding of the steps involved in the application of microarrays. The available microarray platforms are discussed along with their advantages and disadvantages. Additional considerations include study design, quality control and systematic assessment of microarray performance, RNA-labeling strategies, sample allocation, signal amplification schemes, defining the number of appropriate biological replicates, data normalization, statistical approaches to identify differentially regulated genes, and clustering algorithms for data visualization. In this chapter, the underlying principles regarding microarrays are reviewed, to serve as a guide when navigating through this powerful technology. PMID:17332646

  17. [Study toward practical use of oligonucleotide therapeutics].

    PubMed

    Inoue, Takao; Yoshida, Tokuyuki

    2014-01-01

    Over the past decade, oligonucleotide-based therapeutics such as antisense oligonucleotides and small interfering RNAs (siRNAs) have been developed extensively. For example, mipomersen (Kynamro; ISIS Pharmaceuticals), which is a second-generation antisense oligonucleotide administered by subcutaneous injection, has recently been approved by the FDA for the treatment of homozygous familial hypercholesterolemia. On the other hands, methods for the evaluation of quality, efficacy and safety of oligonucleotide therapeutics have not been fully discussed. Furthermore, the regulatory guidance specific for oligonucleotide therapeutics has not been established yet. Under these circumstances, we started to collaborate with Osaka University and PMDA to discuss regulatory science focused on oligonucleotide therapeutics. Through the collaboration, we would like to propose the possible design of quality evaluation and preclinical safety-evaluation of oligonucleotide therapeutics. PMID:25707197

  18. Microarrays--status and prospects.

    PubMed

    Venkatasubbarao, Srivatsa

    2004-12-01

    Microarrays have become an extremely important research tool for life science researchers and are also beginning to be used in diagnostic, treatment and monitoring applications. This article provides a detailed description of microarrays prepared by in situ synthesis, deposition using microspotting methods, nonplanar bead arrays, flow-through microarrays, optical fiber bundle arrays and nanobarcodes. The problems and challenges in the development of microarrays, development of standards and diagnostic microarrays are described. Tables summarizing the vendor list of various derivatized microarray surfaces, commercially sold premade microarrays, bead arrays and unique microarray products in development are also included. PMID:15542153

  19. Design of a Seven-Genome Escherichia coli Microarray for Comparative Genomic Profiling▿

    PubMed Central

    Willenbrock, Hanni; Petersen, Anne; Sekse, Camilla; Kiil, Kristoffer; Wasteson, Yngvild; Ussery, David W.

    2006-01-01

    We describe the design and evaluate the use of a high-density oligonucleotide microarray covering seven sequenced Escherichia coli genomes in addition to several sequenced E. coli plasmids, bacteriophages, pathogenicity islands, and virulence genes. Its utility is demonstrated for comparative genomic profiling of two unsequenced strains, O175:H16 D1 and O157:H7 3538 (Δstx2::cat) as well as two well-known control strains, K-12 W3110 and O157:H7 EDL933. By using fluorescently labeled genomic DNA to query the microarrays and subsequently analyze common virulence genes and phage elements and perform whole-genome comparisons, we observed that O175:H16 D1 is a K-12-like strain and confirmed that its φ3538 (Δstx2::cat) phage element originated from the E. coli 3538 (Δstx2::cat) strain, with which it shares a substantial proportion of phage elements. Moreover, a number of genes involved in DNA transfer and recombination was identified in both new strains, providing a likely explanation for their capability to transfer φ3538 (Δstx2::cat) between them. Analyses of control samples demonstrated that results using our custom-designed microarray were representative of the true biology, e.g., by confirming the presence of all known chromosomal phage elements as well as 98.8 and 97.7% of queried chromosomal genes for the two control strains. Finally, we demonstrate that use of spatial information, in terms of the physical chromosomal locations of probes, improves the analysis. PMID:16963574

  20. Microarray Analysis in Glioblastomas.

    PubMed

    Bhawe, Kaumudi M; Aghi, Manish K

    2016-01-01

    Microarray analysis in glioblastomas is done using either cell lines or patient samples as starting material. A survey of the current literature points to transcript-based microarrays and immunohistochemistry (IHC)-based tissue microarrays as being the preferred methods of choice in cancers of neurological origin. Microarray analysis may be carried out for various purposes including the following: i. To correlate gene expression signatures of glioblastoma cell lines or tumors with response to chemotherapy (DeLay et al., Clin Cancer Res 18(10):2930-2942, 2012). ii. To correlate gene expression patterns with biological features like proliferation or invasiveness of the glioblastoma cells (Jiang et al., PLoS One 8(6):e66008, 2013). iii. To discover new tumor classificatory systems based on gene expression signature, and to correlate therapeutic response and prognosis with these signatures (Huse et al., Annu Rev Med 64(1):59-70, 2013; Verhaak et al., Cancer Cell 17(1):98-110, 2010). While investigators can sometimes use archived tumor gene expression data available from repositories such as the NCBI Gene Expression Omnibus to answer their questions, new arrays must often be run to adequately answer specific questions. Here, we provide a detailed description of microarray methodologies, how to select the appropriate methodology for a given question, and analytical strategies that can be used. Experimental methodology for protein microarrays is outside the scope of this chapter, but basic sample preparation techniques for transcript-based microarrays are included here. PMID:26113463

  1. Rapid genotyping with DNA micro-arrays for high-density linkage mapping and QTL mapping in common buckwheat (Fagopyrum esculentum Moench)

    PubMed Central

    Yabe, Shiori; Hara, Takashi; Ueno, Mariko; Enoki, Hiroyuki; Kimura, Tatsuro; Nishimura, Satoru; Yasui, Yasuo; Ohsawa, Ryo; Iwata, Hiroyoshi

    2014-01-01

    For genetic studies and genomics-assisted breeding, particularly of minor crops, a genotyping system that does not require a priori genomic information is preferable. Here, we demonstrated the potential of a novel array-based genotyping system for the rapid construction of high-density linkage map and quantitative trait loci (QTL) mapping. By using the system, we successfully constructed an accurate, high-density linkage map for common buckwheat (Fagopyrum esculentum Moench); the map was composed of 756 loci and included 8,884 markers. The number of linkage groups converged to eight, which is the basic number of chromosomes in common buckwheat. The sizes of the linkage groups of the P1 and P2 maps were 773.8 and 800.4 cM, respectively. The average interval between adjacent loci was 2.13 cM. The linkage map constructed here will be useful for the analysis of other common buckwheat populations. We also performed QTL mapping for main stem length and detected four QTL. It took 37 days to process 178 samples from DNA extraction to genotyping, indicating the system enables genotyping of genome-wide markers for a few hundred buckwheat plants before the plants mature. The novel system will be useful for genomics-assisted breeding in minor crops without a priori genomic information. PMID:25914583

  2. Rapid genotyping with DNA micro-arrays for high-density linkage mapping and QTL mapping in common buckwheat (Fagopyrum esculentum Moench).

    PubMed

    Yabe, Shiori; Hara, Takashi; Ueno, Mariko; Enoki, Hiroyuki; Kimura, Tatsuro; Nishimura, Satoru; Yasui, Yasuo; Ohsawa, Ryo; Iwata, Hiroyoshi

    2014-12-01

    For genetic studies and genomics-assisted breeding, particularly of minor crops, a genotyping system that does not require a priori genomic information is preferable. Here, we demonstrated the potential of a novel array-based genotyping system for the rapid construction of high-density linkage map and quantitative trait loci (QTL) mapping. By using the system, we successfully constructed an accurate, high-density linkage map for common buckwheat (Fagopyrum esculentum Moench); the map was composed of 756 loci and included 8,884 markers. The number of linkage groups converged to eight, which is the basic number of chromosomes in common buckwheat. The sizes of the linkage groups of the P1 and P2 maps were 773.8 and 800.4 cM, respectively. The average interval between adjacent loci was 2.13 cM. The linkage map constructed here will be useful for the analysis of other common buckwheat populations. We also performed QTL mapping for main stem length and detected four QTL. It took 37 days to process 178 samples from DNA extraction to genotyping, indicating the system enables genotyping of genome-wide markers for a few hundred buckwheat plants before the plants mature. The novel system will be useful for genomics-assisted breeding in minor crops without a priori genomic information. PMID:25914583

  3. Emerging Use of Gene Expression Microarrays in Plant Physiology

    DOE PAGESBeta

    Wullschleger, Stan D.; Difazio, Stephen P.

    2003-01-01

    Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology weremore » selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry.« less

  4. Experimental Approaches to Microarray Analysis of Tumor Samples

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Winter, Michael B.; Meyers, Jacob I.; Furge, Kyle A.

    2008-01-01

    Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has…

  5. Biopolymer synthesis on polypropylene supports. I. Oligonucleotides.

    PubMed

    Matson, R S; Rampal, J B; Coassin, P J

    1994-03-01

    We have modified polypropylene to serve as a new solid-phase support for oligonucleotide synthesis. The plastic is first surface aminated by exposure to an ammonia plasma generated by radiofrequency plasma discharge. The aminated polypropylene has been found to be useful as a support for the in situ synthesis of oligonucleotides from monomers. Furthermore, oligonucleotides synthesized on the surface of the plastic remain attached following deprotection and can be used directly for hybridization. PMID:8203760

  6. A dynamic bead-based microarray for parallel DNA detection

    NASA Astrophysics Data System (ADS)

    Sochol, R. D.; Casavant, B. P.; Dueck, M. E.; Lee, L. P.; Lin, L.

    2011-05-01

    A microfluidic system has been designed and constructed by means of micromachining processes to integrate both microfluidic mixing of mobile microbeads and hydrodynamic microbead arraying capabilities on a single chip to simultaneously detect multiple bio-molecules. The prototype system has four parallel reaction chambers, which include microchannels of 18 × 50 µm2 cross-sectional area and a microfluidic mixing section of 22 cm length. Parallel detection of multiple DNA oligonucleotide sequences was achieved via molecular beacon probes immobilized on polystyrene microbeads of 16 µm diameter. Experimental results show quantitative detection of three distinct DNA oligonucleotide sequences from the Hepatitis C viral (HCV) genome with single base-pair mismatch specificity. Our dynamic bead-based microarray offers an effective microfluidic platform to increase parallelization of reactions and improve microbead handling for various biological applications, including bio-molecule detection, medical diagnostics and drug screening.

  7. Multiplex pairwise assembly of array-derived DNA oligonucleotides

    PubMed Central

    Klein, Jason C.; Lajoie, Marc J.; Schwartz, Jerrod J.; Strauch, Eva-Maria; Nelson, Jorgen; Baker, David; Shendure, Jay

    2016-01-01

    While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens. PMID:26553805

  8. Multiplex pairwise assembly of array-derived DNA oligonucleotides.

    PubMed

    Klein, Jason C; Lajoie, Marc J; Schwartz, Jerrod J; Strauch, Eva-Maria; Nelson, Jorgen; Baker, David; Shendure, Jay

    2016-03-18

    While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens. PMID:26553805

  9. Use of a bacterial antimicrobial resistance gene microarray for the identification of resistant Staphylococcus aureus.

    PubMed

    Garneau, P; Labrecque, O; Maynard, C; Messier, S; Masson, L; Archambault, M; Harel, J

    2010-11-01

    As diagnostic and surveillance activities are vital to determine measures needed to control antimicrobial resistance (AMR), new and rapid laboratory methods are necessary to facilitate this important effort. DNA microarray technology allows the detection of a large number of genes in a single reaction. This technology is simple, specific and high-throughput. We have developed a bacterial antimicrobial resistance gene DNA microarray that will allow rapid antimicrobial resistance gene screening for all Gram-positive and Gram-negative bacteria. A prototype microarray was designed using a 70-mer based oligonucleotide set targeting AMR genes of Gram-negative and Gram-positive bacteria. In the present version, the microarray consists of 182 oligonucleotides corresponding to 166 different acquired AMR gene targets, covering most of the resistance genes found in both Gram-negative and -positive bacteria. A test study was performed on a collection of Staphylococcus aureus isolates from milk samples from dairy farms in Québec, Canada. The reproducibility of the hybridizations was determined, and the microarray results were compared with those obtained by phenotypic resistance tests (either MIC or Kirby-Bauer). The microarray genotyping demonstrated a correlation between penicillin, tetracycline and erythromycin resistance phenotypes with the corresponding acquired resistance genes. The hybridizations showed that the 38 antimicrobial resistant S. aureus isolates possessed at least one AMR gene. PMID:21083822

  10. The prebiotic synthesis of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Oro, J.; Stephen-Sherwood, E.

    1974-01-01

    This paper is primarily a review of recent developments in the abiotic synthesis of nucleotides, short chain oligonucleotides, and their mode of replication in solution. It also presents preliminary results from this laboratory on the prebiotic synthesis of thymidine oligodeoxynucleotides. A discussion, based on the physicochemical properties of RNA and DNA oligomers, relevant to the molecular evolution of these compounds leads to the tentative hypothesis that oligodeoxyribonucleotides of about 12 units may have been of sufficient length to initiate a self replicating coding system. Two models are suggested to account for the synthesis of high molecular weight oligomers using short chain templates and primers.

  11. Detection of human genome mutations associated with pregnancy complications using 3-D microarray based on macroporous polymer monoliths.

    PubMed

    Glotov, A S; Sinitsyna, E S; Danilova, M M; Vashukova, E S; Walter, J G; Stahl, F; Baranov, V S; Vlakh, E G; Tennikova, T B

    2016-01-15

    Analysis of variations in DNA structure using a low-density microarray technology for routine diagnostic in evidence-based medicine is still relevant. In this work the applicability of 3-D macroporous monolithic methacrylate-based platforms for detection of different pathogenic genomic substitutions was studied. The detection of nucleotide replacements in F5 (Leiden G/A, rs6025), MTHFR (C/T, rs1801133) and ITGB3 (T/C, rs5918), involved in coagulation, and COMT (C/G, rs4818), TPH2 (T/A, rs11178997), PON1 (T/A rs854560), AGTR2 (C/A, rs11091046) and SERPINE1 (5G/4G, rs1799889), associated with pregnancy complications, was performed. The effect of such parameters as amount and type of oligonucleotide probe, amount of PCR product on signal-to-noise ratio, as well as mismatch discrimination was analyzed. Sensitivity and specificity of mutation detections were coincided and equal to 98.6%. The analysis of SERPINE1 and MTHFR genotypes by both NGS and developed microarray was performed and compared. PMID:26592644

  12. Cellular Uptake and Intracellular Trafficking of Oligonucleotides: Implications for Oligonucleotide Pharmacology

    PubMed Central

    Ming, Xin; Carver, Kyle; Laing, Brian

    2014-01-01

    One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed. PMID:24383421

  13. Nanotechnologies in protein microarrays.

    PubMed

    Krizkova, Sona; Heger, Zbynek; Zalewska, Marta; Moulick, Amitava; Adam, Vojtech; Kizek, Rene

    2015-01-01

    Protein microarray technology became an important research tool for study and detection of proteins, protein-protein interactions and a number of other applications. The utilization of nanoparticle-based materials and nanotechnology-based techniques for immobilization allows us not only to extend the surface for biomolecule immobilization resulting in enhanced substrate binding properties, decreased background signals and enhanced reporter systems for more sensitive assays. Generally in contemporarily developed microarray systems, multiple nanotechnology-based techniques are combined. In this review, applications of nanoparticles and nanotechnologies in creating protein microarrays, proteins immobilization and detection are summarized. We anticipate that advanced nanotechnologies can be exploited to expand promising fields of proteins identification, monitoring of protein-protein or drug-protein interactions, or proteins structures. PMID:26039143

  14. Electron Transfer Dissociation of Oligonucleotide Cations.

    PubMed

    Smith, Suncerae I; Brodbelt, Jennifer S

    2009-06-01

    Electron transfer dissociation (ETD) of multi-protonated 6 - 20-mer oligonucleotides and 12- and 14-mer duplexes is compared to collision activated dissociation (CAD). ETD causes efficient charge reduction of the multi-protonated oligonucleotides in addition to limited backbone cleavages to yield sequence ions of low abundance. Subsequent CAD of the charge-reduced oligonucleotides formed upon electron transfer, in a net process termed electron transfer collision activated dissociation (ETcaD), results in rich fragmentation in terms of w, a, z, and d products, with a marked decrease in the abundance of base loss ions and internal fragments. Complete sequencing was possible for nearly all oligonucleotides studied. ETcaD of an oligonucleotide duplex resulted in specific backbone cleavages, with conservation of weaker non-covalent bonds. PMID:20161288

  15. Molecular Crowding Effects on Microgel-Tethered Oligonucleotide Probes.

    PubMed

    Ma, Youlong; Libera, Matthew

    2016-06-28

    Microgel tethering is a nontraditional method with which to bind oligonucleotide hybridization probes to a solid surface. Microgel-tethering physically positions the probes away from the underlying hard substrate and maintains them in a highly waterlike environment. This paper addresses the question of whether molecular crowding affects the performance of microgel-tethered molecular beacon probes. The density of probe-tethering sites is controlled experimentally using thin-film blends of biotin-terminated [PEG-B] and hydroxyl-terminated [PEG-OH] poly(ethylene glycol) from which microgels are synthesized and patterned by electron beam lithography. Fluorescence measurements indicate that the number of streptavidins, linear DNA probes, hairpin probes, and molecular beacon probes bound to the microgels increases linearly with increasing PEG-B/PEG-OH ratio. For a given tethering-site concentration, more linear probes can bind than structured probes. Crowding effects emerge during the hybridization of microgel-tethered molecular beacons but not during the hybridization of linear probes, as the tethering density increases. Crowding during hybridization is associated with conformational constraints imposed by the close proximity of closed and hybridized structured probes. The signal-to-background ratio (SBR) of hybridized beacons is highest and roughly constant for low tethering densities and decreases at the highest tethering densities. Despite differences between microgel tethering and traditional oligonucleotide surface-immobilization approaches, these results show that crowding defines an optimum tethering density for molecular beacon probes that is less than the maximum possible, which is consistent with previous studies involving various linear and structured oligonucleotide probes. PMID:27253904

  16. Microarrays for Undergraduate Classes

    ERIC Educational Resources Information Center

    Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

    2006-01-01

    A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by…

  17. Synthesis of 5'-Aldehyde Oligonucleotide.

    PubMed

    Lartia, Rémy

    2016-01-01

    Synthesis of oligonucleotide ending with an aldehyde functional group at their 5'-end (5'-AON) is possible for both DNA (5'-AODN) and RNA (5'-AORN) series irrespectively of the nature of the last nucleobase. The 5'-alcohol of on-support ODN is mildly oxidized under Moffat conditions. Transient protection of the resulting aldehyde by N,N'-diphenylethylenediamine derivatives allows cleavage, deprotection, and RP-HPLC purification of the protected 5'-AON. Finally, 5'-AON is deprotected by usual acetic acid treatment. In the aggregates, 5'-AON can be now synthesized and purified as routinely as non-modified ODNs, following procedures similar to the well-known "DMT-On" strategy. PMID:26967469

  18. Microintaglio Printing for Soft Lithography-Based in Situ Microarrays

    PubMed Central

    Biyani, Manish; Ichiki, Takanori

    2015-01-01

    Advances in lithographic approaches to fabricating bio-microarrays have been extensively explored over the last two decades. However, the need for pattern flexibility, a high density, a high resolution, affordability and on-demand fabrication is promoting the development of unconventional routes for microarray fabrication. This review highlights the development and uses of a new molecular lithography approach, called “microintaglio printing technology”, for large-scale bio-microarray fabrication using a microreactor array (µRA)-based chip consisting of uniformly-arranged, femtoliter-size µRA molds. In this method, a single-molecule-amplified DNA microarray pattern is self-assembled onto a µRA mold and subsequently converted into a messenger RNA or protein microarray pattern by simultaneously producing and transferring (immobilizing) a messenger RNA or a protein from a µRA mold to a glass surface. Microintaglio printing allows the self-assembly and patterning of in situ-synthesized biomolecules into high-density (kilo-giga-density), ordered arrays on a chip surface with µm-order precision. This holistic aim, which is difficult to achieve using conventional printing and microarray approaches, is expected to revolutionize and reshape proteomics. This review is not written comprehensively, but rather substantively, highlighting the versatility of microintaglio printing for developing a prerequisite platform for microarray technology for the postgenomic era.

  19. Tandem oligonucleotide synthesis using linker phosphoramidites

    PubMed Central

    Pon, Richard T.; Yu, Shuyuan

    2005-01-01

    Multiple oligonucleotides of the same or different sequence, linked end-to-end in tandem can be synthesized in a single automated synthesis. A linker phosphoramidite [R. T. Pon and S. Yu (2004) Nucleic Acids Res., 32, 623–631] is added to the 5′-terminal OH end of a support-bound oligonucleotide to introduce a cleavable linkage (succinic acid plus sulfonyldiethanol) and the 3′-terminal base of the new sequence. Conventional phosphoramidites are then used for the rest of the sequence. After synthesis, treatment with ammonium hydroxide releases the oligonucleotides from the support and cleaves the linkages between each sequence. Mixtures of one oligonucleotide with both 5′- and 3′-terminal OH ends and other oligonucleotides with 5′-phosphorylated and 3′-OH ends are produced, which are deprotected and worked up as a single product. Tandem synthesis can be used to make pairs of PCR primers, sets of cooperative oligonucleotides or multiple copies of the same sequence. When tandem synthesis is used to make two self-complementary sequences, double-stranded structures spontaneously form after deprotection. Tandem synthesis of oligonucleotide chains containing up to six consecutive 20mer (120 bases total), various trinucleotide codons and primer pairs for PCR, or self-complementary strands for in situ formation of double-stranded DNA fragments has been demonstrated. PMID:15814811

  20. A tiling microarray for global analysis of chloroplast genome expression in cucumber and other plants

    PubMed Central

    2011-01-01

    Plastids are small organelles equipped with their own genomes (plastomes). Although these organelles are involved in numerous plant metabolic pathways, current knowledge about the transcriptional activity of plastomes is limited. To solve this problem, we constructed a plastid tiling microarray (PlasTi-microarray) consisting of 1629 oligonucleotide probes. The oligonucleotides were designed based on the cucumber chloroplast genomic sequence and targeted both strands of the plastome in a non-contiguous arrangement. Up to 4 specific probes were designed for each gene/exon, and the intergenic regions were covered regularly, with 70-nt intervals. We also developed a protocol for direct chemical labeling and hybridization of as little as 2 micrograms of chloroplast RNA. We used this protocol for profiling the expression of the cucumber chloroplast plastome on the PlasTi-microarray. Owing to the high sequence similarity of plant plastomes, the newly constructed microarray can be used to study plants other than cucumber. Comparative hybridization of chloroplast transcriptomes from cucumber, Arabidopsis, tomato and spinach showed that the PlasTi-microarray is highly versatile. PMID:21952044

  1. Quantitation of phosphorothioate oligonucleotides in human plasma.

    PubMed

    Leeds, J M; Graham, M J; Truong, L; Cummins, L L

    1996-03-01

    Methods are presented for the extraction of phosphorothioate oligonucleotides from human plasma to permit quantitation by capillary gel electrophoresis. Extraction of the phosphorothioate oligonucleotides from plasma was accomplished using two solid-phase extraction columns, a strong anion-exchange column to remove plasma proteins and lipids, followed by a reverse-phase column to remove salts. A second desalting step, achieved by dialysis utilizing a membrane with a molecular weight cutoff of 2500 Da floating on distilled water, was required to remove residual ionic material from the extracted sample. This method should be generally applicable to the analysis and quantitation of phosphorothioate oligonucleotides. PMID:8850544

  2. Structural analysis of hepatitis C RNA genome using DNA microarrays

    PubMed Central

    Martell, María; Briones, Carlos; de Vicente, Aránzazu; Piron, María; Esteban, Juan I.; Esteban, Rafael; Guardia, Jaime; Gómez, Jordi

    2004-01-01

    Many studies have tried to identify specific nucleotide sequences in the quasispecies of hepatitis C virus (HCV) that determine resistance or sensitivity to interferon (IFN) therapy, unfortunately without conclusive results. Although viral proteins represent the most evident phenotype of the virus, genomic RNA sequences determine secondary and tertiary structures which are also part of the viral phenotype and can be involved in important biological roles. In this work, a method of RNA structure analysis has been developed based on the hybridization of labelled HCV transcripts to microarrays of complementary DNA oligonucleotides. Hybridizations were carried out at non-denaturing conditions, using appropriate temperature and buffer composition to allow binding to the immobilized probes of the RNA transcript without disturbing its secondary/tertiary structural motifs. Oligonucleotides printed onto the microarray covered the entire 5′ non-coding region (5′NCR), the first three-quarters of the core region, the E2–NS2 junction and the first 400 nt of the NS3 region. We document the use of this methodology to analyse the structural degree of a large region of HCV genomic RNA in two genotypes associated with different responses to IFN treatment. The results reported here show different structural degree along the genome regions analysed, and differential hybridization patterns for distinct genotypes in NS2 and NS3 HCV regions. PMID:15247323

  3. Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

    PubMed Central

    Linger, Yvonne; Kukhtin, Alexander; Golova, Julia; Perov, Alexander; Qu, Peter; Knickerbocker, Christopher; Cooney, Christopher G.; Chandler, Darrell P.

    2014-01-01

    Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice. PMID:24796567

  4. Navigating Public Microarray Databases

    PubMed Central

    Bähler, Jürg

    2004-01-01

    With the ever-escalating amount of data being produced by genome-wide microarray studies, it is of increasing importance that these data are captured in public databases so that researchers can use this information to complement and enhance their own studies. Many groups have set up databases of expression data, ranging from large repositories, which are designed to comprehensively capture all published data, through to more specialized databases. The public repositories, such as ArrayExpress at the European Bioinformatics Institute contain complete datasets in raw format in addition to processed data, whilst the specialist databases tend to provide downstream analysis of normalized data from more focused studies and data sources. Here we provide a guide to the use of these public microarray resources. PMID:18629145

  5. Microarrays under the microscope.

    PubMed

    Wildsmith, S E; Elcock, F J

    2001-02-01

    Microarray technology is a rapidly advancing area, which is gaining popularity in many biological disciplines from drug target identification to predictive toxicology. Over the past few years, there has been a dramatic increase in the number of methods and techniques available for carrying out this form of gene expression analysis. The techniques and associated peripherals, such as slide types, deposition methods, robotics, and scanning equipment, are undergoing constant improvement, helping to drive the technology forward in terms of robustness and ease of use. These rapid developments, combined with the number of options available and the associated hyperbole, can prove daunting for the new user. This review aims to guide the researcher through the various steps of conducting microarray experiments, from initial strategy to analysing the data, with critical examination of the benefits and disadvantages along the way. PMID:11212888

  6. Navigating public microarray databases.

    PubMed

    Penkett, Christopher J; Bähler, Jürg

    2004-01-01

    With the ever-escalating amount of data being produced by genome-wide microarray studies, it is of increasing importance that these data are captured in public databases so that researchers can use this information to complement and enhance their own studies. Many groups have set up databases of expression data, ranging from large repositories, which are designed to comprehensively capture all published data, through to more specialized databases. The public repositories, such as ArrayExpress at the European Bioinformatics Institute contain complete datasets in raw format in addition to processed data, whilst the specialist databases tend to provide downstream analysis of normalized data from more focused studies and data sources. Here we provide a guide to the use of these public microarray resources. PMID:18629145

  7. An on-chip thin film photodetector for the quantification of DNA probes and targets in microarrays

    PubMed Central

    Fixe, F.; Chu, V.; Prazeres, D. M. F.; Conde, J. P.

    2004-01-01

    A flat microdevice which incorporates a thin-film amorphous silicon (a-Si:H) photodetector with an upper layer of functionalized SiO2 is used to quantify the density of both immobilized and hybridized DNA oligonucleotides labeled with a fluorophore. The device is based on the photoconductivity of hydrogenated amorphous silicon in a coplanar electrode configuration. Excitation, with near UV/blue light, of a single-stranded DNA molecule tagged with the fluorophore 1-(3-(succinimidyloxycarbonyl)benzyl)-4-(5-(4-methoxyphenyl)oxazol-2-yl) pyridinium bromide (PyMPO), results in the emission of visible light. The emitted light is then converted into an electrical signal in the photodetector, thus allowing the optoelectronic detection of the DNA molecules. The detection limit of the present device is of the order of 1 × 1012 molecules/cm2 and is limited by the efficiency of the filtering of the excitation light. A surface density of 33.5 ± 4.0 pmol/cm2 was measured for DNA covalently immobilized to the functionalized SiO2 thin film and a surface density of 3.7 ± 1.5 pmol/cm2 was measured for the complementary DNA hybridized to the bound DNA. The detection concept explored can enable on-chip electronic data acquisition, improving both the speed and the reliability of DNA microarrays. PMID:15148343

  8. Transformation of yeast with synthetic oligonucleotides.

    PubMed Central

    Moerschell, R P; Tsunasawa, S; Sherman, F

    1988-01-01

    Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cyc1 mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonucleotides as short as 20 nucleotides and with amounts as low as 100 micrograms. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated. PMID:2829192

  9. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment.

    PubMed

    Wagner, Victoria E; Bushnell, Daniel; Passador, Luciano; Brooks, Andrew I; Iglewski, Barbara H

    2003-04-01

    Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the las and rhl systems, have been identified previously in the opportunistic pathogen Pseudomonas aeruginosa. High-density oligonucleotide microarrays for the P. aeruginosa PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying las and/or rhl QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (P

  10. Antisense oligonucleotides as therapeutics for malignant diseases.

    PubMed

    Ho, P T; Parkinson, D R

    1997-04-01

    The continued progress in our understanding of the biology of neoplasia and in the identification, cloning, and sequencing of genes critical to tumor cell function permits the exploitation of this information to develop specific agents that may directly modulate the function of these genes or their protein products. Antisense oligonucleotides are being investigated as a potential therapeutic modality that takes direct advantage of molecular sequencing. The antisense approach uses short oligonucleotides designed to hybridize to a target mRNA transcript through Watson-Crick base pairing. The formation of this oligonucleotide: RNA heteroduplex results in mRNA inactivation and consequent inhibition of synthesis of the protein product. A fundamental attraction of the antisense approach is that this method potentially may be applied to any gene product, in theory, for the treatment of malignant and non-malignant diseases. However, this simple and attractive model has proven to be much more complex in practice. A number of important challenges in the preclinical development of antisense oligonucleotides have been identified, including stability, sequence length, cellular uptake, target sequence selection, appropriate negative controls, oligonucleotide: protein interactions, and cost of manufacture. Although the biological activity of an oligonucleotide against its molecular target is theoretically sequence-dependent, the animal pharmacokinetics and toxicology of phosphorothioate analogues directed against vastly disparate gene products appear relatively non-sequence-specific. In oncology, a number of clinical trials have been initiated with antisense oligonucleotides directed against molecular targets including: p53; bcl-2; raf kinase; protein kinase C-alpha; c-myb. The experience gained from these early clinical trials will be applicable to the next generation of antisense agents in development. These may include molecules with novel backbones or other structural

  11. Direct microcontact printing of oligonucleotides for biochip applications

    PubMed Central

    Thibault, C; Le Berre, V; Casimirius, S; Trévisiol, E; François, J; Vieu, C

    2005-01-01

    Background A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. Results Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane) material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. Conclusion The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting technology. PMID:15992404

  12. SuperSAGE array: the direct use of 26-base-pair transcript tags in oligonucleotide arrays.

    PubMed

    Matsumura, Hideo; Bin Nasir, Khairun Hisam; Yoshida, Kentaro; Ito, Akiko; Kahl, Günter; Krüger, Detlev H; Terauchi, Ryohei

    2006-06-01

    We developed a new platform for genome-wide gene expression analysis in any eukaryotic organism, which we called SuperSAGE array. The SuperSAGE array is a microarray onto which 26-bp oligonucleotides corresponding to SuperSAGE tag sequences are directly synthesized. A SuperSAGE array combines the advantages of the highly quantitative SuperSAGE expression analysis with the high-throughput microarray technology. We demonstrated highly reproducible gene expression profiling by the SuperSAGE array for 1,000 genes (tags) in rice. We also applied this technology to the detailed study of expressed genes identified by SuperSAGE in Nicotiana benthamiana, an organism for which sufficient genome sequence information is not available. We propose that the SuperSAGE array system represents a new paradigm for microarray construction, as no genomic or cDNA sequence data are required for its preparation. PMID:16721381

  13. Advances in spotted microarray resources for expression profiling.

    PubMed

    Lyons, Paul

    2003-04-01

    The new millennium has ushered in a new era in human biology. The elucidation of the human genome sequence, together with those of model organisms, provides us with an unprecedented insight into the makeup of our genetic blueprint. The challenge now is to figure out how all the constituent pieces fit together to form the whole picture, and the consequences of what happens when the process goes awry. One experimental tool that has the potential to provide enormous insights into this complex process is expression profiling using microarrays. The past few years have seen a considerable growth in the availability and use of microarrays. Fuelled in part by the many genome projects currently underway, there has been a large increase in the number of organisms for which microarray reagents are available from both commercial and academic sources. In addition to the increasing number of genome-wide probe sets that are available, a significant amount of attention has been focussed on generating more targeted probe sets that focus in on specific pathways or biological processes. Finally, the microarray field is starting to see a shift away from the use of cDNAs or polymerase chain reaction (PCR) products as probes towards the use of 50-70mer oligonucleotide probes with all of the potential advantages that they offer. The aim of this review is to provide an overview of what is currently available in terms of spotted microarray reagents both with respect to pre-made arrays and to probe sets available for arraying. PMID:15239940

  14. Tiling Microarray Analysis Tools

    SciTech Connect

    Nix, Davis Austin

    2005-05-04

    TiMAT is a package of 23 command line Java applications for use in the analysis of Affymetrix tiled genomic microarray data. TiMAT enables: 1) Rebuilding the genome annotation for entire tiled arrays (repeat filtering, chromosomal coordinate assignment). 2) Post processing of oligo intensity values (quantile normalization, median scaling, PMMM transformation), 3) Significance testing (Wilcoxon rank sum and signed rank tests, intensity difference and ratio tests) and Interval refinement (filtering based on multiple statistics, overlap comparisons), 4) Data visualization (detailed thumbnail/zoomed view with Interval Plots and data export to Affymetrix's Integrated Genome Browser) and Data reports (spreadsheet summaries and detailed profiles)

  15. Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

    SciTech Connect

    Proudnikov, D.; Kirillov, E.; Chumakov, K.; Donion, J.; Rezapkin, G.; Mirzabekov, A.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Center for Biologics Evaluation and Research

    2000-01-01

    This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.

  16. Spotting and validation of a genome wide oligonucleotide chip with duplicate measurement of each gene

    SciTech Connect

    Thomassen, Mads . E-mail: mads.thomassen@ouh.fyns-amt.dk; Skov, Vibe; Eiriksdottir, Freyja; Tan, Qihua; Jochumsen, Kirsten; Fritzner, Niels; Brusgaard, Klaus; Dahlgaard, Jesper; Kruse, Torben A.

    2006-06-16

    The quality of DNA microarray based gene expression data relies on the reproducibility of several steps in a microarray experiment. We have developed a spotted genome wide microarray chip with oligonucleotides printed in duplicate in order to minimise undesirable biases, thereby optimising detection of true differential expression. The validation study design consisted of an assessment of the microarray chip performance using the MessageAmp and FairPlay labelling kits. Intraclass correlation coefficient (ICC) was used to demonstrate that MessageAmp was significantly more reproducible than FairPlay. Further examinations with MessageAmp revealed the applicability of the system. The linear range of the chips was three orders of magnitude, the precision was high, as 95% of measurements deviated less than 1.24-fold from the expected value, and the coefficient of variation for relative expression was 13.6%. Relative quantitation was more reproducible than absolute quantitation and substantial reduction of variance was attained with duplicate spotting. An analysis of variance (ANOVA) demonstrated no significant day-to-day variation.

  17. Microarray analysis at single molecule resolution

    PubMed Central

    Mureşan, Leila; Jacak, Jarosław; Klement, Erich Peter; Hesse, Jan; Schütz, Gerhard J.

    2010-01-01

    Bioanalytical chip-based assays have been enormously improved in sensitivity in the recent years; detection of trace amounts of substances down to the level of individual fluorescent molecules has become state of the art technology. The impact of such detection methods, however, has yet not fully been exploited, mainly due to a lack in appropriate mathematical tools for robust data analysis. One particular example relates to the analysis of microarray data. While classical microarray analysis works at resolutions of two to 20 micrometers and quantifies the abundance of target molecules by determining average pixel intensities, a novel high resolution approach [1] directly visualizes individual bound molecules as diffraction limited peaks. The now possible quantification via counting is less susceptible to labeling artifacts and background noise. We have developed an approach for the analysis of high-resolution microarray images. It consists first of a single molecule detection step, based on undecimated wavelet transforms, and second, of a spot identification step via spatial statistics approach (corresponding to the segmentation step in the classical microarray analysis). The detection method was tested on simulated images with a concentration range of 0.001 to 0.5 molecules per square micron and signal-to-noise ratio (SNR) between 0.9 and 31.6. For SNR above 15 the false negatives relative error was below 15%. Separation of foreground/background proved reliable, in case foreground density exceeds background by a factor of 2. The method has also been applied to real data from high-resolution microarray measurements. PMID:20123580

  18. Mechanism of oligonucleotide release from cationic liposomes.

    PubMed Central

    Zelphati, O; Szoka, F C

    1996-01-01

    We propose a mechanism for oligonucleotide (ODN) release from cationic lipid complexes in cells that accounts for various observations on cationic lipid-nucleic acid-cell interactions. Fluorescent confocal microscopy of cells treated with rhodamine-labeled cationic liposome/ fluorescein-labeled ODN (F-ODN) complexes show the F-ODN separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures. ODN displacement from the complex was studied by fluorescent resonance energy transfer. Anionic liposome compositions (e.g., phosphatidylserine) that mimic the cytoplasmic facing monolayer of the cell membrane released ODN from the complex at about a 1:1 (-/+) charge ratio. Release was independent of ionic strength and pH. Physical separation of the F-ODN from monovalent and multivalent cationic lipids was confirmed by gel electrophoresis. Fluid but not solid phase anionic liposomes are required, whereas the physical state of the cationic lipids does not effect the release. Water soluble molecules with a high negative linear charge density, dextran sulfate, or heparin also release ODN. However, ATP, spermidine, spermine, tRNA, DNA, polyglutamic acid, polylysine, bovine serum albumin, or histone did not release ODN, even at 100-fold charge excess (-/+). Based upon these results, we propose that the complex, after internalization by endocytosis, induces flip-flop of anionic lipids from the cytoplasmic facing monolayer. Anionic lipids laterally diffuse into the complex and form a charged neutralized ion-pair with the cationic lipids. This leads to displacement of the ODN from the cationic lipid and its release into the cytoplasm. Images Fig. 1 Fig. 3 PMID:8876163

  19. Compressive Sensing DNA Microarrays

    PubMed Central

    2009-01-01

    Compressive sensing microarrays (CSMs) are DNA-based sensors that operate using group testing and compressive sensing (CS) principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed. PMID:19158952

  20. Caged oligonucleotides for studying biological systems

    PubMed Central

    Ruble, Brittani K.; Yeldell, Sean B.; Dmochowski, Ivan J.

    2015-01-01

    Light-activated (“caged”) compounds have been widely employed for studying biological processes with high spatial and temporal control. In the past decade, several new approaches for caging the structure and function of DNA and RNA oligonucleotides have been developed. This review focuses on caged oligonucleotides that incorporate site-specifically one or two photocleavable linkers, whose photolysis yields oligonucleotides with dramatic structural and functional changes. This technique has been employed by our laboratory and others to photoregulate gene expression in cells and living organisms, typically using near UV-activated organic chromophores. To improve capabilities for in vivo studies, we harnessed the rich inorganic photochemistry of ruthenium bipyridyl complexes to synthesize Ru-caged morpholino antisense oligonucleotides that remain inactive in zebrafish embryos until uncaged with visible light. Expanding into new caged oligonucleotide applications, our lab has developed Transcriptome In Vivo Analysis (TIVA) technology, which provides the first noninvasive, unbiased method for isolating mRNA from single neurons in brain tissues. TIVA-isolated mRNA can be amplified and then analyzed using next-generation sequencing (RNA-seq). PMID:25865001

  1. Microarray-Based Mapping for the Detection of Molecular Markers in Response to Aspergillus flavus Infection in Susceptible and Resistant Maize Lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objectives of this study were (1) to evaluate differential gene expression levels for resistance to A. flavus kernel infection in susceptible (Va35) and resistant (Mp313E) maize lines using Oligonucleotide and cDNA microarray analysis, (2) to evaluate differences in A. flavus accumulation betwee...

  2. Protocol for Gene Expression Profiling Using DNA Microarrays in Neisseria gonorrhoeae

    PubMed Central

    Jackson, Lydgia A.; Dyer, David W.

    2016-01-01

    Gene expression profiling using DNA microarrays has become commonplace in current molecular biology practices, and has dramatically enhanced our understanding of the biology of Neisseria spp., and the interaction of these organisms with the host. With the choice of microarray platforms offered for gene expression profiling and commercially available arrays, investigators must ask several central questions to make decisions based on their research focus. Are arrays on hand for their organism and if not then would it be cost-effective to design custom arrays. Other important considerations; what types of specialized equipment for array hybridization and signal detection are required and is the specificity and sensitivity of the array adequate for your application. Here, we describe the use of a custom 12K CombiMatrix ElectraSense™ oligonucleotide microarray format for assessing global gene expression profiles in Neisseria spp. PMID:22782831

  3. Identifying protein interactions with metal-modified DNA using microarray technology.

    PubMed

    Stansfield, Hope E; Kulczewski, Bethany P; Lybrand, Kyle E; Jamieson, Elizabeth R

    2009-02-01

    Protein microarrays have been used extensively to identify protein-protein interactions; however, this technology has not been widely applied to protein-DNA interactions. In particular, this work demonstrates the utility of this technique for rapidly identifying interactions of proteins with metal-modified DNA. Protein macroarray experiments were carried out with high mobility group protein 1 (HMG-1) and cisplatin- and chromium-modified 50-mer oligonucleotides to demonstrate "proof of principle." Commercially available protein microarrays containing many different classes of human proteins were then employed to search for additional interactions with cisplatin-modified DNA. The results of the microarray experiments confirmed some known interactions and, more importantly, identified many novel protein interactions, demonstrating the utility of this method as a rapid, high-throughput technique to discover proteins that interact with metal-modified DNA. PMID:18936984

  4. A comparative analysis of DNA barcode microarray feature size

    PubMed Central

    Ammar, Ron; Smith, Andrew M; Heisler, Lawrence E; Giaever, Guri; Nislow, Corey

    2009-01-01

    Background Microarrays are an invaluable tool in many modern genomic studies. It is generally perceived that decreasing the size of microarray features leads to arrays with higher resolution (due to greater feature density), but this increase in resolution can compromise sensitivity. Results We demonstrate that barcode microarrays with smaller features are equally capable of detecting variation in DNA barcode intensity when compared to larger feature sizes within a specific microarray platform. The barcodes used in this study are the well-characterized set derived from the Yeast KnockOut (YKO) collection used for screens of pooled yeast (Saccharomyces cerevisiae) deletion mutants. We treated these pools with the glycosylation inhibitor tunicamycin as a test compound. Three generations of barcode microarrays at 30, 8 and 5 μm features sizes independently identified the primary target of tunicamycin to be ALG7. Conclusion We show that the data obtained with 5 μm feature size is of comparable quality to the 30 μm size and propose that further shrinking of features could yield barcode microarrays with equal or greater resolving power and, more importantly, higher density. PMID:19825181

  5. The Genopolis Microarray Database

    PubMed Central

    Splendiani, Andrea; Brandizi, Marco; Even, Gael; Beretta, Ottavio; Pavelka, Norman; Pelizzola, Mattia; Mayhaus, Manuel; Foti, Maria; Mauri, Giancarlo; Ricciardi-Castagnoli, Paola

    2007-01-01

    Background Gene expression databases are key resources for microarray data management and analysis and the importance of a proper annotation of their content is well understood. Public repositories as well as microarray database systems that can be implemented by single laboratories exist. However, there is not yet a tool that can easily support a collaborative environment where different users with different rights of access to data can interact to define a common highly coherent content. The scope of the Genopolis database is to provide a resource that allows different groups performing microarray experiments related to a common subject to create a common coherent knowledge base and to analyse it. The Genopolis database has been implemented as a dedicated system for the scientific community studying dendritic and macrophage cells functions and host-parasite interactions. Results The Genopolis Database system allows the community to build an object based MIAME compliant annotation of their experiments and to store images, raw and processed data from the Affymetrix GeneChip® platform. It supports dynamical definition of controlled vocabularies and provides automated and supervised steps to control the coherence of data and annotations. It allows a precise control of the visibility of the database content to different sub groups in the community and facilitates exports of its content to public repositories. It provides an interactive users interface for data analysis: this allows users to visualize data matrices based on functional lists and sample characterization, and to navigate to other data matrices defined by similarity of expression values as well as functional characterizations of genes involved. A collaborative environment is also provided for the definition and sharing of functional annotation by users. Conclusion The Genopolis Database supports a community in building a common coherent knowledge base and analyse it. This fills a gap between a local

  6. [The use of oligonucleotide aptamers in cancer therapy].

    PubMed

    Odrzywolski, Adrian; Waśko, Adam

    2016-01-01

    Aptamers are a new class of molecules which originated in the 1990s. They are usually RNA or DNA oligonucleotides, the length of which ranges between 20 and 80 nt. They are produced using the SELEX method that allows one to obtain aptamers that bind to virtually any molecule of interest, providing a high specificity. Aptamers are an alternative to antibodies because on the one hand, their sensitivity is at a similar or sometimes even higher level, while on the other hand they do not show immunogenicity, and may be synthesized in vitro. To date, a broad range of different applications of aptamers has been described: as components of biosensors, or use in various laboratory techniques, such as microarrays or chromatography. One of the most important is the use of aptamers in medicine, especially in the fight against cancer. They can be used both for diagnosis and for the eradication of cancers - particularly through the delivery of drugs. Currently, most transport-related research is devoted to the delivery of chemotherapeutic drugs, such as doxorubicin. This was used in research on liver cancer cells, prostate, and acute lymphoblastic leukemia blast cells. Another possibility is to use aptamers to deliver siRNAs. In this way inhibition of the quality control process of the mRNA in tumor cells is possible. An aptamer complex with the drug allows for direct delivery of the active substance in a particular cell type, substantially eliminating the non-specific effect of the drug. PMID:27180958

  7. DNA Microarray-Based Diagnostics.

    PubMed

    Marzancola, Mahsa Gharibi; Sedighi, Abootaleb; Li, Paul C H

    2016-01-01

    The DNA microarray technology is currently a useful biomedical tool which has been developed for a variety of diagnostic applications. However, the development pathway has not been smooth and the technology has faced some challenges. The reliability of the microarray data and also the clinical utility of the results in the early days were criticized. These criticisms added to the severe competition from other techniques, such as next-generation sequencing (NGS), impacting the growth of microarray-based tests in the molecular diagnostic market.Thanks to the advances in the underlying technologies as well as the tremendous effort offered by the research community and commercial vendors, these challenges have mostly been addressed. Nowadays, the microarray platform has achieved sufficient standardization and method validation as well as efficient probe printing, liquid handling and signal visualization. Integration of various steps of the microarray assay into a harmonized and miniaturized handheld lab-on-a-chip (LOC) device has been a goal for the microarray community. In this respect, notable progress has been achieved in coupling the DNA microarray with the liquid manipulation microsystem as well as the supporting subsystem that will generate the stand-alone LOC device.In this chapter, we discuss the major challenges that microarray technology has faced in its almost two decades of development and also describe the solutions to overcome the challenges. In addition, we review the advancements of the technology, especially the progress toward developing the LOC devices for DNA diagnostic applications. PMID:26614075

  8. Development and validation of a 2,000-gene microarray for the fathead minnow (Pimephales promelas)

    SciTech Connect

    Larkin, Patrick; Villeneuve, Daniel L.; Knoebl, Iris; Miracle, Ann L.; Carter, Barbara J.; Liu, Li; Denslow, Nancy D.; Ankley, Gerald T.

    2007-07-01

    Gene microarrays provide the field of ecotoxicology new tools to identify mechanisms of action of chemicals and chemical mixtures. Herein we describe the development and application of a 2,000-gene oligonucleotide microarray for the fathead minnow Pimephales promelas, a species commonly used in ecological risk assessments in North America. The microarrays were developed from various cDNA and subtraction libraries that we constructed. Consistency and reproducibility of the microarrays were documented by examining multiple technical replicates. To test application of the fathead minnow microarrays, gene expression profiles of fish exposed to 17-estradiol, a well-characterized estrogen receptor (ER) agonist, were examined. For these experiments, adult male fathead minnows were exposed for 24 h to waterborne 17-estradiol (40 or 100 ng/L) in a flow-through system, and gene expression in liver samples was characterized. Seventy-one genes were identified as differentially regulated by estradiol exposure. Examination of the gene ontology designations of these genes revealed patterns consistent with estradiol’s expected mechanisms of action and also provided novel insights as to molecular effects of the estrogen. Our studies indicate the feasibility and utility of microarrays as a basis for understanding biological responses to chemical exposure in a model ecotoxicology test species.

  9. Oligonucleotide-based therapy for neurodegenerative diseases.

    PubMed

    Magen, Iddo; Hornstein, Eran

    2014-10-10

    Molecular genetics insight into the pathogenesis of several neurodegenerative diseases, such as Alzheimer׳s disease, Parkinson׳s disease, Huntington׳s disease and amyotrophic lateral sclerosis, encourages direct interference with the activity of neurotoxic genes or the molecular activation of neuroprotective pathways. Oligonucleotide-based therapies are recently emerging as an efficient strategy for drug development and these can be employed as new treatments of neurodegenerative states. Here we review advances in this field in recent years which suggest an encouraging assessment that oligonucleotide technologies for targeting of RNAs will enable the development of new therapies and will contribute to preservation of brain integrity. PMID:24727531

  10. Tiling Microarray Analysis Tools

    Energy Science and Technology Software Center (ESTSC)

    2005-05-04

    TiMAT is a package of 23 command line Java applications for use in the analysis of Affymetrix tiled genomic microarray data. TiMAT enables: 1) Rebuilding the genome annotation for entire tiled arrays (repeat filtering, chromosomal coordinate assignment). 2) Post processing of oligo intensity values (quantile normalization, median scaling, PMMM transformation), 3) Significance testing (Wilcoxon rank sum and signed rank tests, intensity difference and ratio tests) and Interval refinement (filtering based on multiple statistics, overlap comparisons),more » 4) Data visualization (detailed thumbnail/zoomed view with Interval Plots and data export to Affymetrix's Integrated Genome Browser) and Data reports (spreadsheet summaries and detailed profiles)« less