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Sample records for density oligonucleotide microarray

  1. Gene expression profiling in peanut using high density oligonucleotide microarrays

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  2. Development of a 37K high-density oligo-nucleotide microarray for rainbow trout

    USDA-ARS?s Scientific Manuscript database

    We have constructed a rainbow trout high-density oligonucleotide microarray by using all the available tentative consensus (TC) sequences from the Rainbow Trout Gene Index database (The Computational Biology and Functional Genomics Lab., Dana Farber Cancer Institute and Harvard School of Public Heal...

  3. A new approach for filtering noise from high-density oligonucleotide microarray datasets

    PubMed Central

    Mills, Jason C.; Gordon, Jeffrey I.

    2001-01-01

    Although DNA microarrays are powerful tools for profiling gene expression, the dynamic range and the sheer number of signals produced require efficient procedures for distinguishing false positive results (noise) from changes in expression that are ‘real’ (independently reproducible). We have developed an approach to filter noise from datasets generated when high density oligonucleotide-based microarrays are used to compare two distinct RNA populations. First, we performed comparisons between chips hybridized with cRNAs prepared from an identical starting RNA population; an ‘Increase’ or ‘Decrease’ call in such a comparison was defined as a false positive. Plotting the average distribution of these false positive signal intensities across 18 such comparisons of nine independent RNA preparations allowed us to develop a series of noise-filtering look-up tables (LUTs). Using a database of 70 separate chip-to-chip comparisons between distinct RNA preparations prepared by different workers at different sites and at different times, we show that the LUTs can be used to predict the likelihood that a given transcript called Increased or Decreased in one comparison will again be called Increased or Decreased in a replicate comparison. Evidence is presented that this LUT-based scoring system provides greater predictive value for reproducible microarray results than imposition of arbitrary fold-change thresholds and accurately predicts which microarray-identified changes will be validated by independent assays such as quantitative real-time PCR. PMID:11470887

  4. MIMAS: an innovative tool for network-based high density oligonucleotide microarray data management and annotation

    PubMed Central

    Hermida, Leandro; Schaad, Olivier; Demougin, Philippe; Descombes, Patrick; Primig, Michael

    2006-01-01

    Background The high-density oligonucleotide microarray (GeneChip) is an important tool for molecular biological research aiming at large-scale detection of small nucleotide polymorphisms in DNA and genome-wide analysis of mRNA concentrations. Local array data management solutions are instrumental for efficient processing of the results and for subsequent uploading of data and annotations to a global certified data repository at the EBI (ArrayExpress) or the NCBI (GeneOmnibus). Description To facilitate and accelerate annotation of high-throughput expression profiling experiments, the Microarray Information Management and Annotation System (MIMAS) was developed. The system is fully compliant with the Minimal Information About a Microarray Experiment (MIAME) convention. MIMAS provides life scientists with a highly flexible and focused GeneChip data storage and annotation platform essential for subsequent analysis and interpretation of experimental results with clustering and mining tools. The system software can be downloaded for academic use upon request. Conclusion MIMAS implements a novel concept for nation-wide GeneChip data management whereby a network of facilities is centered on one data node directly connected to the European certified public microarray data repository located at the EBI. The solution proposed may serve as a prototype approach to array data management between research institutes organized in a consortium. PMID:16597336

  5. 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.

  6. 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.

  7. 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.

  8. 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). PMID:27605027

  9. ADOA3R as a therapeutic target in experimental colitis: proof by validated high-density oligonucleotide microarray analysis.

    PubMed

    Guzman, Jorge; Yu, Jun Ge; Suntres, Zacharias; Bozarov, Andrey; Cooke, Helen; Javed, Najma; Auer, Herbert; Palatini, Jeff; Hassanain, Hamdy H; Cardounel, Arturo J; Javed, Asad; Grants, Iveta; Wunderlich, Jacqueline E; Christofi, Fievos L

    2006-08-01

    Adenosine A3 receptors (ADOA3Rs) are emerging as novel purinergic targets for treatment of inflammatory diseases. Our goal was to assess the protective effect of the ADOA3R agonist N(6)-(3-iodobenzyl)-adenosine-5-N-methyluronamide (IB-MECA) on gene dysregulation and injury in a rat chronic model of 2,4,6-trinitrobenzene sulfonic acid (TNBS)--induced colitis. It was necessary to develop and validate a microarray technique for testing the protective effects of purine-based drugs in experimental inflammatory bowel disease. High-density oligonucleotide microarray analysis of gene dysregulation was assessed in colons from normal, TNBS-treated (7 days), and oral IB-MECA-treated rats (1.5 mg/kg b.i.d.) using a rat RNU34 neural GeneChip of 724 genes and SYBR green polymerase chain reaction. Analysis included clinical evaluation, weight loss assessment, and electron paramagnetic resonance imaging/spin-trap monitoring of free radicals. Remarkable colitis-induced gene dysregulation occurs in the most exceptional cluster of 5.4% of the gene pool, revealing 2 modes of colitis-related dysregulation. Downregulation occurs in membrane transporter, mitogen-activated protein (MAP) kinase, and channel genes. Upregulation occurs in chemokine, cytokine/inflammatory, stress, growth factor, intracellular signaling, receptor, heat shock protein, retinoid metabolism, neural, remodeling, and redox-sensitive genes. Oral IB-MECA prevented dysregulation in 92% of these genes, histopathology, gut injury, and weight loss. IB-MECA or adenosine suppressed elevated free radicals in ex vivo inflamed gut. Oral IB-MECA blocked the colitis-induced upregulation (90% of genes tested (33 of 37 genes). We conclude that our validated high-density

  10. Mutational analysis using oligonucleotide microarrays

    PubMed Central

    Hacia, J.; Collins, F.

    1999-01-01

    The development of inexpensive high throughput methods to identify individual DNA sequence differences is important to the future growth of medical genetics. This has become increasingly apparent as epidemiologists, pathologists, and clinical geneticists focus more attention on the molecular basis of complex multifactorial diseases. Such undertakings will rely upon genetic maps based upon newly discovered, common, single nucleotide polymorphisms. Furthermore, candidate gene approaches used in identifying disease associated genes necessitate screening large sequence blocks for changes tracking with the disease state. Even after such genes are isolated, large scale mutational analyses will often be needed for risk assessment studies to define the likely medical consequences of carrying a mutated gene.
This review concentrates on the use of oligonucleotide arrays for hybridisation based comparative sequence analysis. Technological advances within the past decade have made it possible to apply this technology to many different aspects of medical genetics. These applications range from the detection and scoring of single nucleotide polymorphisms to mutational analysis of large genes. Although we discuss published scientific reports, unpublished work from the private sector12 could also significantly affect the future of this technology.


Keywords: mutational analysis; oligonucleotide microarrays; DNA chips PMID:10528850

  11. Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis.

    PubMed

    Hehir-Kwa, Jayne Y; Egmont-Petersen, Michael; Janssen, Irene M; Smeets, Dominique; van Kessel, Ad Geurts; Veltman, Joris A

    2007-02-28

    Recently, comparative genomic hybridization onto bacterial artificial chromosome (BAC) arrays (array-based comparative genomic hybridization) has proved to be successful for the detection of submicroscopic DNA copy-number variations in health and disease. Technological improvements to achieve a higher resolution have resulted in the generation of additional microarray platforms encompassing larger numbers of shorter DNA targets (oligonucleotides). Here, we present a novel method to estimate the ability of a microarray to detect genomic copy-number variations of different sizes and types (i.e. deletions or duplications). We applied our method, which is based on statistical power analysis, to four widely used high-density genomic microarray platforms. By doing so, we found that the high-density oligonucleotide platforms are superior to the BAC platform for the genome-wide detection of copy-number variations smaller than 1 Mb. The capacity to reliably detect single copy-number variations below 100 kb, however, appeared to be limited for all platforms tested. In addition, our analysis revealed an unexpected platform-dependent difference in sensitivity to detect a single copy-number loss and a single copy-number gain. These analyses provide a first objective insight into the true capacities and limitations of different genomic microarrays to detect and define DNA copy-number variations.

  12. Microarray oligonucleotide probe designer (MOPeD): A web service.

    PubMed

    Patel, Viren C; Mondal, Kajari; Shetty, Amol Carl; Horner, Vanessa L; Bedoyan, Jirair K; Martin, Donna; Caspary, Tamara; Cutler, David J; Zwick, Michael E

    2010-11-01

    Methods of genomic selection that combine high-density oligonucleotide microarrays with next-generation DNA sequencing allow investigators to characterize genomic variation in selected portions of complex eukaryotic genomes. Yet choosing which specific oligonucleotides to be use can pose a major technical challenge. To address this issue, we have developed a software package called MOPeD (Microarray Oligonucleotide Probe Designer), which automates the process of designing genomic selection microarrays. This web-based software allows individual investigators to design custom genomic selection microarrays optimized for synthesis with Roche NimbleGen's maskless photolithography. Design parameters include uniqueness of the probe sequences, melting temperature, hairpin formation, and the presence of single nucleotide polymorphisms. We generated probe databases for the human, mouse, and rhesus macaque genomes and conducted experimental validation of MOPeD-designed microarrays in human samples by sequencing the human X chromosome exome, where relevant sequence metrics indicated superior performance relative to a microarray designed by the Roche NimbleGen proprietary algorithm. We also performed validation in the mouse to identify known mutations contained within a 487-kb region from mouse chromosome 16, the mouse chromosome 16 exome (1.7 Mb), and the mouse chromosome 12 exome (3.3 Mb). Our results suggest that the open source MOPeD software package and website (http://moped.genetics.emory.edu/) will make a valuable resource for investigators in their sequence-based studies of complex eukaryotic genomes.

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

    PubMed

    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

    2014-03-01

    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. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  14. 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...

  15. 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...

  16. 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.

  17. Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray

    PubMed Central

    2009-01-01

    Background It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Results Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. Conclusion The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of

  18. 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.

  19. A facile method for the construction of oligonucleotide microarrays.

    PubMed

    Sethi, Dalip; Kumar, A; Gupta, K C; Kumar, P

    2008-11-19

    In recent years, the oligonucleotide-based microarray technique has emerged as a powerful and promising tool for various molecular biological studies. Here, a facile protocol for the construction of an oligonucleotide microarray is demonstrated that involves immobilization of oligonucleotide-trimethoxysilyl conjugates onto virgin glass microslides. The projected immobilization strategy reflects high immobilization efficiency ( approximately 36-40%) and signal-to-noise ratio ( approximately 98), and hybridization efficiency ( approximately 32-35%). Using the proposed protocol, aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides were immobilized onto virgin glass microslides. Briefly, modified oligonucleotides were reacted first with 3-glycidyloxypropyltriethoxysilane (GOPTS), and subsequently, the resultant conjugates were directly immobilized onto the virgin glass surface by making use of silanization chemistry. The constructed microarrays were then used for discrimination of base mismatches. On subjecting to different pH and thermal conditions, the microarray showed sufficient stability. Application of this chemistry to manufacture oligonucleotide probe-based microarrays for detection of bacterial meningitis is demonstrated. Single-step reaction for the formation of conjugates with the commercially available reagent (GOPTS), omission of capping step and surface modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are the key features of the proposed strategy.

  20. New protocol for oligonucleotide microarray fabrication using SU-8-coated glass microslides.

    PubMed

    Sethi, D; Kumar, A; Gandhi, R P; Kumar, P; Gupta, K C

    2010-09-15

    Microarray technology has become an important tool for detection and analysis of nucleic acid targets. Immobilization of modified and unmodified oligonucleotides on epoxy-functionalized glass surfaces is often used in microarray fabrication. Here, we demonstrate a protocol that employs coating of SU-8 (glycidyl ether of bisphenol A) onto glass microslides to obtain high density of epoxy functions for efficient immobilization of aminoalkyl-, thiophosphoryl-, and phosphorylated oligonucleotides with uniform spot morphology. The resulting microarrays exhibited high immobilization (∼65%) and hybridization efficiency (30-36%) and were sufficiently stable over a range of temperature and pH conditions. The prominent feature of the protocol is that spots can be visualized distinctly at 0.05 μM probe (a 20-mer oligonucleotide) concentration. The constructed microarrays were subsequently used for detection of base mismatches and bacterial diseases (meningitis and typhoid).

  1. Validation of the Swine Protein-Annotated Oligonucleotide Microarray

    USDA-ARS?s Scientific Manuscript database

    The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed i...

  2. Gene expression profiling in peanut using oligonucleotide microarrays

    USDA-ARS?s Scientific Manuscript database

    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...

  3. H5 avian influenza virus pathotyping using oligonucleotide microarray.

    PubMed

    Wang, Lih-Chiann; Huang, Dean; Cheng, Ming-Chu; Lee, Shu-Hwae; Wang, Ching-Ho

    2015-08-01

    The H5 avian influenza virus subtype has huge impact on the poultry industry. Rapid diagnosis and accurate identification of the highly pathogenic avian influenza virus and low-pathogenicity avian influenza virus is essential, especially during H5 outbreaks and surveillance. To this end, a novel and rapid strategy for H5 virus molecular pathotyping is presented. The specific hemagglutinin gene of the H5 virus and the basic amino acid number of the motif at the hemagglutinin precursor protein cleavage site were detected using oligonucleotide microarray. Highly pathogenic and low-pathogenicity avian influenza viruses in Taiwan were differentiated using 13 microarray probes with the naked eye. The detection limit reached 3.4 viral RNA copies, 1000 times more sensitive than reverse transcription polymerase chain reaction. Thus, the oligonucleotide microarray would provide an alternative H5 pathogenicity determination using the naked eye for laboratories lacking facilities. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Multipathogen oligonucleotide microarray for environmental and biodefense applications.

    PubMed

    Sergeev, Nikolay; Distler, Margaret; Courtney, Shannon; Al-Khaldi, Sufian F; Volokhov, Dmitriy; Chizhikov, Vladimir; Rasooly, Avraham

    2004-11-01

    Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.

  5. New oligonucleotide microarray for rapid diagnosis of avian viral diseases.

    PubMed

    Sultankulova, Kulyaisan T; Kozhabergenov, Nurlan S; Strochkov, Vitaliy M; Burashev, Yerbol D; Shorayeva, Kamshat A; Chervyakova, Olga V; Rametov, Nurkuisa M; Sandybayev, Nurlan T; Sansyzbay, Abylay R; Orynbayev, Mukhit B

    2017-04-05

    We developed a new oligonucleotide microarray comprising 16 identical subarrays for simultaneous rapid detection of avian viruses: avian influenza virus (AIV), Newcastle disease virus (NDV), infection bronchitis virus (IBV), and infectious bursal disease virus (IBDV) in single- and mixed-virus infections. The objective of the study was to develop an oligonucleotide microarray for rapid diagnosis of avian diseases that would be used in the course of mass analysis for routine epidemiological surveillance owing to its ability to test one specimen for several infections. The paper describes the technique for rapid and simultaneous diagnosis of avian diseases such as avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease with use of oligonucleotide microarray, conditions for hybridization of fluorescent-labelled viral cDNA on the microarray and its specificity tested with use of AIV, NDV, IBV, IBDV strains as well as biomaterials from poultry. Sensitivity and specificity of the developed microarray was evaluated with use of 122 specimens of biological material: 44 cloacal swabs from sick birds and 78 tissue specimens from dead wild and domestic birds, as well as with use of 15 AIV, NDV, IBV and IBDV strains, different in their origin, epidemiological and biological characteristics (RIBSP Microbial Collection). This microarray demonstrates high diagnostic sensitivity (99.16% within 95% CI limits 97.36-100%) and specificity (100%). Specificity of the developed technique was confirmed by direct sequencing of NP and M (AIV), VP2 (IBDV), S1 (IBV), NP (NDV) gene fragments. Diagnostic effectiveness of the developed DNA microarray is 99.18% and therefore it can be used in mass survey for specific detection of AIV, NDV, IBV and IBDV circulating in the region in the course of epidemiological surveillance. Rather simple method for rapid diagnosis of avian viral diseases that several times shortens duration of assay versus classical diagnostic

  6. Sex determination of bovine preimplantation embryos by oligonucleotide microarray.

    PubMed

    Yang, Hua; Zhong, Fagang; Yang, Yonglin; Wang, Xinhua; Liu, Shouren; Zhu, Bin

    2013-06-01

    The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and β-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs.

  7. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    PubMed

    Mulle, Jennifer G; Patel, Viren C; Warren, Stephen T; Hegde, Madhuri R; Cutler, David J; Zwick, Michael E

    2010-03-29

    DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  8. Transformation and normalization of oligonucleotide microarray data.

    PubMed

    Geller, Sue C; Gregg, Jeff P; Hagerman, Paul; Rocke, David M

    2003-09-22

    Most methods of analyzing microarray data or doing power calculations have an underlying assumption of constant variance across all levels of gene expression. The most common transformation, the logarithm, results in data that have constant variance at high levels but not at low levels. Rocke and Durbin showed that data from spotted arrays fit a two-component model and Durbin, Hardin, Hawkins, and Rocke, Huber et al. and Munson provided a transformation that stabilizes the variance as well as symmetrizes and normalizes the error structure. We wish to evaluate the applicability of this transformation to the error structure of GeneChip microarrays. We demonstrate in an example study a simple way to use the two-component model of Rocke and Durbin and the data transformation of Durbin, Hardin, Hawkins and Rocke, Huber et al. and Munson on Affymetrix GeneChip data. In addition we provide a method for normalization of Affymetrix GeneChips simultaneous with the determination of the transformation, producing a data set without chip or slide effects but with constant variance and with symmetric errors. This transformation/normalization process can be thought of as a machine calibration in that it requires a few biologically constant replicates of one sample to determine the constant needed to specify the transformation and normalize. It is hypothesized that this constant needs to be found only once for a given technology in a lab, perhaps with periodic updates. It does not require extensive replication in each study. Furthermore, the variance of the transformed pilot data can be used to do power calculations using standard power analysis programs. SPLUS code for the transformation/normalization for four replicates is available from the first author upon request. A program written in C is available from the last author.

  9. Linear model for fast background subtraction in oligonucleotide microarrays

    PubMed Central

    2009-01-01

    Background One important preprocessing step in the analysis of microarray data is background subtraction. In high-density oligonucleotide arrays this is recognized as a crucial step for the global performance of the data analysis from raw intensities to expression values. Results We propose here an algorithm for background estimation based on a model in which the cost function is quadratic in a set of fitting parameters such that minimization can be performed through linear algebra. The model incorporates two effects: 1) Correlated intensities between neighboring features in the chip and 2) sequence-dependent affinities for non-specific hybridization fitted by an extended nearest-neighbor model. Conclusion The algorithm has been tested on 360 GeneChips from publicly available data of recent expression experiments. The algorithm is fast and accurate. Strong correlations between the fitted values for different experiments as well as between the free-energy parameters and their counterparts in aqueous solution indicate that the model captures a significant part of the underlying physical chemistry. PMID:19917117

  10. Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray.

    PubMed

    Wang, Lih-Chiann; Huang, Dean; Pu, Chang-En; Wang, Ching-Ho

    2014-12-15

    Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Oligonucleotide microarray for the identification of potential mycotoxigenic fungi

    PubMed Central

    2010-01-01

    Background Mycotoxins are secondary metabolites which are produced by numerous fungi and pose a continuous challenge to the safety and quality of food commodities in South Africa. These toxins have toxicologically relevant effects on humans and animals that eat contaminated foods. In this study, a diagnostic DNA microarray was developed for the identification of the most common food-borne fungi, as well as the genes leading to toxin production. Results A total of 40 potentially mycotoxigenic fungi isolated from different food commodities, as well as the genes that are involved in the mycotoxin synthetic pathways, were analyzed. For fungal identification, oligonucleotide probes were designed by exploiting the sequence variations of the elongation factor 1-alpha (EF-1 α) coding regions and the internal transcribed spacer (ITS) regions of the rRNA gene cassette. For the detection of fungi able to produce mycotoxins, oligonucleotide probes directed towards genes leading to toxin production from different fungal strains were identified in data available in the public domain. The probes selected for fungal identification and the probes specific for toxin producing genes were spotted onto microarray slides. Conclusions The diagnostic microarray developed can be used to identify single pure strains or cultures of potentially mycotoxigenic fungi as well as genes leading to toxin production in both laboratory samples and maize-derived foods offering an interesting potential for microbiological laboratories. PMID:20307326

  12. 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.

  13. Differentiation of the seven major lyssavirus species by oligonucleotide microarray.

    PubMed

    Xi, Jin; Guo, Huancheng; Feng, Ye; Xu, Yunbin; Shao, Mingfu; Su, Nan; Wan, Jiayu; Li, Jiping; Tu, Changchun

    2012-03-01

    An oligonucleotide microarray, LyssaChip, has been developed and verified as a highly specific diagnostic tool for differentiation of the 7 major lyssavirus species. As with conventional typing microarray methods, the LyssaChip relies on sequence differences in the 371-nucleotide region coding for the nucleoprotein. This region was amplified using nested reverse transcription-PCR primers that bind to the 7 major lyssaviruses. The LyssaChip includes 57 pairs of species typing and corresponding control oligonucleotide probes (oligoprobes) immobilized on glass slides, and it can analyze 12 samples on a single slide within 8 h. Analysis of 111 clinical brain specimens (65 from animals with suspected rabies submitted to the laboratory and 46 of butchered dog brain tissues collected from restaurants) showed that the chip method was 100% sensitive and highly consistent with the "gold standard," a fluorescent antibody test (FAT). The chip method could detect rabies virus in highly decayed brain tissues, whereas the FAT did not, and therefore the chip test may be more applicable to highly decayed brain tissues than the FAT. LyssaChip may provide a convenient and inexpensive alternative for diagnosis and differentiation of rabies and rabies-related diseases.

  14. 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.

  15. Detection of genomic deletions in rice using oligonucleotide microarrays

    PubMed Central

    Bruce, Myron; Hess, Ann; Bai, Jianfa; Mauleon, Ramil; Diaz, M Genaleen; Sugiyama, Nobuko; Bordeos, Alicia; Wang, Guo-Liang; Leung, Hei; Leach, Jan E

    2009-01-01

    Background The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL). However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. Results We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip® allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to ~500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations . Conclusion We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a database saturated with deletions across the rice genome. This community resource can continue

  16. Comparison of gene coverage of mouse oligonucleotide microarray platforms

    PubMed Central

    Verdugo, Ricardo A; Medrano, Juan F

    2006-01-01

    Background The increasing use of DNA microarrays for genetical genomics studies generates a need for platforms with complete coverage of the genome. We have compared the effective gene coverage in the mouse genome of different commercial and noncommercial oligonucleotide microarray platforms by performing an in-house gene annotation of probes. We only used information about probes that is available from vendors and followed a process that any researcher may take to find the gene targeted by a given probe. In order to make consistent comparisons between platforms, probes in each microarray were annotated with an Entrez Gene id and the chromosomal position for each gene was obtained from the UCSC Genome Browser Database. Gene coverage was estimated as the percentage of Entrez Genes with a unique position in the UCSC Genome database that is tested by a given microarray platform. Results A MySQL relational database was created to store the mapping information for 25,416 mouse genes and for the probes in five microarray platforms (gene coverage level in parenthesis): Affymetrix430 2.0 (75.6%), ABI Genome Survey (81.24%), Agilent (79.33%), Codelink (78.09%), Sentrix (90.47%); and four array-ready oligosets: Sigma (47.95%), Operon v.3 (69.89%), Operon v.4 (84.03%), and MEEBO (84.03%). The differences in coverage between platforms were highly conserved across chromosomes. Differences in the number of redundant and unspecific probes were also found among arrays. The database can be queried to compare specific genomic regions using a web interface. The software used to create, update and query the database is freely available as a toolbox named ArrayGene. Conclusion The software developed here allows researchers to create updated custom databases by using public or proprietary information on genes for any organisms. ArrayGene allows easy comparisons of gene coverage between microarray platforms for any region of the genome. The comparison presented here reveals that the

  17. A diagnostic oligonucleotide microarray for simultaneous detection of grapevine viruses.

    PubMed

    Engel, Esteban A; Escobar, Paula F; Rojas, Luis A; Rivera, Paulina A; Fiore, Nicola; Valenzuela, Pablo D T

    2010-02-01

    At least 58 viruses have been reported to infect grapevines causing economic damage globally. Conventional detection strategies based on serological assays, biological indexing and RT-PCR targeting one or few viruses in each assay are widely used. Grapevines are prone to contain mixed infections of several viruses, making the use of these techniques time-consuming. A 70-mer oligonucleotide microarray able to detect simultaneously a broad spectrum of known viruses as well as new viruses by cross-hybridization to highly conserved probes is reported in the present study. The array contains 570 unique probes designed against highly conserved and species-specific regions of 44 plant viral genomes. In addition probes designed against plant housekeeping genes are also included. By using a random primed RT-PCR amplification strategy of grapevine double stranded RNA-enriched samples, viral agents were detected in single and mixed infections. The microarray accuracy to detect 10 grapevine viruses was compared with RT-PCR yielding consistent results. For this purpose, grapevine samples containing single or mixed infections of Grapevine leafroll-associated virus-1, -2, -3, -4, -7, -9, Grapevine fanleaf virus, Grapevine rupestris stem pitting-associated virus, Grapevine virus A, and Grapevine virus B were used. Genomic libraries containing complete viral genomes were also used as part of the validation process. The specific probe hybridization pattern obtained from each virus makes this approach a powerful tool for high throughput plant certification purposes and also for virus discovery if the new viral genomic sequences have partial similarity with the microarray probes. Three Closteroviridae members (Grapevine leafroll-associated virus -4, -7 and -9) were detected for the first time in Chilean grapevines using the microarray. 2009 Elsevier B.V. All rights reserved.

  18. Rapid large-scale oligonucleotide selection for microarrays.

    PubMed

    Rahmann, Sven

    2002-01-01

    We present the first algorithm that selects oligonucleotide probes (e.g. 25-mers) for microarray experiments on a large scale. For example, oligos for human genes can be found within 50 hours. This becomes possible by using the longest common substring as a specificity measure for candidate oligos. We present an algorithm based on a suffix array with additional information that is efficient both in terms of memory usage and running time to rank all candidate oligos according to their specificity. We also introduce the concept of master sequences to describe the sequences from which oligos are to be selected. Constraints such as oligo length, melting temperature, and self-complementarity are incorporated in the master sequence at a preprocessing stage and thus kept separate from the main selection problem. As a result, custom oligos can now be designed for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.

  19. Electrochemically directed synthesis of oligonucleotides for DNA microarray fabrication

    PubMed Central

    Egeland, Ryan D.; Southern, Edwin M.

    2005-01-01

    We demonstrate a new method for making oligonucleotide microarrays by synthesis in situ. The method uses conventional DNA synthesis chemistry with an electrochemical deblocking step. Acid is delivered to specific regions on a glass slide, thus allowing nucleotide addition only at chosen sites. The acid is produced by electrochemical oxidation controlled by an array of independent microelectrodes. Deblocking is complete in a few seconds, when competing side-product reactions are minimal. We demonstrate the successful synthesis of 17mers and discrimination of single base pair mismatched hybrids. Features generated in this study are 40 μm wide, with sharply defined edges. The synthetic technique may be applicable to fabrication of other molecular arrays. PMID:16085751

  20. 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

  1. 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.

  2. Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria.

    PubMed

    Weber, D G; Sahm, K; Polen, T; Wendisch, V F; Antranikian, G

    2008-10-01

    The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

  3. Comparison of nucleic acid targets prepared from total RNA or poly(A) RNA for DNA oligonucleotide microarray hybridization.

    PubMed

    Petersen, Kjell; Oyan, Anne Margrete; Rostad, Kari; Olsen, Sue; Bø, Trond Hellem; Salvesen, Helga B; Gjertsen, Bjørn Tore; Bruserud, Oystein; Halvorsen, Ole Johan; Akslen, Lars Andreas; Steen, Vidar M; Jonassen, Inge; Kalland, Karl-Henning

    2007-07-01

    The aim of this work was to compare DNA microarray results using either total RNA or affinity-purified poly(A) RNA from the same biological sample for target preparation. The high-density oligonucleotide microarrays of both Agilent Technologies (based on two-color detection) and Applied Biosystems (based on single-color detection) were evaluated. Real-time quantitative PCR was used to quantify messenger RNA (mRNA) and ribosomal RNA (rRNA) at different stages of target preparations. Poly(A) RNA versus total RNA target hybridizations exhibited slightly lower correlation coefficients than did self versus self hybridizations (i.e., poly(A) RNA targets vs. poly(A) RNA targets or total RNA targets vs. total RNA targets). Only a small fraction of all transcripts appeared to be significantly over- or underrepresented when total RNA targets or poly(A) RNA targets from the same biological sample were compared. Therefore, the conclusion is that poly(A) affinity purification from total RNA can be omitted during target preparation for routine mRNA expression analysis using high-density oligonucleotide microarrays. Among consistently overrepresented transcripts in total RNA targets were histone mRNAs known to lack poly(A) tails. Therefore, structurally exceptional RNA species can be identified by comparing targets derived from either poly(A) RNA or total RNA using microarray hybridization.

  4. 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

  5. Application of oligonucleotide microarrays for bacterial source tracking of environmental Enterococcus sp. isolates.

    PubMed

    Indest, Karl J; Betts, Kelley; Furey, John S

    2005-04-01

    In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST) of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM) classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

  6. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    PubMed Central

    Indest, Karl J.; Betts, Kelley; Furey, John S.

    2005-01-01

    In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST) of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA) and Hierarchical Cluster Analysis (HCA) were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM) classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN. PMID:16705816

  7. Direct detection of Kitasatospora species with a chaperone oligonucleotide microarray method lacking PCR amplification.

    PubMed

    Guenther, Sebastian; Groth, Ingrid; Schierack, Peter; Grabley, Susanne; Munder, Thomas

    2008-08-01

    Identification of members of the genus Kitasatospora from soil samples has been introduced to evaluate occurrence of potential natural compound producers in different habitats. The microarray hybridization usually involves PCR amplification of the target DNA. Since PCR might lead to biased amplification, a diagnostic Kitasatospora microarray technique was improved by a protocol lacking PCR amplification prior to hybridization. The described advanced hybridization method used chaperone oligonucleotides for direct co-hybridization with genomic DNA on an oligonuclotide microarray with optical readout. (c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer.

    PubMed

    Hughes, T R; Mao, M; Jones, A R; Burchard, J; Marton, M J; Shannon, K W; Lefkowitz, S M; Ziman, M; Schelter, J M; Meyer, M R; Kobayashi, S; Davis, C; Dai, H; He, Y D; Stephaniants, S B; Cavet, G; Walker, W L; West, A; Coffey, E; Shoemaker, D D; Stoughton, R; Blanchard, A P; Friend, S H; Linsley, P S

    2001-04-01

    We describe a flexible system for gene expression profiling using arrays of tens of thousands of oligonucleotides synthesized in situ by an ink-jet printing method employing standard phosphoramidite chemistry. We have characterized the dependence of hybridization specificity and sensitivity on parameters including oligonucleotide length, hybridization stringency, sequence identity, sample abundance, and sample preparation method. We find that 60-mer oligonucleotides reliably detect transcript ratios at one copy per cell in complex biological samples, and that ink-jet arrays are compatible with several different sample amplification and labeling techniques. Furthermore, results using only a single carefully selected oligonucleotide per gene correlate closely with those obtained using complementary DNA (cDNA) arrays. Most of the genes for which measurements differ are members of gene families that can only be distinguished by oligonucleotides. Because different oligonucleotide sequences can be specified for each array, we anticipate that ink-jet oligonucleotide array technology will be useful in a wide variety of DNA microarray applications.

  9. 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.

  10. 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.

  11. The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

    PubMed

    Wang, Lih-Chiann; Kuo, Ya-Ting; Chueh, Ling-Ling; Huang, Dean; Lin, Jiunn-Horng

    2017-05-01

    Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control.

  12. Design and validation of an oligonucleotide microarray for the detection of genomic rearrangements associated with common hereditary cancer syndromes.

    PubMed

    Mancini-DiNardo, Debora; Judkins, Thaddeus; Woolstenhulme, Nick; Burton, Collin; Schoenberger, Jeremy; Ryder, Matthew; Murray, Adam; Gutin, Natalia; Theisen, Aaron; Holladay, Jayson; Craft, Jonathan; Arnell, Christopher; Moyes, Kelsey; Roa, Benjamin

    2014-09-11

    Conventional Sanger sequencing reliably detects the majority of genetic mutations associated with hereditary cancers, such as single-base changes and small insertions or deletions. However, detection of genomic rearrangements, such as large deletions and duplications, requires special technologies. Microarray analysis has been successfully used to detect large rearrangements (LRs) in genetic disorders. We designed and validated a high-density oligonucleotide microarray for the detection of gene-level genomic rearrangements associated with hereditary breast and ovarian cancer (HBOC), Lynch, and polyposis syndromes. The microarray consisted of probes corresponding to the exons and flanking introns of BRCA1 and BRCA2 (≈1,700) and Lynch syndrome/polyposis genes MLH1, MSH2, MSH6, APC, MUTYH, and EPCAM (≈2,200). We validated the microarray with 990 samples previously tested for LR status in BRCA1, BRCA2, MLH1, MSH2, MSH6, APC, MUTYH, or EPCAM. Microarray results were 100% concordant with previous results in the validation studies. Subsequently, clinical microarray analysis was performed on samples from patients with a high likelihood of HBOC mutations (13,124), Lynch syndrome mutations (18,498), and polyposis syndrome mutations (2,739) to determine the proportion of LRs. Our results demonstrate that LRs constitute a substantial proportion of genetic mutations found in patients referred for hereditary cancer genetic testing. The use of microarray comparative genomic hybridization (CGH) for the detection of LRs is well-suited as an adjunct technology for both single syndrome (by Sanger sequencing analysis) and extended gene panel testing by next generation sequencing analysis. Genetic testing strategies using microarray analysis will help identify additional patients carrying LRs, who are predisposed to various hereditary cancers.

  13. Improving signal intensities for genes with low-expression on oligonucleotide microarrays

    PubMed Central

    Ramdas, Latha; Cogdell, David E; Jia, Jack Y; Taylor, Ellen E; Dunmire, Valerie R; Hu, Limei; Hamilton, Stanley R; Zhang, Wei

    2004-01-01

    Background DNA microarrays using long oligonucleotide probes are widely used to evaluate gene expression in biological samples. These oligonucleotides are pre-synthesized and sequence-optimized to represent specific genes with minimal cross-hybridization to homologous genes. Probe length and concentration are critical factors for signal sensitivity, particularly when genes with various expression levels are being tested. We evaluated the effects of oligonucleotide probe length and concentration on signal intensity measurements of the expression levels of genes in a target sample. Results Selected genes of various expression levels in a single cell line were hybridized to oligonucleotide arrays of four lengths and four concentrations of probes to determine how these critical parameters affected the intensity of the signal representing their expression. We found that oligonucleotides of longer length significantly increased the signals of genes with low-expression in the target. High-expressing gene signals were also boosted but to a lesser degree. Increasing the probe concentration, however, did not linearly increase the signal intensity for either low- or high-expressing genes. Conclusions We conclude that the longer the oligonuclotide probe the better the signal intensities of low expressing genes on oligonucleotide arrays. PMID:15196312

  14. Development of an oligonucleotide microarray method for Salmonella serotyping

    PubMed Central

    Tankouo‐Sandjong, B.; Sessitsch, A.; Stralis‐Pavese, N.; Liebana, E.; Kornschober, C.; Allerberger, F.; Hächler, H.; Bodrossy, L.

    2008-01-01

    Summary Adequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time‐consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment. PMID:21261872

  15. 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.

  16. Rapid detection of deletional alpha-thalassemia by an oligonucleotide microarray.

    PubMed

    Zesong, Li; Ruijun, Guo; Wen, Zhang

    2005-12-01

    A simple, fast, and highly reproducible protocol for detection of the three most common deletional alpha-thalassemias (- -SEA, - alpha3.7, -alpha4.2) using an oligonucleotide microarray was developed. PCR products were directly hybridized to the microarrays with different deletion-specific probes. Genotypes were determined by quantitative analysis of the fluorescent signals detected by fluorescence scanning. Blind assays on 400 samples validated the efficiency and specificity of the protocol. This method may be suitable for routine clinical use and population screening for deletional alpha-thalassemia. (c) 2005 Wiley-Liss, Inc.

  17. Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays

    PubMed Central

    Jin, Lian-Qun; Li, Jun-Wen; Wang, Sheng-Qi; Chao, Fu-Huan; Wang, Xin-Wei; Yuan, Zheng-Quan

    2005-01-01

    AIM: To detect the common intestinal pathogenic bacteria quickly and accurately. METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays. RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified. CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost. PMID:16437687

  18. 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

  19. Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

    PubMed Central

    Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

    2005-01-01

    The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

  20. A distribution-free convolution model for background correction of oligonucleotide microarray data

    PubMed Central

    Chen, Zhongxue; McGee, Monnie; Liu, Qingzhong; Kong, Megan; Deng, Youping; Scheuermann, Richard H

    2009-01-01

    Introduction Affymetrix GeneChip® high-density oligonucleotide arrays are widely used in biological and medical research because of production reproducibility, which facilitates the comparison of results between experiment runs. In order to obtain high-level classification and cluster analysis that can be trusted, it is important to perform various pre-processing steps on the probe-level data to control for variability in sample processing and array hybridization. Many proposed preprocessing methods are parametric, in that they assume that the background noise generated by microarray data is a random sample from a statistical distribution, typically a normal distribution. The quality of the final results depends on the validity of such assumptions. Results We propose a Distribution Free Convolution Model (DFCM) to circumvent observed deficiencies in meeting and validating distribution assumptions of parametric methods. Knowledge of array structure and the biological function of the probes indicate that the intensities of mismatched (MM) probes that correspond to the smallest perfect match (PM) intensities can be used to estimate the background noise. Specifically, we obtain the smallest q2 percent of the MM intensities that are associated with the lowest q1 percent PM intensities, and use these intensities to estimate background. Conclusion Using the Affymetrix Latin Square spike-in experiments, we show that the background noise generated by microarray experiments typically is not well modeled by a single overall normal distribution. We further show that the signal is not exponentially distributed, as is also commonly assumed. Therefore, DFCM has better sensitivity and specificity, as measured by ROC curves and area under the curve (AUC) than MAS 5.0, RMA, RMA with no background correction (RMA-noBG), GCRMA, PLIER, and dChip (MBEI) for preprocessing of Affymetrix microarray data. These results hold for two spike-in data sets and one real data set that were

  1. The "Clickable" Method for Oligonucleotide Immobilization Onto Azide-Functionalized Microarrays.

    PubMed

    Ratajczak, Tomasz; Uszczyńska, Barbara; Frydrych-Tomczak, Emilia; Chmielewski, Marcin K

    2016-01-01

    The DNA microarray technique was supposed to help identifying and analyzing the expression level of tens of thousands of genes in the whole genome. But there is a serious problem concerning fabrication of the microarrays by chemical synthesis, such as specific and efficient linking of probes to a solid support. Therefore, we reckon that applying "click" chemistry to covalently anchor oligonucleotides on chemically modified supports may help construct microarrays in applications such as gene identification. Silanization of the glass support with organofunctional silane makes it possible to link azide groups on glass surface and the nucleic acid probe that is equipped with a pentynyl group. This is followed by direct spotting of the nucleic acid on the azide-modified glass support in the presence of copper ions, and this is a frequently applied method of "click" chemistry.

  2. Oligonucleotide Microarray for the Study of Functional Gene Diversity in the Nitrogen Cycle in the Environment

    PubMed Central

    Taroncher-Oldenburg, Gaspar; Griner, Erin M.; Francis, Chris A.; Ward, Bess B.

    2003-01-01

    The analysis of functional diversity and its dynamics in the environment is essential for understanding the microbial ecology and biogeochemistry of aquatic systems. Here we describe the development and optimization of a DNA microarray method for the detection and quantification of functional genes in the environment and report on their preliminary application to the study of the denitrification gene nirS in the Choptank River-Chesapeake Bay system. Intergenic and intragenic resolution constraints were determined by an oligonucleotide (70-mer) microarray approach. Complete signal separation was achieved when comparing unrelated genes within the nitrogen cycle (amoA, nifH, nirK, and nirS) and detecting different variants of the same gene, nirK, corresponding to organisms with two different physiological modes, ammonia oxidizers and denitrifying halobenzoate degraders. The limits of intragenic resolution were investigated with a microarray containing 64 nirS sequences comprising 14 cultured organisms and 50 clones obtained from the Choptank River in Maryland. The nirS oligonucleotides covered a range of sequence identities from approximately 40 to 100%. The threshold values for specificity were determined to be 87% sequence identity and a target-to-probe perfect match-to-mismatch binding free-energy ratio of 0.56. The lower detection limit was 10 pg of DNA (equivalent to approximately 107 copies) per target per microarray. Hybridization patterns on the microarray differed between sediment samples from two stations in the Choptank River, implying important differences in the composition of the denitirifer community along an environmental gradient of salinity, inorganic nitrogen, and dissolved organic carbon. This work establishes a useful set of design constraints (independent of the target gene) for the implementation of functional gene microarrays for environmental applications. PMID:12571043

  3. Ultrahigh density microarrays of solid samples.

    PubMed

    LeBaron, Matthew J; Crismon, Heidi R; Utama, Fransiscus E; Neilson, Lynn M; Sultan, Ahmed S; Johnson, Kevin J; Andersson, Eva C; Rui, Hallgeir

    2005-07-01

    We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.

  4. 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

  5. Identification of chromosomal errors in human preimplantation embryos with oligonucleotide DNA microarray.

    PubMed

    Liang, Lifeng; Wang, Cassie T; Sun, Xiaofang; Liu, Lian; Li, Man; Witz, Craig; Williams, Daniel; Griffith, Jason; Skorupski, Josh; Haddad, Ghassan; Gill, Jimmy; Wang, Wei-Hua

    2013-01-01

    A previous study comparing the performance of different platforms for DNA microarray found that the oligonucleotide (oligo) microarray platform containing 385K isothermal probes had the best performance when evaluating dosage sensitivity, precision, specificity, sensitivity and copy number variations border definition. Although oligo microarray platform has been used in some research fields and clinics, it has not been used for aneuploidy screening in human embryos. The present study was designed to use this new microarray platform for preimplantation genetic screening in the human. A total of 383 blastocysts from 72 infertility patients with either advanced maternal age or with previous miscarriage were analyzed after biopsy and microarray. Euploid blastocysts were transferred to patients and clinical pregnancy and implantation rates were measured. Chromosomes in some aneuploid blastocysts were further analyzed by fluorescence in-situ hybridization (FISH) to evaluate accuracy of the results. We found that most (58.1%) of the blastocysts had chromosomal abnormalities that included single or multiple gains and/or losses of chromosome(s), partial chromosome deletions and/or duplications in both euploid and aneuploid embryos. Transfer of normal euploid blastocysts in 34 cycles resulted in 58.8% clinical pregnancy and 54.4% implantation rates. Examination of abnormal blastocysts by FISH showed that all embryos had matching results comparing microarray and FISH analysis. The present study indicates that oligo microarray conducted with a higher resolution and a greater number of probes is able to detect not only aneuploidy, but also minor chromosomal abnormalities, such as partial chromosome deletion and/or duplication in human embryos. Preimplantation genetic screening of the aneuploidy by DNA microarray is an advanced technology used to select embryos for transfer and improved embryo implantation can be obtained after transfer of the screened normal embryos.

  6. Development of a gold nanoparticle-based oligonucleotide microarray for simultaneous detection of seven swine viruses.

    PubMed

    Wang, Xiaoqiang; Dang, Erle; Gao, Jinzhuai; Guo, Sen; Li, Zheng

    2013-07-01

    A gold nanoparticle (GNP) based oligonucleotide microarray assay (GNMA) that combined GNP-based multiplex asymmetric PCR with silver enhancement detection, was developed for simultaneous detection of seven important swine viruses in intensive swine production. Multiplex PCR was first performed to enable the target fragments of seven viruses containing a universal sequence, which were labeled simultaneously with GNPs by multiplex asymmetric PCR in the presence of excess GNP-conjugated primer. Target labeled products were captured by virus-specific oligonucleotide probes immobilized on microarrays, followed by silver staining for signal enhancement. Black image of microarray spots was easily detected by the naked eye or a simple flatbed scanner and quantified. The results for purified plasmid constructs indicated that the assay was highly specific for detecting the seven viruses in single or multiple infections, and as few as 6-80 copies/μl of specific viral target fragments were detected successfully. Fifty-seven archived samples were tested by this assay, and the results were 100% consistent with previous results based on real-time PCR and those obtained by conventional PCR/RT-PCR and sequencing. The assay is appropriate for the routine diagnosis of viral infections in pigs due to its simplicity, low-cost, high specificity and sensitivity. Copyright © 2013 Elsevier B.V. All rights reserved.

  7. Stability of a Surface-Bound Oligonucleotide Duplex Inferred from Molecular Dynamics: A Study of Single Nucleotide Defects Using DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Naiser, Thomas; Kayser, Jona; Mai, Timo; Michel, Wolfgang; Ott, Albrecht

    2009-05-01

    Microarray technology uses the sequence dependent hybridization (binding) affinity of surface-bound oligonucleotide strands for the quantification of complex nucleic acid mixtures. In spite of its huge potential in life science and medicine, microarray oligonucleotide hybridization remains far from being understood. Taking advantage of microarray combinatorial possibilities we show that, although surface bound, the hybridization affinities of single-base mismatched oligonucleotides can be derived from first principles using parameters from bulk.

  8. Oligonucleotide microarrays with stem-loop probes: enhancing the hybridization of nucleic acids for sensitive analysis.

    PubMed

    Mahajan, Shweta; Swami, Archana; Sethi, Dalip; Kumar, P; Gupta, K C

    2008-06-15

    We have demonstrated that the dynamics of nucleic acid hybridization in microarrays depend on the physical structure of immobilized probes. We have immobilized oligonucleotide-3'-phosphates with and without stem-loop structure on epoxylated glass surface, followed by hybridization under different conditions, viz., hybridization buffer, pH condition, temperature and ionic strength. In a comparative study, we have established that array constructed using probes with stem-loop structure displayed approximately 2.2 times higher hybridization signals than the probes without it. The stem-loop DNA array format is simple and flexible in design and thus potentially useful in various DNA diagnostic tests.

  9. 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.

  10. Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology

    PubMed Central

    Ruegger, Paul M.; Bent, Elizabeth; Li, Wei; Jeske, Daniel R.; Cui, Xinping; Braun, Jonathan; Jiang, Tao; Borneman, James

    2012-01-01

    Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized. PMID:22640891

  11. Detection of honey bee (Apis mellifera) viruses with an oligonucleotide microarray.

    PubMed

    Glover, Rachel H; Adams, Ian P; Budge, Giles; Wilkins, Selwyn; Boonham, Neil

    2011-07-01

    In recent years, declines in honey bee (Apis mellifera L.) colonies have been observed to varying degrees worldwide with the worst losses in the USA being termed Colony Collapse Disorder (CCD). Pathogen load and the prevalence of honey bee viruses have been implicated in these losses and many diseased hives have multiple viruses present. We have designed and tested an oligonucleotide microarray which enables the simultaneous detection of nine honey bee viruses: Acute bee paralysis virus, Black queen cell virus, Chronic bee paralysis virus, Deformed wing virus, Kashmir bee virus, Sacbrood virus, Israel acute paralysis virus, Varroa destructor virus 1 and Slow paralysis virus. The microarray can be used to robustly diagnose nine viruses in one test.

  12. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Simultaneous detection and differentiation of Newcastle disease and avian influenza viruses using oligonucleotide microarrays.

    PubMed

    Wang, Lih-Chiann; Pan, Chu-Hsiang; Severinghaus, Lucia Liu; Liu, Lu-Yuan; Chen, Chi-Tsong; Pu, Chang-En; Huang, Dean; Lir, Jihn-Tsair; Chin, Shih-Chien; Cheng, Ming-Chu; Lee, Shu-Hwae; Wang, Ching-Ho

    2008-03-18

    Newcastle disease (ND) and avian influenza (AI) are two of the most important zoonotic viral diseases of birds throughout the world. These two viruses often have a great impact upon the poultry industry. Both viruses are associated with transmission from wild to domestic birds, and often display similar signs that need to be differentiated. A rapid surveillance among wild and domestic birds is important for early disease detection and intervention, and is the basis for what measures should be taken. The surveillance, thus, should be able to differentiate the diseases and provide a detailed analysis of the virus strains. Here, we described a fast, simultaneous and inexpensive approach to the detection of Newcastle disease virus (NDV) and avian influenza virus (AIV) using oligonucleotide microarrays. The NDV pathotypes and the AIV haemagglutinin subtypes H5 and H7 were determined at the same time. Different probes on a microarray targeting the same gene were implemented in order to encompass the diversified virus strains or provide multiple confirmations of the genotype. This ensures good sensitivity and specificity among divergent viruses. Twenty-four virus isolates and twenty-four various combinations of the viruses were tested in this study. All viruses were successfully detected and typed. The hybridization results on microarrays were clearly identified with the naked eyes, with no further imaging equipment needed. The results demonstrate that the detection and typing of multiple viruses can be performed simultaneously and easily using oligonucleotide microarrays. The proposed method may provide potential for rapid surveillance and differential diagnosis of these two important zoonoses in both wild and domestic birds.

  14. Multi-gene detection and identification of mosquito-borne RNA viruses using an oligonucleotide microarray.

    PubMed

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

    2013-01-01

    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). 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. 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 priorities, detect disease outbreaks, and evaluate control programs.

  15. 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

  16. 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.

  17. Optimal design of oligonucleotide microarrays for measurement of DNA copy-number.

    PubMed

    Sharp, Andrew J; Itsara, Andy; Cheng, Ze; Alkan, Can; Schwartz, Stuart; Eichler, Evan E

    2007-11-15

    Copy-number variants (CNVs) occur frequently within the human genome, and may be associated with many human phenotypes. If disease association studies of CNVs are to be performed routinely, it is essential that the copy-number status be accurately genotyped. We systematically assessed the dynamic range response of an oligonucleotide microarray platform to accurately predict copy-number in a set of seven patients who had previously been shown to carry between 1 and 6 copies of an approximately 4 Mb region of 15q12.2-q13.1. We identify probe uniqueness, probe length, uniformity of probe melting temperature, overlap with SNPs and common repeats (particularly Alu elements) and guanine homopolymer content as parameters that significantly affect probe performance. Further, we prove the influence of these criteria on array performance by using these parameters to prospectively filter data from a second array design covering an independent genomic region and observing significant improvements in data quality. The informed selection of probes which have superior performance characteristics allows the prospective design of oligonucleotide arrays which show increased sensitivity and specificity compared with current designs. Although based on the analysis of data from comparative genomic hybridization experiments, we anticipate that our results are relevant to the design of improved oligonucleotide arrays for high-throughput copy-number genotyping of complex regions of the human genome.

  18. Sequencing by hybridization with the generic 6-mer oligonucleotide microarray : an advanced scheme for data processing.

    SciTech Connect

    Chechetkin, V. R.; Turygin, A. Y.; Proudnikov, D. Y.; Prokopenko, D. V.; Kirillov, E. V.; Mirzabekov, A. D.; Biochip Technology Center; Russian Academy of Sciences

    2000-08-01

    DNA sequencing by hybridization was carried out with a microarray of all 4{sup 6} = 4,096 hexadeoxyribonucleotides (the generic microchip). The oligonucleotides immobilized in 100 x 100 x 20-{mu}m polyacrylamide gel pads of the generic microchip were hybridized with fluorescently labeled ssDNA, providing perfect and mismatched duplexes. Melting curves were measured in parallel for all microchip duplexes with a fluorescence microscope equipped with CCD camera. This allowed us to discriminate the perfect duplexes formed by the oligonucleotides, which are complementary to the target DNA. The DNA sequence was reconstructed by overlapping the complementary oligonucleotide probes. We developed a data processing scheme to heighten the discrimination of perfect duplexes from mismatched ones. The procedure was united with a reconstruction of the DNA sequence. The scheme includes the proper definition of a discriminant signal, preprocessing, and the variational principle for the sequence indicator function. The effectiveness of the procedure was confirmed by sequencing, proofreading, and nucleotide polymorphism (mutation) analysis of 13 DNA fragments from 31 to 70 nucleotides long.

  19. MPrime: efficient large scale multiple primer and oligonucleotide design for customized gene microarrays

    PubMed Central

    Rouchka, Eric C; Khalyfa, Abdelnaby; Cooper, Nigel GF

    2005-01-01

    Background Enhancements in sequencing technology have recently yielded assemblies of large genomes including rat, mouse, human, fruit fly, and zebrafish. The availability of large-scale genomic and genic sequence data coupled with advances in microarray technology have made it possible to study the expression of large numbers of sequence products under several different conditions in days where traditional molecular biology techniques might have taken months, or even years. Therefore, to efficiently study a number of gene products associated with a disease, pathway, or other biological process, it is necessary to be able to design primer pairs or oligonucleotides en masse rather than using a time consuming and laborious gene-by-gene method. Results We have developed an integrated system, MPrime, in order to efficiently calculate primer pairs or specific oligonucleotides for multiple genic regions based on a keyword, gene name, accession number, or sequence fasta format within the rat, mouse, human, fruit fly, and zebrafish genomes. A set of products created for mouse housekeeping genes from MPrime-designed primer pairs has been validated using both PCR-amplification and DNA sequencing. Conclusion These results indicate MPrime accurately incorporates standard PCR primer design characteristics to produce high scoring primer pairs for genes of interest. In addition, sequence similarity for a set of oligonucleotides constructed for the same set of genes indicates high specificity in oligo design. PMID:16014168

  20. Selection of optimal oligonucleotide probes for microarrays usingmultiple criteria, global alignment and parameter estimation.

    SciTech Connect

    Li, Xingyuan; He, Zhili; Zhou, Jizhong

    2005-10-30

    The oligonucleotide specificity for microarray hybridizationcan be predicted by its sequence identity to non-targets, continuousstretch to non-targets, and/or binding free energy to non-targets. Mostcurrently available programs only use one or two of these criteria, whichmay choose 'false' specific oligonucleotides or miss 'true' optimalprobes in a considerable proportion. We have developed a software tool,called CommOligo using new algorithms and all three criteria forselection 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) andmelting temperature (Tm), are used to check each possibleoligonucleotide. A sequence identity is calculated based on gapped globalalignments. A traversal algorithm is used to generate alignments for freeenergy calculation. The optimal Tm interval is determined based on probecandidates that have passed all other filters. Final probes are pickedusing a combination of user-configurable piece-wise linear functions andan iterative process. The thresholds for identity, stretch and freeenergy filters are automatically determined from experimental data by anaccessory software tool, CommOligo_PE (CommOligo Parameter Estimator).The program was used to design probes for both whole-genome and highlyhomologous sequence data. CommOligo and CommOligo_PE are freely availableto academic users upon request.

  1. 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

  2. A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

    PubMed

    Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

    2014-01-01

    Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

  3. Stability of double-stranded oligonucleotide DNA with a bulged loop: a microarray study

    PubMed Central

    2011-01-01

    Background DNA is a carrier of biological information. The hybridization process, the formation of the DNA double-helix from single-strands with complementary sequences, is important for all living cells. DNA microarrays, among other biotechnologies such as PCR, rely on DNA hybridization. However, to date the thermodynamics of hybridization is only partly understood. Here we address, experimentally and theoretically, the hybridization of oligonucleotide strands of unequal lengths, which form a bulged loop upon hybridization. For our study we use in-house synthesized DNA microarrays. Results We synthesize a microarray with additional thymine bases in the probe sequence motifs so that bulged loops occur upon target hybridization. We observe a monotonic decrease of the fluorescence signal of the hybridized strands with increasing length of the bulged loop. This corresponds to a decrease in duplex binding affinity within the considered loop lengths of one to thirteen bases. By varying the position of the bulged loop along the DNA duplex, we observe a symmetric signal variation with respect to the center of the strand. We reproduce the experimental results well using a molecular zipper model at thermal equilibrium. However, binding states between both strands, which emerge through duplex opening at the position of the bulged loop, need to be taken into account. Conclusions We show that stable DNA duplexes with a bulged loop can form from short strands of unequal length and they contribute substantially to the fluorescence intensity from the hybridized strands on a microarray. In order to reproduce the result with the help of equilibrium thermodynamics, it is essential (and to a good approximation sufficient) to consider duplex opening not only at the ends but also at the position of the bulged loop. Although the thermodynamic parameters used in this study are taken from hybridization experiments in solution, these parameters fit our DNA microarray data well. PMID

  4. Design and evaluation of an oligonucleotide-microarray for the detection of different species of the genus Kitasatospora.

    PubMed

    Günther, Sebastian; Groth, Ingrid; Grabley, Susanne; Munder, Thomas

    2006-05-01

    An oligonucleotide-microarray method was developed for the detection of Kitasatospora species in soil samples. The 16S-23S rDNA internal transcribed spacer (ITS) sequence of these antibiotics-producing actinomycetes was applied to design short oligonucleotide probes. Two different 26-mers were synthesized, specific to each species used. Additionally, four oligonucleotide probes were designed to evaluate the system. The oligonucleotides were spotted onto slides of the ArrayTube microarray system and examined with a new silver-labeling detection technique. Prior to hybridization analysis, the 16S-23S rDNA were amplified by polymerase chain reaction both from bacterial cells and environmental samples using two actinomycetes specific primers containing a 5' biotin labeling. The type strains of eight Kitasatospora species included in this study were K. phosalacinea DSM 43860, K. setae DSM 43861, K. cochleata DSM 41652, K. cystarginea DSM 41680, K. azatica DSM 41650, K. mediocidica DSM 43929, K. paracochleata DSM 41656, and K. griseola DSM 43859. The actinomycetes-specific primers were shown to amplify the entire 16S-23S rDNA ITS region from all tested strains. More importantly, the described technique allows the detection of Kitasatospora strains from soil samples by extracting metagenomic DNA followed by a PCR amplification step. This indicates that the oligonucleotide-microarray method developed in this study is a reliable tool for the detection of Kitasatospora species in environmental samples.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  6. Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

    PubMed Central

    2009-01-01

    Background In forest ecosystems, communities of ectomycorrhizal fungi (ECM) are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species) have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. Results We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS) regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. Conclusion For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot signal intensity were

  7. An Oligonucleotide Microarray for High-Throughput Sequencing of the Mitochondrial Genome

    PubMed Central

    Zhou, Shaoyu; Kassauei, Keyaunoosh; Cutler, David J.; Kennedy, Giulia C.; Sidransky, David; Maitra, Anirban; Califano, Joseph

    2006-01-01

    Previously we developed an oligonucleotide sequencing microarray (MitoChip) as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The first generation MitoChip, however, was not tiled with probes for the noncoding D-loop region, a site frequently mutated in human cancers. Here we report the development of a second-generation MitoChip (v2.0) with oligonucleotide probes to sequence the entire mitochondrial genome. In addition, the MitoChip v2.0 contains redundant tiling of sequences for 500 of the most common haplotypes including single-nucleotide changes, insertions, and deletions. Sequencing results from 14 primary head and neck tumor tissues demonstrated that the v2.0 MitoChips detected a larger number of variants than the original version. Multiple coding region variants detected only in the second generation MitoChips, but not the earlier chip version, were further confirmed with conventional sequencing. Moreover, 31 variations in noncoding region were identified using MitoChips v2.0. Replicate experiments demonstrated >99.99% reproducibility in the second generation MitoChip. In seven head and neck cancer samples with matched lymphocyte DNA, the MitoChip v2.0 detected at least one cancer-associated mitochondrial mutation in four (57%) samples. These results indicate that the second generation MitoChip is a high-throughput platform for identification of mitochondrial DNA mutations in primary tumors. PMID:16931588

  8. In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication

    PubMed Central

    Phillips, Margaret F.; Lockett, Matthew R.; Rodesch, Matthew J.; Shortreed, Michael R.; Cerrina, Franco; Smith, Lloyd M.

    2008-01-01

    Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired. PMID:18084027

  9. Gene expression profiling in Salmonella Choleraesuis-infected porcine lung using a long oligonucleotide microarray.

    PubMed

    Zhao, Shu-Hong; Kuhar, Daniel; Lunney, Joan K; Dawson, Harry; Guidry, Catherine; Uthe, Jolita J; Bearson, Shawn M D; Recknor, Justin; Nettleton, Dan; Tuggle, Christopher K

    2006-07-01

    Understanding the transcriptional response to pathogenic bacterial infection within food animals is of fundamental and applied interest. To determine the transcriptional response to Salmonella enterica serovar Choleraesuis (SC) infection, a 13,297-oligonucleotide swine array was used to analyze RNA from control, 24-h postinoculation (hpi), and 48-hpi porcine lung tissue from pigs infected with SC. In total, 57 genes showed differential expression (p < 0.001; false discovery rate = 12%). Quantitative real-time PCR (qRT-PCR) of 61 genes was used to confirm the microarray results and to identify pathways responding to infection. Of the 33 genes identified by microarray analysis as differentially expressed, 23 were confirmed by qRT-PCR results. A novel finding was that two transglutaminase family genes (TGM1 and TGM3) showed dramatic increases in expression postinoculation; combined with several other apoptotic genes, they indicated the induction of apoptotic pathways during SC infection. A predominant T helper 1-type immune response occurred during infection, with interferon gamma (IFNG) significantly increased at 48 hpi. Genes induced by IFNs (GBP1, GBP2, C1S, C1R, MHC2TA, PSMB8, TAP1, TAP2) showed increased expression during porcine lung infection. These data represent the first thorough investigation of gene regulation pathways that control an important porcine respiratory and foodborne bacterial infection.

  10. 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.

  11. 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.

  12. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

    PubMed Central

    Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

    2001-01-01

    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 μg of total RNA, representing approximately 7.5 × 106 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

  13. Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

    PubMed Central

    Lodes, Michael J.; Suciu, Dominic; Wilmoth, Jodi L.; Ross, Marty; Munro, Sandra; Dix, Kim; Bernards, Karen; Stöver, Axel G.; Quintana, Miguel; Iihoshi, Naomi; Lyon, Wanda J.; Danley, David L.; McShea, Andrew

    2007-01-01

    Bacterial and viral upper respiratory infections (URI) produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD) on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae) and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluinza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format. PMID:17895966

  14. Oligonucleotide microarray identifies genes differentially expressed during tumorigenesis of DMBA-induced pancreatic cancer in rats.

    PubMed

    Guo, Jun-Chao; Li, Jian; Yang, Ying-Chi; Zhou, Li; Zhang, Tai-Ping; Zhao, Yu-Pei

    2013-01-01

    The extremely dismal prognosis of pancreatic cancer (PC) is attributed, at least in part, to lack of early diagnosis. Therefore, identifying differentially expressed genes in multiple steps of tumorigenesis of PC is of great interest. In the present study, a 7,12-dimethylbenzanthraene (DMBA)-induced PC model was established in male Sprague-Dawley rats. The gene expression profile was screened using an oligonucleotide microarray, followed by real-time quantitative polymerase chain reaction (qRT-PCR) and immunohistochemical staining validation. A total of 661 differentially expressed genes were identified in stages of pancreatic carcinogenesis. According to GO classification, these genes were involved in multiple molecular pathways. Using two-way hierarchical clustering analysis, normal pancreas, acute and chronic pancreatitis, PanIN, early and advanced pancreatic cancer were completely discriminated. Furthermore, 11 upregulated and 142 downregulated genes (probes) were found by Mann-Kendall trend Monotone test, indicating homologous genes of rat and human. The qRT-PCR and immunohistochemistry analysis of CXCR7 and UBe2c, two of the identified genes, confirmed the microarray results. In human PC cell lines, knockdown of CXCR7 resulted in decreased migration and invasion. Collectively, our data identified several promising markers and therapeutic targets of PC based on a comprehensive screening and systemic validation.

  15. 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. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  16. Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes.

    PubMed

    Yang, Dan-Hui; Barari, Mehrnoosh; Arif, Basil M; Krell, Peter J

    2007-08-01

    A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

  17. Gel-based oligonucleotide microarray approach to analyze protein–ssDNA binding specificity

    PubMed Central

    Zasedateleva, Olga A.; Mikheikin, Andrey L.; Turygin, Alexander Y.; Prokopenko, Dmitry V.; Chudinov, Alexander V.; Belobritskaya, Elena E.; Chechetkin, Vladimir R.; Zasedatelev, Alexander S.

    2008-01-01

    Gel-based oligonucleotide microarray approach was developed for quantitative profiling of binding affinity of a protein to single-stranded DNA (ssDNA). To demonstrate additional capabilities of this method, we analyzed the binding specificity of ribonuclease (RNase) binase from Bacillus intermedius (EC 3.1.27.3) to ssDNA using generic hexamer oligodeoxyribonucleotide microchip. Single-stranded octamer oligonucleotides were immobilized within 3D hemispherical gel pads. The octanucleotides in individual pads 5′-{N}N1N2N3N4N5N6{N}-3′ consisted of a fixed hexamer motif N1N2N3N4N5N6 in the middle and variable parts {N} at the ends, where {N} represent A, C, G and T in equal proportions. The chip has 4096 pads with a complete set of hexamer sequences. The affinity was determined by measuring dissociation of the RNase–ssDNA complexes with the temperature increasing from 0°C to 50°C in quasi-equilibrium conditions. RNase binase showed the highest sequence-specificity of binding to motifs 5′-NNG(A/T/C)GNN-3′ with the order of preference: GAG > GTG > GCG. High specificity towards G(A/T/C)G triplets was also confirmed by measuring fluorescent anisotropy of complexes of binase with selected oligodeoxyribonucleotides in solution. The affinity of RNase binase to other 3-nt sequences was also ranked. These results demonstrate the applicability of the method and provide the ground for further investigations of nonenzymatic functions of RNases. PMID:18474529

  18. Development of a short oligonucleotide microarray for the detection and identification of multiple potyviruses.

    PubMed

    Wei, Ting; Pearson, Michael N; Blohm, Dietmar; Nölte, Manfred; Armstrong, Karen

    2009-12-01

    The genus Potyvirus is the largest and one of the most economically important virus genera infecting plants. However, current diagnostic techniques are limited in their ability to identify multiple potyvirus infections. An assay that can identify multiple potyviruses simultaneously, with good specificity and sensitivity, is therefore highly desirable. To determine the feasibility of simultaneous detection of multiple potyviruses a 25-mer oligonucleotide microarray was developed targeting four distinct potyviruses: Dasheen mosaic virus (DsMV), Leek yellow stripe virus (LYSV), Potato virus Y (PVY) and Zucchini yellow mosaic virus (ZYMV). A total of 85 probes including 33 perfect-match and 52 mismatch probes were designed from conserved and variable sequence regions of the nuclear inclusion b (NIb) gene, RNA-dependent RNA polymerase (RdRp) gene, coat protein (CP) gene and the 3' untranslated region (UTR), representing the four targeted potyviruses at both species and strain levels. Each probe was synthesized with spacers of either 6 or 12 poly-cytosine or poly-thymine at the 5' terminus. The array showed high specificity when tested with nineteen different geographically diverse potyvirus isolates of the four target species, four distinct but closely related potyviruses, and four healthy plant species. The approaches and protocols developed in this study form a useful basis for developing other potyviruses arrays and the results also provide useful insights into generic issues for the development of arrays for detecting other pathogens.

  19. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    El Fantroussi, Said; Urakawa, Hidetoshi; Bernhard, Anne E.; Kelly, John J.; Noble, Peter A.; Smidt, H.; Yershov, G. M.; Stahl, David A.

    2003-01-01

    Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

  20. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays

    NASA Technical Reports Server (NTRS)

    El Fantroussi, Said; Urakawa, Hidetoshi; Bernhard, Anne E.; Kelly, John J.; Noble, Peter A.; Smidt, H.; Yershov, G. M.; Stahl, David A.

    2003-01-01

    Oligonucleotide microarrays were used to profile directly extracted rRNA from environmental microbial populations without PCR amplification. In our initial inspection of two distinct estuarine study sites, the hybridization patterns were reproducible and varied between estuarine sediments of differing salinities. The determination of a thermal dissociation curve (i.e., melting profile) for each probe-target duplex provided information on hybridization specificity, which is essential for confirming adequate discrimination between target and nontarget sequences.

  1. 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.

  2. Specific discrimination of three pathogenic Salmonella enterica subsp. enterica serotypes by carB-based oligonucleotide microarray.

    PubMed

    Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun; Cha, Hyung Joon

    2014-01-01

    It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.

  3. Specific Discrimination of Three Pathogenic Salmonella enterica subsp. enterica Serotypes by carB-Based Oligonucleotide Microarray

    PubMed Central

    Shin, Hwa Hui; Hwang, Byeong Hee; Seo, Jeong Hyun

    2014-01-01

    It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes. PMID:24185846

  4. 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.

  5. 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

  6. Differential analysis for high density tiling microarray data.

    PubMed

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

    2007-09-24

    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. 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. 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].

  7. 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.

  8. 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.

  9. 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.

  10. Subtype identification of the novel A H1N1 and other human influenza A viruses using an oligonucleotide microarray.

    PubMed

    Kang, Xiaoping; Li, Yongqiang; Sun, Honghe; Wu, Weili; Liu, Hong; Lin, Fang; Qing, Chenfeng; Chang, Guohui; Zhu, Qingyu; Chen, Weijun; Yang, Yinhui

    2010-01-01

    A novel strain of influenza A (H1N1) virus was isolated in Mexico and the US in March and April 2009. This novel virus spread to many countries and regions in a few months, and WHO raised the level of pandemic alert from phase 5 to phase 6 on June 11, 2009. The accurate identification of H1N1 virus and other human seasonal influenza A viruses is very important for further treatment and control of their infections. In this study, we developed an oligonucleotide microarray to subtype human H1N1, H3N2 and H5N1 influenza viruses, which could distinguish the novel H1N1 from human seasonal H1N1 influenza viruses and swine H1N1 influenza viruses. The microarray utilizes a panel of primers for multiplex PCR amplification of the hemagglutinin (HA), neuraminidase (NA) and matrix (MP) genes of human influenza A viruses. The 59-mer oligonucleotides were designed to distinguish different subtypes of human influenza A viruses. With this microarray, we accurately identified and correctly subtyped the reference virus strains. Moreover, we confirmed 4 out of 39 clinical throat swab specimens from suspected cases of novel H1N1.

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

    PubMed

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

    2015-02-07

    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 high complexity and 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-nucleotide sequence-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

  12. 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

  13. Automated and Multiplexed Soft Lithography for the Production of Low-Density DNA Microarrays

    PubMed Central

    Fredonnet, Julie; Foncy, Julie; Cau, Jean-Christophe; Séverac, Childérick; François, Jean Marie; Trévisiol, Emmanuelle

    2016-01-01

    Microarrays are established research tools for genotyping, expression profiling, or molecular diagnostics in which DNA molecules are precisely addressed to the surface of a solid support. This study assesses the fabrication of low-density oligonucleotide arrays using an automated microcontact printing device, the InnoStamp 40®. This automate allows a multiplexed deposition of oligoprobes on a functionalized surface by the use of a MacroStampTM bearing 64 individual pillars each mounted with 50 circular micropatterns (spots) of 160 µm diameter at 320 µm pitch. Reliability and reuse of the MacroStampTM were shown to be fast and robust by a simple washing step in 96% ethanol. The low-density microarrays printed on either epoxysilane or dendrimer-functionalized slides (DendriSlides) showed excellent hybridization response with complementary sequences at unusual low probe and target concentrations, since the actual probe density immobilized by this technology was at least 10-fold lower than with the conventional mechanical spotting. In addition, we found a comparable hybridization response in terms of fluorescence intensity between spotted and printed oligoarrays with a 1 nM complementary target by using a 50-fold lower probe concentration to produce the oligoarrays by the microcontact printing method. Taken together, our results lend support to the potential development of this multiplexed microcontact printing technology employing soft lithography as an alternative, cost-competitive tool for fabrication of low-density DNA microarrays. PMID:27681742

  14. Development of mitochondria-specific mouse oligonucleotide microarray and validation of data by real-time PCR.

    PubMed

    Desai, Varsha G; Lee, Taewon; Delongchamp, Robert R; Moland, Carrie L; Branham, William S; Fuscoe, James C; Leakey, Julian E A

    2007-09-01

    This study describes the development of a mitochondria-specific microarray, MitoChip, to measure transcripts of mitochondria-associated genes in various diseases and drug-induced toxicities in the mouse. The array consists of 542 oligonucleotides that represent genes from the mitochondrial and nuclear genomes associated with mitochondrial structure and functions. The expression of mitochondrial genes was measured in the liver of both p53 haplodeficient (+/-) and wild-type (+/+) C3B6F(1) female mice exposed to antiretroviral agents, Zidovudine (AZT) and Lamivudine (3TC). Among genes whose expression was significantly altered, a set was selected for real-time PCR analysis to verify their differential gene expression. The real-time PCR data confirmed the observations by microarray analysis suggesting that the MitoChip may be an important tool for examining mitochondrial involvement in diseases and drug-induced toxicities.

  15. Osprey: a comprehensive tool employing novel methods for the design of oligonucleotides for DNA sequencing and microarrays

    PubMed Central

    Gordon, Paul M. K.; Sensen, Christoph W.

    2004-01-01

    We have developed a software package called Osprey for the calculation of optimal oligonucleotides for DNA sequencing and the creation of microarrays based on either PCR-products or directly spotted oligomers. It incorporates a novel use of position-specific scoring matrices, for the sensitive and specific identification of secondary binding sites anywhere in the target sequence. Using accelerated hardware is faster and more efficient than the traditional pairwise alignments used in most oligo-design software. Osprey consists of a module for target site selection based on user input, novel utilities for dealing with problematic sequences such as repeats, and a common code base for the identification of optimal oligonucleotides from the target list. Overall, these improvements provide a program that, without major increases in run time, reflects current DNA thermodynamics models, improves specificity and reduces the user's data preprocessing and parameterization requirements. Using a TimeLogic™ hardware accelerator, we report up to 50-fold reduction in search time versus a linear search strategy. Target sites may be derived from computer analysis of DNA sequence assemblies in the case of sequencing efforts, or genome or EST analysis in the case of microarray development in both prokaryotes and eukaryotes. PMID:15456895

  16. Osprey: a comprehensive tool employing novel methods for the design of oligonucleotides for DNA sequencing and microarrays.

    PubMed

    Gordon, Paul M K; Sensen, Christoph W

    2004-09-29

    We have developed a software package called Osprey for the calculation of optimal oligonucleotides for DNA sequencing and the creation of microarrays based on either PCR-products or directly spotted oligomers. It incorporates a novel use of position-specific scoring matrices, for the sensitive and specific identification of secondary binding sites anywhere in the target sequence. Using accelerated hardware is faster and more efficient than the traditional pairwise alignments used in most oligo-design software. Osprey consists of a module for target site selection based on user input, novel utilities for dealing with problematic sequences such as repeats, and a common code base for the identification of optimal oligonucleotides from the target list. Overall, these improvements provide a program that, without major increases in run time, reflects current DNA thermodynamics models, improves specificity and reduces the user's data preprocessing and parameterization requirements. Using a TimeLogic hardware accelerator, we report up to 50-fold reduction in search time versus a linear search strategy. Target sites may be derived from computer analysis of DNA sequence assemblies in the case of sequencing efforts, or genome or EST analysis in the case of microarray development in both prokaryotes and eukaryotes.

  17. Light-directed 5′→3′ synthesis of complex oligonucleotide microarrays

    PubMed Central

    Albert, Thomas J.; Norton, Jason; Ott, Markus; Richmond, Todd; Nuwaysir, Kate; Nuwaysir, Emile F.; Stengele, Klaus-Peter; Green, Roland D.

    2003-01-01

    Light-directed synthesis of high-density microarrays is currently performed in the 3′→5′ direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5′→3′ direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5′→3′ direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150 000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R2 = 0.998). We have also shown that the 3′ ends of array probes are available for sequence-specific primer extension and ligation reactions. PMID:12655023

  18. 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.

  19. 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.

  20. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  2. 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...

  3. 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...

  4. Detection of genetically modified canola using multiplex PCR coupled with oligonucleotide microarray hybridization.

    PubMed

    Schmidt, Anna-Mary; Sahota, Robert; Pope, Derek S; Lawrence, Tracy S; Belton, Mark P; Rott, Michael E

    2008-08-27

    A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

  5. An oligonucleotide-based microarray for detection of plant RNA viruses.

    PubMed

    Nicolaisen, Mogens

    2011-04-01

    Currently, some of the methods used most widely for diagnosis and detection of plant viruses are ELISA, PCR, bioassays and electron microscopy. These methods only target one or a few species in each assay or they are time consuming and require expertise. Microarray-based approaches offer an alternative to these methods as microarrays with virus-specific probes could be capable of detecting an almost unlimited number of virus species in one assay. In the present study, the feasibility of this strategy was studied by constructing a microarray with 150 probes potentially capable of detecting 52 viruses from a broad range of genera. The array was printed in 16 subarrays to allow testing of several samples on each slide. Hybridizations with cDNA from plants infected with 52 different virus species showed that out of the 52 species tested, 49 were positive and identified correctly to species level. This array represents the largest published microarray for plant virus detection in terms of the number of targeted species and is thus an important milestone towards the construction of a generic microarray able to detect most, if not all, plant RNA viruses.

  6. Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

    2005-05-18

    To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

  7. Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray.

    PubMed

    Xu, Jia; Miao, Haizhen; Wu, Houfei; Huang, Wensheng; Tang, Rong; Qiu, Minyan; Wen, Jianguo; Zhu, Shuifang; Li, Yao

    2006-07-15

    In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95% of currently commercial GMO plants and the limits of detection are 0.5% for soybean and 1% for maize. This method is proved to be a new method for routine analysis of GMOs.

  8. [The second generation universal oligonucleotide microarray for subtyping of influenza virus A].

    PubMed

    Kostina, E V; Riabinin, V A; Maksakova, G A; Siniakov, A N

    2012-01-01

    The microchip for influenza A subtyping was developed, functioning on a principle "one spot--one subtype". Each spot contains the set of oligonucleotide probes, specific for a particular subtype of hemagglutinin, neuraminidase or matrix gene. Reliability of the proposed chip version is the same as for earlier created in our group full-size microchip for separate hemagglutinin and neuraminidase subtyping. To visualize the image, analyzed DNA can be labeled by either fluorescent dye or biotin with the further fixation in system streptavidin-gold nanoparticles and image development by silver precipitation. In the second case common version of scanner can be used for the image analysis, that essentially simplifies procedure of influenza A subtyping.

  9. Identification of differentially regulated transcripts in mouse striatum following methamphetamine treatment--an oligonucleotide microarray approach.

    PubMed

    Thomas, David M; Francescutti-Verbeem, Dina M; Liu, Xiuli; Kuhn, Donald M

    2004-01-01

    Methamphetamine is an addictive drug of abuse that can produce neurotoxic effects in dopamine nerve endings of the striatum. The purpose of this study was to identify new genes that may play a role in the highly complex cascade of events associated with methamphetamine intoxication. Using Affymetrix oligonucleotide arrays, 12 488 genes were simultaneously interrogated and there were 152 whose expression levels were changed following methamphetamine treatment. The genes are grouped into broad functional categories with inflammatory/immune response elements, receptor/signal transduction components and ion channel/transport proteins among the most populated. Many genes within these categories can be linked to ion regulation and apoptosis, both of which have been implicated in methamphetamine toxicity, and numerous factors associated with microglial activation emerged with significant changes in expression. For example, brain-derived neurotrophic factor (BDNF), chemokine (C-C) receptor 6 (CCr6) and numerous chemokine transcripts were increased or decreased in expression more than 2.8-fold. These results point to activated microglia as a potential source of the reactive oxygen/nitrogen species and cytokines that have been previously associated with methamphetamine toxicity and other neurotoxic conditions.

  10. Understanding the physics of oligonucleotide microarrays: the Affymetrix spike-in data reanalysed.

    PubMed

    Burden, Conrad J

    2008-03-27

    The Affymetrix U95 and U133 Latin-Square spike-in datasets are reanalysed, together with a dataset from a version of the U95 spike-in experiment without a complex non-specific background. The approach uses a physico-chemical model which includes the effects of the specific and non-specific hybridization and probe folding at the microarray surface, target folding and hybridization in the bulk RNA target solution and duplex dissociation during the post-hybridization washing phase. The model predicts a three-parameter hyperbolic response function that fits well with fluorescence intensity data from all the three datasets. The importance of the various hybridization and washing effects in determining each of the three parameters is examined, and some guidance is given as to how a practical algorithm for determining specific target concentrations might be developed.

  11. Understanding the physics of oligonucleotide microarrays: the Affymetrix spike-in data reanalysed

    NASA Astrophysics Data System (ADS)

    Burden, Conrad J.

    2008-03-01

    The Affymetrix U95 and U133 Latin-Square spike-in datasets are reanalysed, together with a dataset from a version of the U95 spike-in experiment without a complex non-specific background. The approach uses a physico-chemical model which includes the effects of the specific and non-specific hybridization and probe folding at the microarray surface, target folding and hybridization in the bulk RNA target solution and duplex dissociation during the post-hybridization washing phase. The model predicts a three-parameter hyperbolic response function that fits well with fluorescence intensity data from all the three datasets. The importance of the various hybridization and washing effects in determining each of the three parameters is examined, and some guidance is given as to how a practical algorithm for determining specific target concentrations might be developed.

  12. 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.

  13. 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

  14. Oligonucleotide microarray analysis reveals dysregulation of energy-related metabolism in insulin-sensitive tissues of type 2 diabetes patients.

    PubMed

    Wang, M; Wang, X C; Zhao, L; Zhang, Y; Yao, L L; Lin, Y; Peng, Y D; Hu, R M

    2014-06-17

    Impaired insulin action within skeletal muscle, adipose tissue, and the liver is an important characteristic of type 2 diabetes (T2D). In order to identify common underlying defects in insulin-sensitive tissues that may be involved in the pathogenesis of T2D, the gene expression profiles of skeletal muscle, visceral adipose tissue, and liver from autopsy donors with or without T2D were examined using oligonucleotide microarrays and quantitative reverse transcriptase-PCR. Compared with controls, 691 genes were commonly dysregulated in these three insulin-sensitive tissues of humans with T2D. These co-expressed genes were enriched within the mitochondrion, with suggested involvement in energy metabolic processes such as glycolysis and gluconeogenesis, fatty acid beta oxidative, tricarboxylic acid cycle, and electron transport. Genes related to energy metabolism were mostly downregulated in diabetic skeletal muscle and visceral adipose tissue, while they were upregulated in the diabetic liver. This observed dysregulation in energy-related metabolism may be the underlying factor leading to the molecular mechanisms responsible for the insulin resistance of patients with T2D.

  15. 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.

  16. 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.

  17. Oligonucleotide Microarray Analysis of Dietary-Induced Hyperlipidemia Gene Expression Profiles in Miniature Pigs

    PubMed Central

    Takahashi, Junko; Waki, Shiori; Matsumoto, Rena; Odake, Junji; Miyaji, Takayuki; Tottori, Junichi; Iwanaga, Takehiro; Iwahashi, Hitoshi

    2012-01-01

    Background Hyperlipidemia animal models have been established, but complete gene expression profiles of the transition from normal lipid levels have not been obtained. Miniature pigs are useful model animals for gene expression studies on dietary-induced hyperlipidemia because they have a similar anatomy and digestive physiology to humans, and blood samples can be obtained from them repeatedly. Methodology Two typical dietary treatments were used for dietary-induced hyperlipidemia models, by using specific pathogen-free (SPF) Clawn miniature pigs. One was a high-fat and high-cholesterol diet (HFCD) and the other was a high-fat, high-cholesterol, and high-sucrose diet (HFCSD). Microarray analyses were conducted from whole blood samples during the dietary period and from white blood cells at the end of the dietary period to evaluate the transition of expression profiles of the two dietary models. Principal Findings Variations in whole blood gene expression intensity within the HFCD or the HFCSD group were in the same range as the controls provide with normal diet at all periods. This indicates uniformity of dietary-induced hyperlipidemia for our dietary protocols. Gene ontology- (GO) based functional analyses revealed that characteristics of the common changes between HFCD and HFCSD were involved in inflammatory responses and reproduction. The correlation coefficient between whole blood and white blood cell expression profiles at 27 weeks with the HFCSD diet was significantly lower than that of the control and HFCD diet groups. This may be due to the effects of RNA originating from the tissues and/or organs. Conclusions No statistically significant differences in fasting plasma lipids and glucose levels between the HFCD and HFCSD groups were observed. However, blood RNA analyses revealed different characteristics corresponding to the dietary protocols. In this study, whole blood RNA analyses proved to be a useful tool to evaluate transitions in dietary

  18. Binding specificity and stability of duplexes formed by modified oligonucleotides with a 4096-hexanucleotide microarray

    PubMed Central

    Timofeev, Edward; Mirzabekov, Andrei

    2001-01-01

    The binding of oligodeoxynucleotides modified with adenine 2′-O-methyl riboside, 2,6-diaminopurine 2′-O-methyl riboside, cytosine 2′-O-methyl riboside, 2,6-diaminopurine deoxyriboside or 5-bromodeoxyuridine was studied with a microarray containing all possible (4096) polyacrylamide-bound hexadeoxynucleotides (a generic microchip). The generic microchip was manufactured by using reductive immobilization of aminooligonucleotides in the activated copolymer of acrylamide, bis-acrylamide and N-(2,2-dimethoxyethyl) acrylamide. The binding of the fluorescently labeled modified octanucleotides to the array was analyzed with the use of both melting profiles and the fluorescence distribution at selected temperatures. Up to three substitutions of adenosines in the octamer sequence by adenine 2′-O-methyl ribosides (Am), 2,6-diaminopurine 2′-O-methyl ribosides (Dm) or 2,6-diaminopurine deoxyribosides (D) resulted in increased mismatch discrimination measured at the melting temperature of the corresponding perfect duplex. The stability of complexes formed by 2′-O-methyl-adenosine-modified oligodeoxynucleotides was slightly decreased with every additional substitution, yielding ∼4°C of total loss in melting temperature for three modifications, as followed from microchip thermal denaturation experiments. 2,6-Diaminopurine 2′-O-methyl riboside modifications led to considerable duplex stabilization. The cytosine 2′-O-methyl riboside and 5-bromodeoxyuridine modifications generally did not change either duplex stability or mismatch resolution. Denaturation experiments conducted with selected perfect duplexes on microchips and in solution showed similar results on thermal stabilities. Some hybridization artifacts were observed that might indicate the formation of parallel DNA. PMID:11410672

  19. An oligonucleotide prokaryotic acidophile microarray: its validation and its use to monitor seasonal variations in extreme acidic environments with total environmental RNA.

    PubMed

    Garrido, Patricia; González-Toril, Elena; García-Moyano, Antonio; Moreno-Paz, Mercedes; Amils, Ricardo; Parro, Víctor

    2008-04-01

    An oligonucleotide microarray that monitors prokaryotic diversity in extremely acidic environments has been developed. The oligonucleotide probes target most known acidophilic microorganisms, including members of the Nitrospira phylum, Acidithiobacillus genus, acidobacteria, sulfur reducing bacteria, Actinobacteria and Archaea of the Ferroplasma and Thermoplasma genera. The probes were tested for their specificity against the corresponding type strain by microarray hybridization using PCR-amplified fluorescent DNA of the 16S rRNA genes. The microarray was tested and validated against well-established molecular ecology techniques such as molecular cloning and sequencing and FISH by using samples obtained from a natural extremely acidic environment, the Río Tinto (SW Spain). Also, fluorescent labelled total environmental RNA from Río Tinto samples were used as targets for microarray hybridizations. This approach allowed the detection of the most metabolically active prokaryotes of the ecosystem by simultaneously checking probes against 16S and 23S rRNAs as well as other functional genes. Seasonal and spatial variations in the relative expression of specific rRNA genes have been detected between two sampling sites that differ in several physicochemical parameters, mainly iron and sulfur content.

  20. 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.

  1. An oligonucleotide microarray for the identification and differentiation of trichothecene producing and non-producing Fusarium species occurring on cereal grain.

    PubMed

    Nicolaisen, Mogens; Justesen, Annemarie F; Thrane, Ulf; Skouboe, Pernille; Holmstrøm, Kim

    2005-07-01

    Cereal grain may be infected with a number of Fusarium species some of which are producers of highly toxic compounds such as the trichothecenes. Correct identification of these species is essential for risk assessment of cereal grain for human or animal consumption. Most of the available methods for identification are either time consuming or aimed at only one or a few target species. Microarray technology offers parallel analysis of a high number of DNA targets. In this study 57 capture oligonucleotides (CO) were designed based upon Fusarium ITS2 rDNA sequences, and used for microarray production. From this array COs could be selected that were able to hybridise specifically to labelled PCR products from the ITS region of Fusarium graminearum/Fusarium culmorum, Fusarium pseudograminearum, Fusarium poae, Fusarium sporotrichioides, Fusarium equiseti, Fusarium langsethiae and Fusarium tricinctum/Fusarium avenaceum. A few COs showed some cross hybridisation to non-target species. In a preliminary experiment it was shown that this cross hybridisation could be eliminated by increasing hybridisation stringency. The array could be used to detect individual Fusarium species in mixed samples and in environmental samples. This study demonstrates the feasibility of oligonucleotide microarrays for parallel detection of a number of Fusarium species.

  2. 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…

  3. 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…

  4. High-density tiling microarray analysis of the full transcriptional activity of yeast.

    PubMed

    David, Lior; Clauder-Münster, Sandra; Steinmetz, Lars M

    2014-01-01

    Understanding the relationship between DNA sequence variation and phenotypic variation in complex or quantitative traits is one of the major challenges in modern biology. We are witnessing a deluge of DNA sequence information and association studies of genetic polymorphisms with phenotypes of interest in families and populations. In addition, it has become clear that large portions of eukaryotic genomes beyond protein-coding genes are transcribed, generating numerous noncoding RNA (ncRNA) molecules whose functions remain mostly unknown.DNA oligonucleotide microarrays constitute a powerful technology for studying the expression of genes in different organisms. The Saccharomyces cerevisiae tiling array presents a significant advance over previous array-based platforms. It has a high density of overlapping probes that start on average every 8 bp along each strand of the genome, enabling precise definition of transcript structure. Furthermore, the array includes probes specific for the polymorphic positions of another, distantly related yeast strain, allowing accurate measurement of allele-specific expression in a hybrid of the two strains. This technology thus allows high-resolution, quantitative, strand- and allele-specific measurements of transcription from a full eukaryotic genome. In this chapter, we describe the methods for extracting RNA, synthesizing first-strand cDNA, fragmenting, and labeling of samples for hybridization to the tiling array. Combining genome-wide information on variation in DNA sequence with variation in transcript structure and levels promises to increase our understanding of the genotype-to-phenotype relationship.

  5. 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.

  6. Sensitive detection of SARS coronavirus RNA by a novel asymmetric multiplex nested RT-PCR amplification coupled with oligonucleotide microarray hybridization.

    PubMed

    Zhang, Zhi-wei; Zhou, Yi-ming; Zhang, Yan; Guo, Yong; Tao, Sheng-ce; Li, Ze; Zhang, Qiong; Cheng, Jing

    2005-01-01

    We have developed a sensitive method for the detection of specific genes simultaneously. First, DNA was amplified by a novel asymmetric multiplex PCR with universal primer(s). Second, the 6-carboxytetramethylrhodamine (TAMRA)-labeled PCR products were hybridized specifically with oligonucleotide microarrays. Finally, matched duplexes were detected by using a laser-induced fluorescence scanner. The usefulness of this method was illustrated by analyzing severe acute respiratory syndrome (SARS) coronavirus RNA. The detection limit was 10(0) copies/microL. The results of the asymmetric multiplex nested reverse transcription-PCR were in agreement with the results of the microarray hybridization; no hybridization signal was lost as happened with applicons from symmetric amplifications. This reliable method can be used to the identification of other microorganisms, screening of genetic diseases, and other applications.

  7. Inferring Loss-of-Heterozygosity from Unpaired Tumors Using High-Density Oligonucleotide SNP Arrays

    PubMed Central

    Park, Yuhyun; Hao, Ke; Zhao, Xiaojun; Garraway, Levi A; Fox, Edward A; Hochberg, Ephraim P; Mellinghoff, Ingo K; Hofer, Matthias D; Descazeaud, Aurelien; Rubin, Mark A; Meyerson, Matthew; Wong, Wing Hung; Sellers, William R; Li, Cheng

    2006-01-01

    Loss of heterozygosity (LOH) of chromosomal regions bearing tumor suppressors is a key event in the evolution of epithelial and mesenchymal tumors. Identification of these regions usually relies on genotyping tumor and counterpart normal DNA and noting regions where heterozygous alleles in the normal DNA become homozygous in the tumor. However, paired normal samples for tumors and cell lines are often not available. With the advent of oligonucleotide arrays that simultaneously assay thousands of single-nucleotide polymorphism (SNP) markers, genotyping can now be done at high enough resolution to allow identification of LOH events by the absence of heterozygous loci, without comparison to normal controls. Here we describe a hidden Markov model-based method to identify LOH from unpaired tumor samples, taking into account SNP intermarker distances, SNP-specific heterozygosity rates, and the haplotype structure of the human genome. When we applied the method to data genotyped on 100 K arrays, we correctly identified 99% of SNP markers as either retention or loss. We also correctly identified 81% of the regions of LOH, including 98% of regions greater than 3 megabases. By integrating copy number analysis into the method, we were able to distinguish LOH from allelic imbalance. Application of this method to data from a set of prostate samples without paired normals identified known regions of prevalent LOH. We have developed a method for analyzing high-density oligonucleotide SNP array data to accurately identify of regions of LOH and retention in tumors without the need for paired normal samples. PMID:16699594

  8. Oligonucleotide microarrays for identification of microbial pathogens and detection of their virulence-associated or drug-resistance determinants.

    PubMed

    Volokhov, Dmitriy V; Kong, Hyesuk; Herold, Keith; Chizhikov, Vladimir E; Rasooly, Avraham

    2011-01-01

    Microarrays are spatially ordered arrays with ligands chemically immobilized in discrete spots on a solid matrix, usually a microscope slide. Microarrays are a high-throughput large-scale screening system enabling simultaneous identification of a large number of labeled target molecules (up to several hundred thousand) that bind specifically to the immobilized ligands of the array. DNA microarrays represent a promising tool for clinical, environmental, and industrial microbiology since the technology allows relatively rapid identification of large number of genetic determinants simultaneously, providing detailed genomic level information regarding the pathogen species, including identification of their virulence-associated factors and the presence of antibiotic resistance genes. In this chapter, we describe key aspects and methodologies important for the development and use of DNA microarrays for microbial diagnostics.

  9. chipD: a web tool to design oligonucleotide probes for high-density tiling arrays

    PubMed Central

    Dufour, Yann S.; Wesenberg, Gary E.; Tritt, Andrew J.; Glasner, Jeremy D.; Perna, Nicole T.; Mitchell, Julie C.; Donohue, Timothy J.

    2010-01-01

    chipD is a web server that facilitates design of DNA oligonucleotide probes for high-density tiling arrays, which can be used in a number of genomic applications such as ChIP-chip or gene-expression profiling. The server implements a probe selection algorithm that takes as an input, in addition to the target sequences, a set of parameters that allow probe design to be tailored to specific applications, protocols or the array manufacturer’s requirements. The algorithm optimizes probes to meet three objectives: (i) probes should be specific; (ii) probes should have similar thermodynamic properties; and (iii) the target sequence coverage should be homogeneous and avoid significant gaps. The output provides in a text format, the list of probe sequences with their genomic locations, targeted strands and hybridization characteristics. chipD has been used successfully to design tiling arrays for bacteria and yeast. chipD is available at http://chipd.uwbacter.org/. PMID:20529880

  10. Massively parallel pathogen identification using high‐density microarrays

    PubMed Central

    Berthet, Nicolas; Dickinson, Philip; Filliol, Ingrid; Reinhardt, Anita K.; Batejat, Christophe; Vallaeys, Tatiana; Kong, Katherine A.; Davies, Christopher; Lee, Walter; Zhang, Shenglan; Turpaz, Yaron; Heym, Beate; Coralie, Gilberte; Dacheux, Laurent; Burguière, Ana Maria; Bourhy, Hervé; Old, Iain G.; Manuguerra, Jean‐Claude; Cole, Stewart T.; Kennedy, Giulia C.

    2008-01-01

    Summary Identification of microbial pathogens in clinical specimens is still performed by phenotypic methods that are often slow and cumbersome, despite the availability of more comprehensive genotyping technologies. We present an approach based on whole‐genome amplification and resequencing microarrays for unbiased pathogen detection. This 10 h process identifies a broad spectrum of bacterial and viral species and predicts antibiotic resistance and pathogenicity and virulence profiles. We successfully identify a variety of bacteria and viruses, both in isolation and in complex mixtures, and the high specificity of the microarray distinguishes between different pathogens that cause diseases with overlapping symptoms. The resequencing approach also allows identification of organisms whose sequences are not tiled on the array, greatly expanding the repertoire of identifiable organisms and their variants. We identify organisms by hybridization of their DNA in as little as 1–4 h. Using this method, we identified Monkeypox virus and drug‐resistant Staphylococcus aureus in a skin lesion taken from a child suspected of an orthopoxvirus infection, despite poor transport conditions of the sample, and a vast excess of human DNA. Our results suggest this technology could be applied in a clinical setting to test for numerous pathogens in a rapid, sensitive and unbiased manner. PMID:21261824

  11. 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.

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

    PubMed

    Ramirez, Lisa S; Wang, Jun

    2016-01-06

    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.

  13. 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.

  14. Discrimination of bacillus anthracis and closely related microorganisms by analysis of 16S and 23S rRNA with oligonucleotide microarray.

    SciTech Connect

    Bavykin, S. G.; Mikhailovich, V. M.; Zakharyev, V. M.; Lysov, Y. P.; Kelly, J. J.; Alferov, O. S.; Jackman, J.; Stahl, D. A.; Mirzabekov, A. D.; Gavin, I. M.; Kukhtin, A. V.; Chandler, D.

    2008-01-30

    Analysis of 16S rRNA sequences is a commonly used method for the identification and discrimination of microorganisms. However, the high similarity of 16S and 23S rRNA sequences of Bacillus cereus group organisms (up to 99-100%) and repeatedly failed attempts to develop molecular typing systems that would use DNA sequences to discriminate between species within this group have resulted in several suggestions to consider B. cereus and B. thuringiensis, or these two species together with B. anthracis, as one species. Recently, we divided the B. cereus group into seven subgroups, Anthracis, Cereus A and B, Thuringiensis A and B, and Mycoides A and B, based on 16S rRNA, 23S rRNA and gyrB gene sequences and identified subgroup-specific makers in each of these three genes. Here we for the first time demonstrated discrimination of these seven subgroups, including subgroup Anthracis, with a 3D gel element microarray of oligonucleotide probes targeting 16S and 23S rRNA markers. This is the first microarray enabled identification of B. anthracis and discrimination of these seven subgroups in pure cell cultures and in environmental samples using rRNA sequences. The microarray bearing perfect match/mismatch (p/mm) probe pairs was specific enough to discriminate single nucleotide polymorphisms (SNPs) and was able to identify targeted organisms in 5 min. We also demonstrated the ability of the microarray to determine subgroup affiliations for B. cereus group isolates without rRNA sequencing. Correlation of these seven subgroups with groupings based on multilocus sequence typing (MLST), fluorescent amplified fragment length polymorphism analysis (AFLP) and multilocus enzyme electrophoresis (MME) analysis of a wide spectrum of different genes, and the demonstration of subgroup-specific differences in toxin profiles, psychrotolerance, and the ability to harbor some plasmids, suggest that these seven subgroups are not based solely on neutral genomic polymorphisms, but instead reflect

  15. Empirical Establishment of Oligonucleotide Probe Design Criteria; Use of Microarrays with Different Probe Sizes for Monitoring Gene Expression; Temporal Transcriptomic Analysis as Desulfovibrio vulgaris Hildenborough Transitions into Stationary Phase during Electron Donor Depletion

    SciTech Connect

    He, Q.; He, Z.; Huang, K. H.; Alm, E. J.; Wan, X. F.; Hazen, T. C.; Arkin, A. P.; Wall, J. D.; Zhou, J. Z.; Fields, M. W.

    2005-07-15

    In order to experimentally establish the criteria for designing gene-specific and group-specific oligonucleotide probes, an oligonucleotide array was constructed that contained perfect match (PM) and mismatch (MM) probes (50mers and 70mers) based upon 4 genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were examined. Little hybridization was observed at a probe-target identity of <85% for both 50mer and 70mer probes........Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and these criteria should provide valuable information for the development of new software and algorithms for microarray-based studies.; Microarrays with oligonucleotides of different lengths were used to monitor gene expression at a wholegenome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from Shewanella oneidensis MR-1 in terms of overall signal intensity, numbers of genes detected, specificity, sensitivity, and differential gene expression under experimental conditions. .......To evaluate differential gene expression under experimental conditions, S. oneidensis MR-1 cells were exposed to low- or high-pH conditions for 30 and 60 min, and the transcriptional profiles detected by oligonucleotide probes (50-mer, 60-mer, and 70-mer) were closely correlated with those detected by the PCR probes. The results demonstrated that 70-mer oligonucleotides can provide the performance most comparable to the performance obtained with PCR-generated probes.; Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the

  16. Response to drought and salt stress in leaves of poplar (Populus alba × Populus glandulosa): expression profiling by oligonucleotide microarray analysis.

    PubMed

    Yoon, Seo-Kyung; Park, Eung-Jun; Choi, Young-Im; Bae, Eun-Kyung; Kim, Joon-Hyeok; Park, So-Young; Kang, Kyu-Suk; Lee, Hyoshin

    2014-11-01

    Drought and salt stresses are major environmental constraints on forest productivity. To identify genes responsible for stress tolerance, we conducted a genome-wide analysis in poplar (Populus alba × Populus glandulosa) leaves exposed to drought and salt (NaCl) stresses. We investigated gene expression at the mRNA level using oligonucleotide microarrays containing 44,718 genes from Populus trichocarpa. A total of 1604 and 1042 genes were up-regulated (≥2-fold; P value < 0.05) by drought and salt stresses, respectively, and 765 genes were up-regulated by both stresses. In addition, 2742 and 1685 genes were down-regulated by drought and salt stresses, respectively, and 1564 genes were down-regulated by both stresses. The large number of genes regulated by both stresses suggests that crosstalk occurs between the drought and salt stress responses. Most up-regulated genes were involved in functions such as subcellular localization, signal transduction, metabolism, and transcription. Among the up-regulated genes, we identified 47 signaling proteins, 65 transcription factors, and 43 abiotic stress-related genes. Several genes were modulated by only one of the two stresses. About 25% of the genes significantly regulated by these stresses are of unknown function, suggesting that poplar may provide an opportunity to discover novel stress-related genes.

  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

    USDA-ARS?s Scientific Manuscript database

    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. UPS 2.0: unique probe selector for probe design and oligonucleotide microarrays at the pangenomic/genomic level.

    PubMed

    Chen, Shu-Hwa; Lo, Chen-Zen; Su, Sheng-Yao; Kuo, Bao-Han; Hsiung, Chao A; Lin, Chung-Yen

    2010-12-02

    Nucleic acid hybridization is an extensively adopted principle in biomedical research, in which the performance of any hybridization-based method depends on the specificity of probes to their targets. To determine the optimal probe(s) for detecting target(s) from a sample cocktail, we developed a novel algorithm, which has been implemented into a web platform for probe designing. This probe design workflow is now upgraded to satisfy experiments that require a probe designing tool to take the increasing volume of sequence datasets. Algorithms and probe parameters applied in UPS 2.0 include GC content, the secondary structure, melting temperature (Tm), the stability of the probe-target duplex estimated by the thermodynamic model, sequence complexity, similarity of probes to non-target sequences, and other empirical parameters used in the laboratory. Several probe background options,Unique probe within a group,Unique probe in a specific Unigene set,Unique probe based on the pangenomic level, and Unique Probe in the user-defined genome/transcriptome, are available to meet the scenarios that the experiments will be conducted. Parameters, such as salt concentration and the lower-bound Tm of probes, are available for users to optimize their probe design query. Output files are available for download on the result page. Probes designed by the UPS algorithm are suitable for generating microarrays, and the performance of UPS-designed probes has been validated by experiments. The UPS 2.0 evaluates probe-to-target hybridization under a user-defined condition to ensure high-performance hybridization with minimal chance of non-specific binding at the pangenomic and genomic levels. The UPS algorithm mimics the target/non-target mixture in an experiment and is very useful in developing diagnostic kits and microarrays. The UPS 2.0 website has had more than 1,300 visits and 360,000 sequences performed the probe designing task in the last 30 months. It is freely accessible at http

  20. Analysis of differentially expressed genes in colorectal adenocarcinoma with versus without metastasis by three-dimensional oligonucleotide microarray

    PubMed Central

    Moura Franco, Rita MAM; Linhares, Marcelo M; Lustosa, Suzana S; Silva, Ismael DGC; Souza, Naiara CN; Matos, Delcio

    2014-01-01

    Background: Our objective was to examine how the gene expression profile of tumor tissue correlates with lymph node metastasis in patients with advanced colorectal adenocarcinoma (CRAC). Methods: We studied 36 patients (20 men and 16 women, 22-90 years of age) treated for CRAC (classifications of T2, T3, or T4; histological grade of G1 or G2). Amplified tumor mRNA samples were exposed to 20,000 human sequence probes and digitized images of the hybridized samples were analyzed. Results: On average, 2389 probes were detected above the background, with an average correlation R value of 0.19 between data from different patient groups (with or without lymph node invasion, colon or rectal, with or without angio-lymphatic invasion, with or without recurrence). Lymph node metastasis had a statistically significant signature according to Significance Analysis of Microarrays (SAM) and parametric t-tests, with a false discovery rate (FDR) = 0.1% and p = 0.001, respectively. Cross-correlation of these two tests identified 102 transcripts as being potentially related to node metastases, with fold changes in the range of 2.182-12.960. Conclusion: We identified 102 differentially expressed genes related to the presence of lymph node metastases in patients with advanced colorectal cancer. PMID:24427346

  1. Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

    PubMed Central

    Kamalakaran, Sitharthan; Kendall, Jude; Zhao, Xiaoyue; Tang, Chunlao; Khan, Sohail; Ravi, Kandasamy; Auletta, Theresa; Riggs, Michael; Wang, Yun; Helland, Åslaug; Naume, Bjørn; Dimitrova, Nevenka; Børresen-Dale, Anne-Lise; Hicks, Jim; Lucito, Robert

    2009-01-01

    Methylation of CpG islands associated with genes can affect the expression of the proximal gene, and methylation of non-associated CpG islands correlates to genomic instability. This epigenetic modification has been shown to be important in many pathologies, from development and disease to cancer. We report the development of a novel high-resolution microarray that detects the methylation status of over 25 000 CpG islands in the human genome. Experiments were performed to demonstrate low system noise in the methodology and that the array probes have a high signal to noise ratio. Methylation measurements between different cell lines were validated demonstrating the accuracy of measurement. We then identified alterations in CpG islands, both those associated with gene promoters, as well as non-promoter-associated islands in a set of breast and ovarian tumors. We demonstrate that this methodology accurately identifies methylation profiles in cancer and in principle it can differentiate any CpG methylation alterations and can be adapted to analyze other species. PMID:19474344

  2. A low density microarray method for the identification of human papillomavirus type 18 variants.

    PubMed

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

    2013-09-26

    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.

  3. 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

  4. High-density polymer microarrays: identifying synthetic polymers that control human embryonic stem cell growth.

    PubMed

    Hansen, Anne; Mjoseng, Heidi K; Zhang, Rong; Kalloudis, Michail; Koutsos, Vasileios; de Sousa, Paul A; Bradley, Mark

    2014-06-01

    The fabrication of high-density polymer microarray is described, allowing the simultaneous and efficient evaluation of more than 7000 different polymers in a single-cellular-based screen. These high-density polymer arrays are applied in the search for synthetic substrates for hESCs culture. Up-scaling of the identified hit polymers enables long-term cellular cultivation and promoted successful stem-cell maintenance.

  5. 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.

  6. 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.

  7. Genomic Analysis of Vaccine-Derived Poliovirus Strains in Stool Specimens by Combination of Full-Length PCR and Oligonucleotide Microarray Hybridization

    PubMed Central

    Laassri, Majid; Dragunsky, Eugenia; Enterline, Joan; Eremeeva, Tatiana; Ivanova, Olga; Lottenbach, Kathleen; Belshe, Robert; Chumakov, Konstantin

    2005-01-01

    Sabin strains of poliovirus used in the manufacture of oral poliovirus vaccine (OPV) are prone to genetic variations that occur during growth in cell cultures and the organisms of vaccine recipients. Such derivative viruses often have increased neurovirulence and transmissibility, and in some cases they can reestablish chains of transmission in human populations. Monitoring for vaccine-derived polioviruses is an important part of the worldwide campaign to eradicate poliomyelitis. Analysis of vaccine-derived polioviruses requires, as a first step, their isolation in cell cultures, which takes significant time and may yield viral stocks that are not fully representative of the strains present in the original sample. Here we demonstrate that full-length viral cDNA can be PCR amplified directly from stool samples and immediately subjected to genomic analysis by oligonucleotide microarray hybridization and nucleotide sequencing. Most fecal samples from healthy children who received OPV were found to contain variants of Sabin vaccine viruses. Sequence changes in the 5′ untranslated region were common, as were changes in the VP1-coding region, including changes in a major antigenic site. Analysis of stool samples taken from cases of acute flaccid paralysis revealed the presence of mixtures of recombinant polioviruses, in addition to the emergence of new sequence variants. Avoiding the need for cell culture isolation dramatically shortened the time needed for identification and analysis of vaccine-derived polioviruses and could be useful for preliminary screening of clinical samples. The amplified full-length viral cDNA can be archived and used to recover live virus for further virological studies. PMID:15956413

  8. The proximal chromosome 14q microdeletion syndrome: delineation of the phenotype using high resolution SNP oligonucleotide microarray analysis (SOMA) and review of the literature.

    PubMed

    Torgyekes, Edina; Shanske, Alan L; Anyane-Yeboa, Kwame; Nahum, Odelia; Pirzadeh, Sara; Blumfield, Einat; Jobanputra, Vaidehi; Warburton, Dorothy; Levy, Brynn

    2011-08-01

    We report on two patients with overlapping small interstitial deletions involving regions 14q12 to 14q13.1. Both children had severe developmental delay, failure to thrive, microcephaly, and distinctive facial features, including abnormal spacing of the eyes, epicanthal folds, sloping forehead, low-set ears, rounded eyebrows with triangular media aspect and outer tapering, depressed and broad nasal bridge, small mouth, a long philtrum, and a prominent Cupid's bow. Brain MRI of both children showed partial agenesis of the corpus callosum. Our first patient had bilateral hypoplastic optic nerves causing blindness, mild hearing impairment, sinus arrhythmia, abnormal temperature regulation, frequent apneic episodes, myoclonic jerks, and opisthotonus. Our second patient had a seizure disorder confirmed by EEG, sleep apnea, chronic interstitial lung disease, and several episodes of pneumonia and gastroenteritis. Cytogenetic analysis showed a normal karyotype in Patient 1 and a unique apparently balanced three-way translocation in Patient 2 involving chromosomes 4, 14, and 11. High resolution SNP Oligonucleotide Microarray Analysis (SOMA) revealed a deletion in the proximal region of chromosome 14q overlapping with the deletion of our first patient, and no copy number changes in chromosomes 4 and 11. Here, we review and compare published cases with a deletion involving the 14q12-22.1 chromosomal region in an effort to correlate phenotype and genotype. We also examine the underlying genomic architecture to identify the possible mechanism of the chromosomal abnormality. Our review found a patient with a mirror duplication of our first patient's deletion, confirming the existence of an underlying genomic structural instability in the region. © 2011 Wiley-Liss, Inc.

  9. Detection of CFTR mutations using ARMS and low-density microarrays.

    PubMed

    Eaker, Shannon; Johnson, Matt; Jenkins, Josh; Bauer, Martin; Little, Stephen

    2005-12-15

    The amplification refractory mutation system (ARMS) is routinely used for the identification of specific mutations within genomes. This PCR-based assay, although simple, is performed at a low-throughput scale, usually requiring gel-electrophoresis for the identification of specific mutations. We have applied the ARMS technology to a low-density microarray system to facilitate the needs of the medical clinic; high-throughput capabilities and ease-of-use. Mutations within the cystic fibrosis transmembrane regulator (CFTR) gene (DeltaF508, 1717-1G>A, G542X, 621+1G>T, and N1303K) were detected by multiplex-ARMS-PCR, and fragments were post-PCR labeled with Cy5. Amine-modified probes specific for both the wild-type and mutant forms of each mutation site were attached to glass substrates. Following hybridization of the PCR fragments to the attached probes (in a low-density microarray format), confirmation of the presence of specific sequences was achieved using a commercial scanner, as well as a fabricated low-cost fluorescent detector and applicable software. The novel combination of the ARMS and low-density microarray technologies allows for a high-throughput, simple means to rapidly identify multiple known mutations for many genetic diseases including cystic fibrosis.

  10. Superhydrophilic-Superhydrophobic Patterned Surfaces as High-Density Cell Microarrays: Optimization of Reverse Transfection.

    PubMed

    Ueda, Erica; Feng, Wenqian; Levkin, Pavel A

    2016-10-01

    High-density microarrays can screen thousands of genetic and chemical probes at once in a miniaturized and parallelized manner, and thus are a cost-effective alternative to microwell plates. Here, high-density cell microarrays are fabricated by creating superhydrophilic-superhydrophobic micropatterns in thin, nanoporous polymer substrates such that the superhydrophobic barriers confine both aqueous solutions and adherent cells within each superhydrophilic microspot. The superhydrophobic barriers confine and prevent the mixing of larger droplet volumes, and also control the spreading of droplets independent of the volume, minimizing the variability that arises due to different liquid and surface properties. Using a novel liposomal transfection reagent, ScreenFect A, the method of reverse cell transfection is optimized on the patterned substrates and several factors that affect transfection efficiency and cytotoxicity are identified. Higher levels of transfection are achieved on HOOC- versus NH2 -functionalized superhydrophilic spots, as well as when gelatin and fibronectin are added to the transfection mixture, while minimizing the amount of transfection reagent improves cell viability. Almost no diffusion of the printed transfection mixtures to the neighboring microspots is detected. Thus, superhydrophilic-superhydrophobic patterned surfaces can be used as cell microarrays and for optimizing reverse cell transfection conditions before performing further cell screenings. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. 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…

  12. 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…

  13. Density Variant Glycan Microarray for Evaluating Cross-Linking of Mucin-like Glycoconjugates by Lectins

    PubMed Central

    2012-01-01

    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 A1-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

  14. [Using high-density universal 16S rRNA microarray in microbial molecular ecology research].

    PubMed

    Duan, Liang; Xia, Si-qing; Song, Yong-hui; Piceno, Yvette M; Hermanowicz, Slawomir W

    2009-12-01

    High-density universal 16S rRNA Microarray was used to detect the microbial community in membrane bioreactor (MBR). The results showed MBR had high microbial diversity. 1019 bacteria were detected by Microarray in MBR. Proteobacteria was the predominant phylum. It had 657 OTUs, occupying 64.5% of the total bacteria, gamma Proteobacteria was the main class of Proteobacteria, which occupied 35.8% of Proteobacteria, but the average fluorescence intensity of was not very high. Despite the bacteria diversity of beta Proteobacteria was lower than gamma Proteobacteria. However, it occupied 40% and 36% in the 25 and 50 dominant bacteria according to relative average fluorescence intensity. Clostridia had a relative high concentration when compare the average fluorescence intensity of the whole bacteria in MBR. Some common nitrify bacteria such as Nitrosomonadaceae and Nitrospiraceae were also detected and had high fluorescence intensity in the system. Microarray is a newer molecular method and has some advantages such as real time, high efficiency and exact results. It's very suitable for investigation of microbial diversity in wastewater treatment.

  15. Representational oligonucleotide microarray analysis (ROMA) and comparison of binning and change-point methods of analysis: application to detection of del22q11.2 (DiGeorge) syndrome.

    PubMed

    Stanczak, Christopher M; Chen, Zugen; Nelson, Stanley F; Suchard, Marc; McCabe, Edward R B; McGhee, Sean

    2008-01-01

    DiGeorge (del22q11.2) syndrome is estimated to occur in 1:4,000 births, is the most common contiguous-gene deletion syndrome in humans, and is caused by autosomal dominant deletions in the 22q11.2 DiGeorge syndrome critical region (DGCR). Multiple microarray methods have been developed recently for analyzing such copy number changes, but data analysis and accurate deletion detection remains challenging. Clinical use of these microarray methods would have many advantages, particularly when the possibility of a chromosomal disorder cannot be determined simply on the basis of history and physical examination data alone. We investigated the use of the microarray technique, representational oligonucleotide microarray analysis (ROMA), in the detection of del22q11.2 syndrome. Genomic DNA was isolated from three well-characterized cell lines with 22q11.2 DGCR deletions and from the blood of a patient suspected of having del22q11.2 syndrome, and analyzed using both the binning and change-point model algorithms. Though the 22q11.2 deletion was easily identified with either method, change-point models provide clearer identification of deleted regions, with the potential for fewer false-positive results. For circumstances in which a clear, a priori, copy-number change hypothesis is not present, such as in many clinical samples, change-point methods of analysis may be easier to interpret.

  16. Microarrays with Varying Carbohydrate Density Reveal Distinct Subpopulations of Serum Antibodies

    PubMed Central

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

    2009-01-01

    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

  17. Density based pruning for identification of differentially expressed genes from microarray data

    PubMed Central

    2010-01-01

    Motivation Identification of differentially expressed genes from microarray datasets is one of the most important analyses for microarray data mining. Popular algorithms such as statistical t-test rank genes based on a single statistics. The false positive rate of these methods can be improved by considering other features of differentially expressed genes. Results We proposed a pattern recognition strategy for identifying differentially expressed genes. Genes are mapped to a two dimension feature space composed of average difference of gene expression and average expression levels. A density based pruning algorithm (DB Pruning) is developed to screen out potential differentially expressed genes usually located in the sparse boundary region. Biases of popular algorithms for identifying differentially expressed genes are visually characterized. Experiments on 17 datasets from Gene Omnibus Database (GEO) with experimentally verified differentially expressed genes showed that DB pruning can significantly improve the prediction accuracy of popular identification algorithms such as t-test, rank product, and fold change. Conclusions Density based pruning of non-differentially expressed genes is an effective method for enhancing statistical testing based algorithms for identifying differentially expressed genes. It improves t-test, rank product, and fold change by 11% to 50% in the numbers of identified true differentially expressed genes. The source code of DB pruning is freely available on our website http://mleg.cse.sc.edu/degprune PMID:21047384

  18. A high-density transcript linkage map with 1,845 expressed genes positioned by microarray-based Single Feature Polymorphisms (SFP) in Eucalyptus

    PubMed Central

    2011-01-01

    Background Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers. Results SFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome. Conclusions The Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping

  19. Clustering by fast search and merge of local density peaks for gene expression microarray data.

    PubMed

    Mehmood, Rashid; El-Ashram, Saeed; Bie, Rongfang; Dawood, Hussain; Kos, Anton

    2017-04-19

    Clustering is an unsupervised approach to classify elements based on their similarity, and it is used to find the intrinsic patterns of data. There are enormous applications of clustering in bioinformatics, pattern recognition, and astronomy. This paper presents a clustering approach based on the idea that density wise single or multiple connected regions make a cluster, in which density maxima point represents the center of the corresponding density region. More precisely, our approach firstly finds the local density regions and subsequently merges the density connected regions to form the meaningful clusters. This idea empowers the clustering procedure, in which outliers are automatically detected, higher dense regions are intuitively determined and merged to form clusters of arbitrary shape, and clusters are identified regardless the dimensionality of space in which they are embedded. Extensive experiments are performed on several complex data sets to analyze and compare our approach with the state-of-the-art clustering methods. In addition, we benchmarked the algorithm on gene expression microarray data sets for cancer subtyping; to distinguish normal tissues from tumor; and to classify multiple tissue data sets.

  20. Clustering by fast search and merge of local density peaks for gene expression microarray data

    PubMed Central

    Mehmood, Rashid; El-Ashram, Saeed; Bie, Rongfang; Dawood, Hussain; Kos, Anton

    2017-01-01

    Clustering is an unsupervised approach to classify elements based on their similarity, and it is used to find the intrinsic patterns of data. There are enormous applications of clustering in bioinformatics, pattern recognition, and astronomy. This paper presents a clustering approach based on the idea that density wise single or multiple connected regions make a cluster, in which density maxima point represents the center of the corresponding density region. More precisely, our approach firstly finds the local density regions and subsequently merges the density connected regions to form the meaningful clusters. This idea empowers the clustering procedure, in which outliers are automatically detected, higher dense regions are intuitively determined and merged to form clusters of arbitrary shape, and clusters are identified regardless the dimensionality of space in which they are embedded. Extensive experiments are performed on several complex data sets to analyze and compare our approach with the state-of-the-art clustering methods. In addition, we benchmarked the algorithm on gene expression microarray data sets for cancer subtyping; to distinguish normal tissues from tumor; and to classify multiple tissue data sets. PMID:28422088

  1. Self-assembled random arrays: high-performance imaging and genomics applications on a high-density microarray platform

    NASA Astrophysics Data System (ADS)

    Barker, David L.; Theriault, Greg; Che, Diping; Dickinson, Todd; Shen, Richard; Kain, Robert C.

    2003-07-01

    Illumina is developing a BeadArrayTM technology that supports SNP genotyping, mRNA expression analysis and protein expression analysis on the same platform. We use fiber-optic bundles with a density of approximately 40,000 fibers/mm2. At hte end of each fiber, a derivatized silica bead forms an array element for reading out a genotyping or expression assay data point. Each bead contains oligonucleotide probes that hybridize with high specificity to complementary sequences in a complex nucleic acid mixture. We derivatize the beads in bulk, pool them to form a quality-controlled source of microarray elements, and allow them to assemble spontaneously into pits etched into the end of each optical fiber bundle. We load our fiber bundles, containing 49,777 fibers, with up to 1520 different bead types. The presence of many beads of each type greatly improves the accuracy of each assay. As the final step in our manufacturing process, we decode the identity of each bead by a series of rapid hybridizations with fluroescent oligos. Decoding accuracy and the number of beads of each type is recorded for each array. Decoding also serves as a quality control procedure for the performance of each element in the array. To facilitate high-throughput analysis of many samples, the fiber bundles are arranged in an array matrix (SentrixTM arrays). Using a 96-bundle array matrix, up to 1520 assays can be performed on each of 96 samples simultaneously for a total of 145,920 assays. Using a 384-bundle array matrix, up to 583,680 assays can be performed simultaneously. The BeadArray platform is the highest density microarray in commercial use, requiring development of a high-performance array scanner. To meet this need, we developed the SherlockTM system, a laser-scanning confocal imaging system that automatically scans all 96 bundles of an array matrix at variable resolution down to 0.8 micron. The system scans with both 532 and 635 nm lasers simultaneously, collecting two fluorescence

  2. Comparing three methods for variance estimation with duplicated high density oligonucleotide arrays.

    PubMed

    Huang, Xiaohong; Pan, Wei

    2002-08-01

    Microarray experiments are being increasingly used in molecular biology. A common task is to detect genes with differential expression across two experimental conditions, such as two different tissues or the same tissue at two time points of biological development. To take proper account of statistical variability, some statistical approaches based on the t-statistic have been proposed. In constructing the t-statistic, one needs to estimate the variance of gene expression levels. With a small number of replicated array experiments, the variance estimation can be challenging. For instance, although the sample variance is unbiased, it may have large variability, leading to a large mean squared error. For duplicated array experiments, a new approach based on simple averaging has recently been proposed in the literature. Here we consider two more general approaches based on nonparametric smoothing. Our goal is to assess the performance of each method empirically. The three methods are applied to a colon cancer data set containing 2,000 genes. Using two arrays, we compare the variance estimates obtained from the three methods. We also consider their impact on the t-statistics. Our results indicate that the three methods give variance estimates close to each other. Due to its simplicity and generality, we recommend the use of the smoothed sample variance for data with a small number of replicates.

  3. Specific mutation screening of TP53 gene by low-density DNA microarray

    PubMed Central

    Rangel-López, Angélica; Méndez-Tenorio, Alfonso; Beattie, Kenneth L; Maldonado, Rogelio; Mendoza, Patricia; Vázquez, Guelaguetza; Pérez-Plasencia, Carlos; Sánchez, Martha; Navarro, Guillermo; Salcedo, Mauricio

    2009-01-01

    TP53 is the most commonly mutated gene in human cancers. Approximately 90% of mutations in this gene are localized between domains encoding exons 5 to 8. The aim of this investigation was to examine the ability of the low density DNA microarray with the assistance of double tandem hybridization platform to characterize TP53 mutational hotspots in exons 5, 7, and 8 of the TP53. Nineteen capture probes specific to each potential mutation site were designed to hybridize to specific site. Virtual hybridization was used to predict the stability of hybridization of each capture probe with the target. Thirty-three DNA samples from different sources were analyzed for mutants in these exons. A total of 32 codon substitutions were found by DNA sequencing. 24 of them a showed a perfect correlation with the hybridization pattern system and DNA sequencing analysis of the regions scanned. Although in this work we directed our attention to some of the most representative mutations of the TP53 gene, the results suggest that this microarray system proved to be a rapid, reliable, and effective method for screening all the mutations in TP53 gene. PMID:24198462

  4. Peptide microarray with ligands at high density based on symmetrical carrier landscape phage for detection of cellulase.

    PubMed

    Qi, Huan; Wang, Fei; Petrenko, Valery A; Liu, Aihua

    2014-06-17

    Peptide microarrays evolved recently as a routine analytical implementation in various research areas due to their unique characteristics. However, the immobilization of peptides with high density in each spot during the fabricating process remains a problem, which will affect the performance of the resultant microarray greatly. To respond to this challenge, a novel peptide immobilization method using symmetrical phage carrier was developed in this work. The cellulytic enzyme endoglucanase I (EG I) was used as a model for selection of its specific peptide ligands from the f8/8 landscape library. Three phage monoclones were selected and identified by the specificity array, of which one phage monoclone displaying the fusion peptide EGSDPRMV (phage EGSDPRMV) could bind EG I specifically with highest affinity. Subsequently, the phage EGSDPRMV was used directly to construct peptide microarray. For comparison, major coat protein pVIII fused EG I specific peptide EGSDPRMV (pVIII-fused EGSDPRMV) which was isolated from phage EGSDPRMV was also immobilized by traditional method to fabricate peptide microarray. The fluorescent signal of the phage EGSDPRMV-mediated peptide microarray was more reproducible and about four times higher than the value for pVIII-fused EGSDPRMV-based microarray, suggesting the high efficiency of the proposed phage EGSDPRMV-mediated peptide immobilization method. Further, the phage EGSDPRMV based microarray not only simplified the procedure of microarray construction but also exhibited significantly enhanced sensitivity due to the symmetrical carrier landscape phage, which dramatically increased the density and sterical regularity of immobilized peptides in each spot. Thus, the proposed strategy has the advantages that the immobilizing peptide ligands were not disturbed by their composition and the immobilized peptides were highly regular with free amino-terminal.

  5. Generalized Logical Network Modeling of Interactions Among Bacteria in Aerosols Under Meteorological Factors Using High Density Phylogenetic Microarrays

    NASA Astrophysics Data System (ADS)

    Song, J.; Luce, C.; Desantis, T.; Arkin, A.; Brodie, E.; Andersen, G.

    2007-12-01

    The generalized logical network model utilizes temporal information in the 16S rRNA gene concentration time- course to examine interactions of bacteria within a microbial community under meteorological factors. The Biowatch aerosol bacterial community data set (Brodie et al., PNAS 104[1]:299-304, 2007) of 8,763 taxa intensities was generated using 237 16S rRNA oligonucleotide phylogenetic microarrays at 30 locations throughout the U.S. over time-courses of up to 20 weeks at each location. Each microarray contains about 9,000 probe sets, with an average of 24 probes per set. Seventy-two meteorological factors were measured at the time each microarray was analyzed. In a generalized logical network, a generalized truth table, associated with every node representing either a bacterial taxon or a meteorological factor, describes the represented bacterial behavior dictated by some environmental factors in addition to associations with other bacterial taxa. The optimal generalized logics at each bacterial node in the network will be searched so that they best explain the observed time-course data. Determination of an optimal logic will involve parent node selection and generalized truth-table generation. The maximum number of parents is set to a given number. If the current node shows consistent behavior during transition from one state to another given the parent nodes, then the parent nodes are kept. The actual goodness of the transition is calculated using the chi-square test. In addition to the dependency of the concentrations of bacteria on meteorological factors, with various time delays, the initial generalized logical network modeling results indicate that the concentrations of specific bacterial taxa are also associated with concentrations of other bacteria.

  6. Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses

    PubMed Central

    Sforzini, Susanna; Arlt, Volker M.; Barranger, Audrey; Dallas, Lorna J.; Oliveri, Caterina; Aminot, Yann; Pacchioni, Beniamina; Millino, Caterina; Lanfranchi, Gerolamo; Readman, James W.; Moore, Michael N.; Viarengo, Aldo; Jha, Awadhesh N.

    2017-01-01

    Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new ‘STressREsponse Microarray’ (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 μg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels

  7. Optimization of high-density cDNA-microarray protocols by ‘design of experiments’

    PubMed Central

    Wrobel, Gunnar; Schlingemann, Joerg; Hummerich, Lars; Kramer, Heidi; Lichter, Peter; Hahn, Meinhard

    2003-01-01

    Expression analysis using microarray technology implies a complex experimental procedure with a large number of parameters affecting the final result. We have demonstrated that optimization of such a complex protocol can be far better handled using design of experiments (DOE) than by working on a single parameter at a time. Based on the results of a screening design, we developed a spotting buffer composed of formamide, betaine and nitrocellulose. This buffer provides a 2-fold increase in signal-to-background ratio compared to 3× SSC. Comparison to seven other buffers tested on 10 different substrates revealed it had the highest sensitivity. DNA dissolved in this buffer can be spotted on epoxysilane-coated microscope slides at a density of up to 70 000 spots per slide. A second DOE approach characterized the RNA labeling process with regard to the concentration of fluorescent dyes, dNTPs and reverse transcriptase. Adjust ments of the concentrations of dNTPs, as well as reverse transcriptase, towards the optimum, produced an improvement in the performance of the labeling procedure by a factor of 3 (Cy3) and 10 (Cy5). These results demonstrate that the process of establishing a stable expression profiling protocol and its further optimization can be significantly shortened and improved by DOE. PMID:12799456

  8. Improved porous silicon microarray based prostate specific antigen immunoassay by optimized surface density of the capture antibody.

    PubMed

    Lee, SangWook; Kim, Soyoun; Malm, Johan; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas

    2013-09-24

    Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5 ng mL(-1), 80 pg mL(-1), and 800 fg mL(-1) when arraying the PSA antibody, H117 at the concentration 15 μg mL(-1), 35 μg mL(-1), and 154 μg mL(-1), respectively. We further investigated PSA spiked into human female serum in the range of 800 fg mL(-1) to 500 ng mL(-1). The microarray showed a LOD of 800 fg mL(-1) and a dynamic range of 800 fg mL(-1) to 80 ng mL(-1) in serum spiked samples. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Microbial diversity in uranium mining-impacted soils as revealed by high-density 16S microarray and clone library.

    PubMed

    Rastogi, Gurdeep; Osman, Shariff; Vaishampayan, Parag A; Andersen, Gary L; Stetler, Larry D; Sani, Rajesh K

    2010-01-01

    Microbial diversity was characterized in mining-impacted soils collected from two abandoned uranium mine sites, the Edgemont and the North Cave Hills, South Dakota, using a high-density 16S microarray (PhyloChip) and clone libraries. Characterization of the elemental compositions of soils by X-ray fluorescence spectroscopy revealed higher metal contamination including uranium at the Edgemont than at the North Cave Hills mine site. Microarray data demonstrated extensive phylogenetic diversity in soils and confirmed nearly all clone-detected taxonomic levels. Additionally, the microarray exhibited greater diversity than clone libraries at each taxonomic level at both the mine sites. Interestingly, the PhyloChip detected the largest number of taxa in Proteobacteria phylum for both the mine sites. However, clone libraries detected Acidobacteria and Bacteroidetes as the most numerically abundant phyla in the Edgemont and North Cave Hills mine sites, respectively. Several 16S rDNA signatures found in both the microarrays and clone libraries displayed sequence similarities with yet-uncultured bacteria representing a hitherto unidentified diversity. Results from this study demonstrated that highly diverse microbial populations were present in these uranium mine sites. Diversity indices indicated that microbial communities at the North Cave Hills mine site were much more diverse than those at the Edgemont mine site.

  10. Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice.

    PubMed

    Joseph, Ajay; Lee, Taewon; Moland, Carrie L; Branham, William S; Fuscoe, James C; Leakey, Julian E A; Allaben, William T; Lewis, Sherry M; Ali, Akhtar A; Desai, Varsha G

    2009-04-01

    Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F(1) female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I-IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.

  11. 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

  12. 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

  13. Polyion oligonucleotide-decorated gold nanoparticles with tunable surface charge density for amplified signal output of potentiometric immunosensor.

    PubMed

    Lv, Shuzhen; Lin, Zhenzhen; Zhang, Kangyao; Lu, Minghua; Tang, Dianping

    2017-04-29

    Methods based on nanostructures have been developed for potentiometric immunosensors, but most involve low sensitivity or weak signal output and are unsuitable for routine use in diagnosis. Herein, we devise an in-situ signal-amplification strategy for enhanced electrical readout of potentiometric immunosensor toward target prostate-specific antigen (PSA, one kind of cancer biomarkers), based on polyion oligonucleotide-labeled gold nanoparticles (AuNPs). To decrease the background signal, monoclonal anti-human PSA capture antibody was covalently conjugated onto an activated glassy carbon electrode via typical carbodiimide coupling. AuNPs heavily functionalized with the polyion oligonucleotides and polyclonal anti-PSA detection antibodies (pAb2-AuNP-DNA) were utilized as the signal-generation nanotags. In the presence of target PSA, a sandwich-type immunoreaction was executed between capture antibody and detection antibody on the electrode. The detectable signal derived from the shift in the electric potential as a result of the change in the surface charge before and after the antigen-antibody reaction. With target PSA increased, the captured pAb2-AuNP-DNA to the electrode accompanying detection antibody increased, thereby resulting in the change of the electrode potential. Due to numerous polyion oligonucleotides with the negative charge, the signal readout amplified. Under the optimal conditions, the shift in the output potential was proportional to the logarithm of target PSA concentration and displayed a dynamic linear range from 0.05 to 20 ng mL(-1) with a detection limit of 13.6 pg mL(-1). An intermediate precision of ≤13.2% was accomplished with the batch-to-batch identification. The selectivity was acceptable. The method accuracy was evaluated for human serum specimens, and gave the consistent results between the potentiometric immunosensor and the referenced enzyme-linked immunosorbent assay (ELISA). Copyright © 2017 Elsevier B.V. All rights

  14. 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

  15. Construction of high-density tissue microarrays at low cost by using self-made manual microarray kits and recipient paraffin blocks.

    PubMed

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

    2012-12-01

    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. 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. 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. Construction of TMAs using self-made TMA kits is an inexpensive alternative to construction of TMAs using commercial devices.

  16. 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

  17. 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.

  18. 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.

  19. Detection of genome-wide polymorphisms in the AT-rich Plasmodium falciparum genome using a high-density microarray

    PubMed Central

    Jiang, Hongying; Yi, Ming; Mu, Jianbing; Zhang, Louie; Ivens, Al; Klimczak, Leszek J; Huyen, Yentram; Stephens, Robert M; Su, Xin-zhuan

    2008-01-01

    Background Genetic mapping is a powerful method to identify mutations that cause drug resistance and other phenotypic changes in the human malaria parasite Plasmodium falciparum. For efficient mapping of a target gene, it is often necessary to genotype a large number of polymorphic markers. Currently, a community effort is underway to collect single nucleotide polymorphisms (SNP) from the parasite genome. Here we evaluate polymorphism detection accuracy of a high-density 'tiling' microarray with 2.56 million probes by comparing single feature polymorphisms (SFP) calls from the microarray with known SNP among parasite isolates. Results We found that probe GC content, SNP position in a probe, probe coverage, and signal ratio cutoff values were important factors for accurate detection of SFP in the parasite genome. We established a set of SFP calling parameters that could predict mSFP (SFP called by multiple overlapping probes) with high accuracy (≥ 94%) and identified 121,087 mSFP genome-wide from five parasite isolates including 40,354 unique mSFP (excluding those from multi-gene families) and ~18,000 new mSFP, producing a genetic map with an average of one unique mSFP per 570 bp. Genomic copy number variation (CNV) among the parasites was also cataloged and compared. Conclusion A large number of mSFP were discovered from the P. falciparum genome using a high-density microarray, most of which were in clusters of highly polymorphic genes at chromosome ends. Our method for accurate mSFP detection and the mSFP identified will greatly facilitate large-scale studies of genome variation in the P. falciparum parasite and provide useful resources for mapping important parasite traits. PMID:18724869

  20. High-throughput immuno-profiling of mamba (Dendroaspis) venom toxin epitopes using high-density peptide microarrays

    PubMed Central

    Engmark, Mikael; Andersen, Mikael R.; Laustsen, Andreas H.; Patel, Jigar; Sullivan, Eric; de Masi, Federico; Hansen, Christian S.; Kringelum, Jens V.; Lomonte, Bruno; Gutiérrez, José María; Lund, Ole

    2016-01-01

    Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated. PMID:27824133

  1. Defining the humoral immune response to infectious agents using high-density protein microarrays

    PubMed Central

    Vigil, Adam; Davies, D Huw; Felgner, Philip L

    2010-01-01

    A major component of the adaptive immune response to infection is the generation of protective and long-lasting humoral immunity. Traditional approaches to understanding the host’s humoral immune response are unable to provide an integrated understanding of the antibody repertoire generated in response to infection. By studying multiple antigenic responses in parallel, we can learn more about the breadth and dynamics of the antibody response to infection. Measurement of antibody production following vaccination is also a gauge for efficacy, as generation of antibodies can protect from future infections and limit disease. Protein microarrays are well suited to identify, quantify and compare individual antigenic responses following exposure to infectious agents. This technology can be applied to the development of improved serodiagnostic tests, discovery of subunit vaccine antigen candidates, epidemiologic research and vaccine development, as well as providing novel insights into infectious disease and the immune system. In this review, we will discuss the use of protein microarrays as a powerful tool to define the humoral immune response to bacteria and viruses. PMID:20143947

  2. The prognostic implications of microvascular density and lymphatic vessel density in esophageal squamous cell carcinoma: Comparative analysis between the traditional whole sections and the tissue microarray.

    PubMed

    Chen, Bo; Fang, Wang-Kai; Wu, Zhi-Yong; Xu, Xiu-E; Wu, Jian-Yi; Fu, Jun-Hui; Yao, Xiao-Dong; Huang, Jian-Hao; Chen, Jie-Xin; Shen, Jin-Hui; Zheng, Chun-Peng; Wang, Shao-Hong; Li, En-Min; Xu, Li-Yan

    2014-05-01

    Focal distribution of microvascular and lymphatic vessels is a critical issue in cancer, and is measured by tissue microarray (TMA) construction from paraffin-embedded surgically obtained tissues, a process that may not accurately reflect true focal distribution. The aim of this study was to assess the concordance of microvascular density (MVD) and lymphatic vessel density (LVD) in TMAs with corresponding whole sections, and to correlate the MVD or LVD with clinicopathological parameters in 124 cases of esophageal squamous cell carcinoma (ESCC). MVD, determined by CD105 immunohistochemistry of whole sections, was strongly associated with lymph node metastasis (p=0.000) and pTNM stage (p=0.001). Kaplan-Meier survival analysis showed that increasing CD105 microvessel count correlated with decreasing survival (p<0.001). The same result was acquired when MVD was calculated from tissue microarrays. Analysis of continuous data showed a highly significant correlation between whole sections and TMA data (Pearson r=0.522, p<0.001). Increasing LVD, as determined by D2-40 immunohistochemistry of whole sections, correlated with decreasing survival, but this relationship was undetectable using TMAs. In conclusion, we demonstrate that for the selected endothelial markers, TMAs can provide a realistic and reliable estimate of the extent of MVD, but not LVD in ESCC samples. Copyright © 2013 Elsevier GmbH. All rights reserved.

  3. 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.

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

    PubMed

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

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2014-04-01

    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.

  6. Microarrays, antiobesity and the liver

    PubMed Central

    Castro-Chávez, Fernando

    2013-01-01

    In this review, the microarray technology and especially oligonucleotide arrays are exemplified with a practical example taken from the perilipin−/− mice and using the dChip software, available for non-lucrative purposes. It was found that the liver of perilipin−/− mice was healthy and normal, even under high-fat diet when compared with the results published for the scd1−/− mice, which under high-fat diets had a darker liver, suggestive of hepatic steatosis. Scd1 is required for the biosynthesis of monounsaturated fatty acids and plays a key role in the hepatic synthesis of triglycerides and of very-low-density lipoproteins. Both models of obesity resistance share many similar phenotypic antiobesity features, however, the perilipin−/− mice had a significant downregulation of stearoyl CoA desaturases scd1 and scd2 in its white adipose tissue, but a normal level of both genes inside the liver, even under high-fat diet. Here, different microarray methodologies are discussed, and also some of the most recent discoveries and perspectives regarding the use of microarrays, with an emphasis on obesity gene expression, and a personal remark on my findings of increased expression for hemoglobin transcripts and other hemo related genes (hemo-like), and for leukocyte like (leuko-like) genes inside the white adipose tissue of the perilipin−/− mice. In conclusion, microarrays have much to offer in comparative studies such as those in antiobesity, and also they are methodologies adequate for new astounding molecular discoveries [free full text of this article PMID:15657555

  7. Testing and Validation of High Density Resequencing Microarray for Broad Range Biothreat Agents Detection

    PubMed Central

    Leski, Tomasz A.; Lin, Baochuan; Malanoski, Anthony P.; Wang, Zheng; Long, Nina C.; Meador, Carolyn E.; Barrows, Brian; Ibrahim, Sofi; Hardick, Justin P.; Aitichou, Mohamed; Schnur, Joel M.; Tibbetts, Clark; Stenger, David A.

    2009-01-01

    Rapid and effective detection and identification of emerging microbiological threats and potential biowarfare agents is very challenging when using traditional culture-based methods. Contemporary molecular techniques, relying upon reverse transcription and/or polymerase chain reaction (RT-PCR/PCR) provide a rapid and effective alternative, however, such assays are generally designed and optimized to detect only a limited number of targets, and seldom are capable of differentiation among variants of detected targets. To meet these challenges, we have designed a broad-range resequencing pathogen microarray (RPM) for detection of tropical and emerging infectious agents (TEI) including biothreat agents: RPM-TEI v 1.0 (RPM-TEI). The scope of the RPM-TEI assay enables detection and differential identification of 84 types of pathogens and 13 toxin genes, including most of the class A, B and C select agents as defined by the Centers for Disease Control and Prevention (CDC, Atlanta, GA). Due to the high risks associated with handling these particular target pathogens, the sensitivity validation of the RPM-TEI has been performed using an innovative approach, in which synthetic DNA fragments are used as templates for testing the assay's limit of detection (LOD). Assay specificity and sensitivity was subsequently confirmed by testing with full-length genomic nucleic acids of selected agents. The LOD for a majority of the agents detected by RPM-TEI was determined to be at least 104 copies per test. Our results also show that the RPM-TEI assay not only detects and identifies agents, but is also able to differentiate near neighbors of the same agent types, such as closely related strains of filoviruses of the Ebola Zaire group, or the Machupo and Lassa arenaviruses. Furthermore, each RPM-TEI assay results in specimen-specific agent gene sequence information that can be used to assess pathogenicity, mutations, and virulence markers, results that are not generally available from

  8. A comprehensive hybridization model allows whole HERV transcriptome profiling using high density microarray.

    PubMed

    Becker, Jérémie; Pérot, Philippe; Cheynet, Valérie; Oriol, Guy; Mugnier, Nathalie; Mommert, Marine; Tabone, Olivier; Textoris, Julien; Veyrieras, Jean-Baptiste; Mallet, François

    2017-04-08

    Human endogenous retroviruses (HERVs) have received much attention for their implications in the etiology of many human diseases and their profound effect on evolution. Notably, recent studies have highlighted associations between HERVs expression and cancers (Yu et al., Int J Mol Med 32, 2013), autoimmunity (Balada et al., Int Rev Immunol 29:351-370, 2010) and neurological (Christensen, J Neuroimmune Pharmacol 5:326-335, 2010) conditions. Their repetitive nature makes their study particularly challenging, where expression studies have largely focused on individual loci (De Parseval et al., J Virol 77:10414-10422, 2003) or general trends within families (Forsman et al., J Virol Methods 129:16-30, 2005; Seifarth et al., J Virol 79:341-352, 2005; Pichon et al., Nucleic Acids Res 34:e46, 2006). To refine our understanding of HERVs activity, we introduce here a new microarray, HERV-V3. This work was made possible by the careful detection and annotation of genomic HERV/MaLR sequences as well as the development of a new hybridization model, allowing the optimization of probe performances and the control of cross-reactions. RESULTS: HERV-V3 offers an almost complete coverage of HERVs and their ancestors (mammalian apparent LTR-retrotransposons, MaLRs) at the locus level along with four other repertoires (active LINE-1 elements, lncRNA, a selection of 1559 human genes and common infectious viruses). We demonstrate that HERV-V3 analytical performances are comparable with commercial Affymetrix arrays, and that for a selection of tissue/pathological specific loci, the patterns of expression measured on HERV-V3 is consistent with those reported in the literature. Given its large HERVs/MaLRs coverage and additional repertoires, HERV-V3 opens the door to multiple applications such as enhancers and alternative promoters identification, biomarkers identification as well as the characterization of genes and HERVs/MaLRs modulation caused by viral infection.

  9. Membrane-based microarrays

    NASA Astrophysics Data System (ADS)

    Dawson, Elliott P.; Hudson, James; Steward, John; Donnell, Philip A.; Chan, Wing W.; Taylor, Richard F.

    1999-11-01

    Microarrays represent a new approach to the rapid detection and identification of analytes. Studies to date have shown that the immobilization of receptor molecules (such as DNA, oligonucleotides, antibodies, enzymes and binding proteins) onto silicon and polymeric substrates can result in arrays able to detect hundreds of analytes in a single step. The formation of the receptor/analyte complex can, itself, lead to detection, or the complex can be interrogated through the use of fluorescent, chemiluminescent or radioactive probes and ligands.

  10. Microarray-based ultra-high resolution discovery of genomic deletion mutations

    PubMed Central

    2014-01-01

    Background Oligonucleotide microarray-based comparative genomic hybridization (CGH) offers an attractive possible route for the rapid and cost-effective genome-wide discovery of deletion mutations. CGH typically involves comparison of the hybridization intensities of genomic DNA samples with microarray chip representations of entire genomes, and has widespread potential application in experimental research and medical diagnostics. However, the power to detect small deletions is low. Results Here we use a graduated series of Arabidopsis thaliana genomic deletion mutations (of sizes ranging from 4 bp to ~5 kb) to optimize CGH-based genomic deletion detection. We show that the power to detect smaller deletions (4, 28 and 104 bp) depends upon oligonucleotide density (essentially the number of genome-representative oligonucleotides on the microarray chip), and determine the oligonucleotide spacings necessary to guarantee detection of deletions of specified size. Conclusions Our findings will enhance a wide range of research and clinical applications, and in particular will aid in the discovery of genomic deletions in the absence of a priori knowledge of their existence. PMID:24655320

  11. Identification of EGFR expression status association with metastatic lymph node density (ND) by expression microarray analysis of advanced gastric cancer.

    PubMed

    Ema, Akira; Waraya, Mina; Yamashita, Keishi; Kokubo, Kenichi; Kobayashi, Hirosuke; Hoshi, Keika; Shinkai, Yoshiko; Kawamata, Hiroshi; Nakamura, Kazunori; Nishimiya, Hiroshi; Katada, Natsuya; Watanabe, Masahiko

    2015-01-01

    Metastatic lymph node density (ND) has been reproducibly proven to be a prognostic factor in gastric cancer. The molecular mechanisms that underlie this aggressiveness are underexplored. Here, we aimed to identify molecules associated with this unique phenotype. Tumor specimens from patients with stage III gastric cancer with high or low ND (n = 4 for both) were compared at the mRNA level using Affymetrix microarray (harboring 54,675 genes). The expression data were prioritized, and genes that correlated with ND were selected. Ultimately, the EGFR was validated as such a candidate molecule in patients with primary advanced gastric cancer who underwent standard treatment (n = 167). Expression data of the microarray were prioritized based on gene expression ratio and frequency of gene expression. The first priority genes to be selected were genes that are known to be amplified in cancer, which included NKX2.1, CHST9, CTNND2, SLC25A27, FGFR2, EGFR, and PTGER1. Of these genes, the EGFR gene was of particular interest. EGFR expression in primary gastric cancer was examined using immunohistochemistry (IHC). The Student's t-test elucidated a significant difference in EGFR expression between IHC 2+/3+ and IHC 1+ according to ND (P = 0.0035). The Chi-square test also indicated a significant difference between high and low levels of EGFR immunohistochemical staining (IHC2+/3+ and IHC1+, respectively) and ND status (P = 0.0023). According to the least squares method, as ND increased, the risk that EGFR staining levels changed from IHC 1+ to IHC 2+ also increased. In this study, we determined that high EGFR expression may underlie the aggressive mechanism of advanced gastric cancer with high ND. © 2014 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

  12. Identification of EGFR expression status association with metastatic lymph node density (ND) by expression microarray analysis of advanced gastric cancer

    PubMed Central

    Ema, Akira; Waraya, Mina; Yamashita, Keishi; Kokubo, Kenichi; Kobayashi, Hirosuke; Hoshi, Keika; Shinkai, Yoshiko; Kawamata, Hiroshi; Nakamura, Kazunori; Nishimiya, Hiroshi; Katada, Natsuya; Watanabe, Masahiko

    2015-01-01

    Metastatic lymph node density (ND) has been reproducibly proven to be a prognostic factor in gastric cancer. The molecular mechanisms that underlie this aggressiveness are underexplored. Here, we aimed to identify molecules associated with this unique phenotype. Tumor specimens from patients with stage III gastric cancer with high or low ND (n = 4 for both) were compared at the mRNA level using Affymetrix microarray (harboring 54,675 genes). The expression data were prioritized, and genes that correlated with ND were selected. Ultimately, the EGFR was validated as such a candidate molecule in patients with primary advanced gastric cancer who underwent standard treatment (n = 167). Expression data of the microarray were prioritized based on gene expression ratio and frequency of gene expression. The first priority genes to be selected were genes that are known to be amplified in cancer, which included NKX2.1, CHST9, CTNND2, SLC25A27, FGFR2, EGFR, and PTGER1. Of these genes, the EGFR gene was of particular interest. EGFR expression in primary gastric cancer was examined using immunohistochemistry (IHC). The Student's t-test elucidated a significant difference in EGFR expression between IHC 2+/3+ and IHC 1+ according to ND (P = 0.0035). The Chi-square test also indicated a significant difference between high and low levels of EGFR immunohistochemical staining (IHC2+/3+ and IHC1+, respectively) and ND status (P = 0.0023). According to the least squares method, as ND increased, the risk that EGFR staining levels changed from IHC 1+ to IHC 2+ also increased. In this study, we determined that high EGFR expression may underlie the aggressive mechanism of advanced gastric cancer with high ND. PMID:25154973

  13. "Harshlighting" small blemishes on microarrays

    PubMed Central

    Suárez-Fariñas, Mayte; Haider, Asifa; Wittkowski, Knut M

    2005-01-01

    Background Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs). Results We present a method that harnesses the statistical power provided by having several HDONAs available, which are obtained under similar conditions except for the experimental factor. This method "harshlights" blemishes and renders them evident. We find empirically that about 25% of our chips are blemished, and we analyze the impact of masking them on screening for differentially expressed genes. Conclusion Experiments attempting to assess subtle expression changes should be carefully screened for blemishes on the chips. The proposed method provides investigators with a novel robust approach to improve the sensitivity of microarray analyses. By utilizing topological information to identify and mask blemishes prior to model based analyses, the method prevents artefacts from confounding the process of background correction, normalization, and summarization. PMID:15784152

  14. "Harshlighting" small blemishes on microarrays.

    PubMed

    Suárez-Fariñas, Mayte; Haider, Asifa; Wittkowski, Knut M

    2005-03-22

    Microscopists are familiar with many blemishes that fluorescence images can have due to dust and debris, glass flaws, uneven distribution of fluids or surface coatings, etc. Microarray scans show similar artefacts, which affect the analysis, particularly when one tries to detect subtle changes. However, most blemishes are hard to find by the unaided eye, particularly in high-density oligonucleotide arrays (HDONAs). We present a method that harnesses the statistical power provided by having several HDONAs available, which are obtained under similar conditions except for the experimental factor. This method "harshlights" blemishes and renders them evident. We find empirically that about 25% of our chips are blemished, and we analyze the impact of masking them on screening for differentially expressed genes. Experiments attempting to assess subtle expression changes should be carefully screened for blemishes on the chips. The proposed method provides investigators with a novel robust approach to improve the sensitivity of microarray analyses. By utilizing topological information to identify and mask blemishes prior to model based analyses, the method prevents artefacts from confounding the process of background correction, normalization, and summarization.

  15. 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

  16. High-density functionalization and cross-linking of DNA: "click" and "bis-click" cycloadditions performed on alkynylated oligonucleotides with fluorogenic anthracene azides.

    PubMed

    Pujari, Suresh S; Ingale, Sachin A; Seela, Frank

    2014-10-15

    High density functionalization of DNA with ethynyl and octadiynyl side chains followed by CuAAC "click labeling" with 9-azidomethylanthracene was performed. Alkynyl DNA was also cross-linked with fluorogenic 9,10-bis-azidomethylanthracene employing the "bis-click" reaction. By this means the fluorescence of the anthracene moiety was imparted to the virtually nonfluorescent DNA. Phosphoramidites of 8-aza-7-deaza-2'-deoxyadenosine with short and long linker arms in a steric nondemanding 7-position were utilized in solid phase oligodeoxynucleotide synthesis. High density alkynylated DNA-without anthracene residues-was found to be of comparable stability with both long and short linker arms. High density anthracene functionalized DNA became less stable with the short linker compared to that with the long linker connectivity. Interstrand cross-linked homodimers constructed from alkynylated oligonucleotides with fluorogenic 9,10-bis-azidomethylanthracene were hybridized with complementary strands to form double helices. They are more stable when the linker was located at a terminus than in a central position. Short linker anthracene adducts were destabilizing compared to long linker adducts. The fluorogenic anthracene residues not only have a significant effect on the duplex stability, but also impart fluorescence to the species. Fluorescence of cross-linked double helices with long linker connectivity was quenched when the cross-link was in a terminal position and was dequenched when the linker was connecting the two double helices at the center of the molecule. The fluorescence of the anthracene cross-linked double helices was strongly increased (dequenched) when the correct base pair was formed, while no change occurred upon mismatch formation.

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

    PubMed

    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.

  18. 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

  19. Efficiency clustering for low-density microarrays and its application to QPCR

    PubMed Central

    2010-01-01

    Background Pathway-targeted or low-density arrays are used more and more frequently in biomedical research, particularly those arrays that are based on quantitative real-time PCR. Typical QPCR arrays contain 96-1024 primer pairs or probes, and they bring with it the promise of being able to reliably measure differences in target levels without the need to establish absolute standard curves for each and every target. To achieve reliable quantification all primer pairs or array probes must perform with the same efficiency. Results Our results indicate that QPCR primer-pairs differ significantly both in reliability and efficiency. They can only be used in an array format if the raw data (so called CT values for real-time QPCR) are transformed to take these differences into account. We developed a novel method to obtain efficiency-adjusted CT values. We introduce transformed confidence intervals as a novel measure to identify unreliable primers. We introduce a robust clustering algorithm to combine efficiencies of groups of probes, and our results indicate that using n < 10 cluster-based mean efficiencies is comparable to using individually determined efficiency adjustments for each primer pair (N = 96-1024). Conclusions Careful estimation of primer efficiency is necessary to avoid significant measurement inaccuracies. Transformed confidence intervals are a novel method to assess and interprete the reliability of an efficiency estimate in a high throughput format. Efficiency clustering as developed here serves as a compromise between the imprecision in assuming uniform efficiency, and the computational complexity and danger of over-fitting when using individually determined efficiencies. PMID:20646303

  20. Diversity of bacterioplankton in contrasting Tibetan lakes revealed by high-density microarray and clone library analysis.

    PubMed

    Zhang, Rui; Wu, Qinglong; Piceno, Yvette M; Desantis, Todd Z; Saunders, F Michael; Andersen, Gary L; Liu, Wen-Tso

    2013-11-01

    Tibetan lakes represent a unique microbial environment and are a good ecosystem to investigate the microbial diversity of high mountain lakes and their relationship with environmental factors. The diversity and community structure of bacterioplankton in Tibetan lakes was determined using DNA fingerprinting analysis, high-density 16S rRNA gene microarray (PhyloChip) analysis, and extensive clone library analysis of bacterial 16S rRNA genes. A previously unseen high microbial diversity (1732 operational taxonomic units based on PhyloChip data) and numerous novel bacterial 16S rRNA gene sequences were observed. Abundant SAR11-like sequences retrieved from saline Lake Qinghai demonstrated a unique SAR11 phylogenetic sister clade related to the freshwater LD12 clade. Water chemistry (e.g. salinity) and altitude played important roles in the selection of bacterial taxa (both presence and relative abundance) in Tibetan lakes. The ubiquity and uniqueness of bacterial taxa, as well as the correlation between environmental factors and bacterial taxa, was observed to vary gradually with different phylogenetic levels. Our study suggested high microbial cosmopolitanism and high endemicity observed at higher and lower phylogenetic levels, respectively. © 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  1. BOND: Basic OligoNucleotide Design

    PubMed Central

    2013-01-01

    Background DNA microarrays have become ubiquitous in biological and medical research. The most difficult problem that needs to be solved is the design of DNA oligonucleotides that (i) are highly specific, that is, bind only to the intended target, (ii) cover the highest possible number of genes, that is, all genes that allow such unique regions, and (iii) are computed fast. None of the existing programs meet all these criteria. Results We introduce a new approach with our software program BOND (Basic OligoNucleotide Design). According to Kane’s criteria for oligo design, BOND computes highly specific DNA oligonucleotides, for all the genes that admit unique probes, while running orders of magnitude faster than the existing programs. The same approach enables us to introduce also an evaluation procedure that correctly measures the quality of the oligonucleotides. Extensive comparison is performed to prove our claims. BOND is flexible, easy to use, requires no additional software, and is freely available for non-commercial use from http://www.csd.uwo.ca/∼ilie/BOND/. Conclusions We provide an improved solution to the important problem of oligonucleotide design, including a thorough evaluation of oligo design programs. We hope BOND will become a useful tool for researchers in biological and medical sciences by making the microarray procedures faster and more accurate. PMID:23444904

  2. 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.

  3. Improved porous silicon (P-Si) microarray based PSA (prostate specific antigen) immunoassay by optimized surface density of the capture antibody

    PubMed Central

    Lee, SangWook; Kim, Soyoun; Malm, Johan; Jeong, Ok Chan; Lilja, Hans; Laurell, Thomas

    2014-01-01

    Enriching the surface density of immobilized capture antibodies enhances the detection signal of antibody sandwich microarrays. In this study, we improved the detection sensitivity of our previously developed P-Si (porous silicon) antibody microarray by optimizing concentrations of the capturing antibody. We investigated immunoassays using a P-Si microarray at three different capture antibody (PSA - prostate specific antigen) concentrations, analyzing the influence of the antibody density on the assay detection sensitivity. The LOD (limit of detection) for PSA was 2.5ngmL−1, 80pgmL−1, and 800fgmL−1 when arraying the PSA antibody, H117 at the concentration 15µgmL−1, 35µgmL−1 and 154µgmL−1, respectively. We further investigated PSA spiked into human female serum in the range of 800fgmL−1 to 500ngmL−1. The microarray showed a LOD of 800fgmL−1 and a dynamic range of 800 fgmL−1 to 80ngmL−1 in serum spiked samples. PMID:24016590

  4. DNA Microarray-based Ecotoxicological Biomarker Discovery in a Small Fish Model Species

    EPA Science Inventory

    This paper addresses several issues critical to use of zebrafish oligonucleotide microarrays for computational toxicology research on endocrine disrupting chemicals using small fish models, and more generally, the use of microarrays in aquatic toxicology.

  5. DNA Microarray-based Ecotoxicological Biomarker Discovery in a Small Fish Model Species

    EPA Science Inventory

    This paper addresses several issues critical to use of zebrafish oligonucleotide microarrays for computational toxicology research on endocrine disrupting chemicals using small fish models, and more generally, the use of microarrays in aquatic toxicology.

  6. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Wagner, Thomas; Huyuh, Hoan K.; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2002-01-01

    Beta-D-Xylopyranosyl-(4 - 2 )-oligonucleotides containing adenine and thymine as nucleohases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative conformational criteria, Beta-D-xylopyranosyl(4 - 2 )- oligonucleotides show Watson-Crick pairing comparable in strength to that shown by pyranosyl-RNA.

  7. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Wagner, Thomas; Huyuh, Hoan K.; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2002-01-01

    Beta-D-Xylopyranosyl-(4 - 2 )-oligonucleotides containing adenine and thymine as nucleohases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative conformational criteria, Beta-D-xylopyranosyl(4 - 2 )- oligonucleotides show Watson-Crick pairing comparable in strength to that shown by pyranosyl-RNA.

  8. A whole-genome mouse BAC microarray with 1-Mb resolution for analysis of DNA copy number changes by array comparative genomic hybridization.

    PubMed

    Chung, Yeun-Jun; Jonkers, Jos; Kitson, Hannah; Fiegler, Heike; Humphray, Sean; Scott, Carol; Hunt, Sarah; Yu, Yuejin; Nishijima, Ichiko; Velds, Arno; Holstege, Henne; Carter, Nigel; Bradley, Allan

    2004-01-01

    Microarray-based comparative genomic hybridization (CGH) has become a powerful method for the genome-wide detection of chromosomal imbalances. Although BAC microarrays have been used for mouse CGH studies, the resolving power of these analyses was limited because high-density whole-genome mouse BAC microarrays were not available. We therefore developed a mouse BAC microarray containing 2803 unique BAC clones from mouse genomic libraries at 1-Mb intervals. For the general amplification of BAC clone DNA prior to spotting, we designed a set of three novel degenerate oligonucleotide-primed (DOP) PCR primers that preferentially amplify mouse genomic sequences while minimizing unwanted amplification of contaminating Escherichia coli DNA. The resulting 3K mouse BAC microarrays reproducibly identified DNA copy number alterations in cell lines and primary tumors, such as single-copy deletions, regional amplifications, and aneuploidy.

  9. Identification of a submicroscopic 3.2 Mb chromosomal 16q12.2-13 deletion in a child with short stature, mild developmental delay, and craniofacial anomalies, by high-density oligonucleotide array-a recognizable syndrome.

    PubMed

    Chang, Ching-Fen; Li, Ling-Hui; Wang, Chung-Hsing; Tsai, Fuu-Jen; Chen, Tsai-Chuan; Wu, Jer-Yuarn; Chen, Yuan-Tsong; Tsai, Anne Chun-Hui

    2010-09-01

    Interstitial deletion of 16q has emerged into a recognizable pattern of congenital malformation. We report on a 9-year-old boy with short stature, psychomotor retardation, high forehead, broad flat nasal bridge, hypertelorism, cup-shaped ears, short neck, and a normal karyotype. Using high-density oligonucleotide array chip (Affymetrix 6.0) to perform parental and proband samples concurrently on three chips and interpreted as a trio set, a de novo 3.2 Mb deletion from bands q12.2 to q13 on chromosome 16 (from 52.08 to 55.3 Mb) of paternal origin was identified. The deletion was confirmed by quantitative genomic PCR and the break points were defined by junction PCR. Our study demonstrated the power of high-density oligonucleotide array chip in identifying novel submicroscopic deletions that were not detectable using G-banding cytogenetic technology. Furthermore, our result narrowed down the critical region for craniofacial features in interstitial 16q11.2-q13 deletion syndrome. In patients who have high forehead, broad flat nasal bridge, hypertelorism, cup-shaped ears, short neck and short stature, high-density array should be included in initial work up.

  10. 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.

  11. 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

  12. Identification of Tumor-associated Autoantigens for the Diagnosis of Colorectal Cancer in Serum Using High Density Protein Microarrays*

    PubMed Central

    Babel, Ingrid; Barderas, Rodrigo; Díaz-Uriarte, Ramón; Martínez-Torrecuadrada, Jorge Luis; Sánchez-Carbayo, Marta; Casal, J. Ignacio

    2009-01-01

    There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers. PMID:19638618

  13. Identification of tumor-associated autoantigens for the diagnosis of colorectal cancer in serum using high density protein microarrays.

    PubMed

    Babel, Ingrid; Barderas, Rodrigo; Díaz-Uriarte, Ramón; Martínez-Torrecuadrada, Jorge Luis; Sánchez-Carbayo, Marta; Casal, J Ignacio

    2009-10-01

    There is a mounting evidence of the existence of autoantibodies associated to cancer progression. Antibodies are the target of choice for serum screening because of their stability and suitability for sensitive immunoassays. By using commercial protein microarrays containing 8000 human proteins, we examined 20 sera from colorectal cancer (CRC) patients and healthy subjects to identify autoantibody patterns and associated antigens. Forty-three proteins were differentially recognized by tumoral and reference sera (p value <0.04) in the protein microarrays. Five immunoreactive antigens, PIM1, MAPKAPK3, STK4, SRC, and FGFR4, showed the highest prevalence in cancer samples, whereas ACVR2B was more abundant in normal sera. Three of them, PIM1, MAPKAPK3, and ACVR2B, were used for further validation. A significant increase in the expression level of these antigens on CRC cell lines and colonic mucosa was confirmed by immunoblotting and immunohistochemistry on tissue microarrays. A diagnostic ELISA based on the combination of MAPKAPK3 and ACVR2B proteins yielded specificity and sensitivity values of 73.9 and 83.3% (area under the curve, 0.85), respectively, for CRC discrimination after using an independent sample set containing 94 sera representative of different stages of progression and control subjects. In summary, these studies confirmed the presence of specific autoantibodies for CRC and revealed new individual markers of disease (PIM1, MAPKAPK3, and ACVR2B) with the potential to diagnose CRC with higher specificity and sensitivity than previously reported serum biomarkers.

  14. Microarray profiling of gene expression in aging and its alteration by caloric restriction in mice.

    PubMed

    Weindruch, R; Kayo, T; Lee, C K; Prolla, T A

    2001-03-01

    An active research area in biological gerontology concerns the mechanisms by which caloric restriction (CR) retards the aging process in laboratory rodents. We used high density oligonucleotide arrays representing 6347 genes to determine the gene expression profile of the aging process in gastrocnemius muscle of male C57BL/6 mice. Aging resulted in a differential gene expression pattern indicative of a marked stress response and lower expression of metabolic and biosynthetic genes. Most alterations were completely or partially prevented by CR. Transcriptional patterns of muscle from calorie-restricted animals suggest that CR retards the aging process by causing a metabolic shift toward increased protein turnover and decreased macromolecular damage. The use of high density oligonucleotide microarrays provides a new tool to measure biological age on a tissue-specific basis and to evaluate at the molecular level the efficacy of nutritional interventions designed to retard the aging process.

  15. 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.

  16. Increasing hybridization rate and sensitivity of DNA microarrays using isotachophoresis.

    PubMed

    Han, Crystal M; Katilius, Evaldas; Santiago, Juan G

    2014-08-21

    We present an on-chip electrokinetic method to increase the reaction kinetics and sensitivity of DNA microarray hybridization. We use isotachophoresis (ITP) to preconcentrate target molecules in solution and transport them over the immobilized probe sites of a microarray, greatly increasing the binding reaction rate. We show theoretically and experimentally that ITP-enhanced microarrays can be hybridized much faster and with higher sensitivity than conventional methods. We demonstrate our assay using a microfluidic system consisting of a PDMS microchannel superstructure bonded onto a glass slide on which 60 spots of 20-27 nt ssDNA oligonucleotide probes are immobilized. Our 30 min assay results in an 8.2 fold higher signal than the conventional overnight hybridization at 100 fM target concentration. We show rapid and quantitative detection over 4 orders of magnitude dynamic range of target concentration with no increase in the nonspecific signal. Our technique can be further multiplexed for higher density microarrays and extended for other reactions of target-surface immobilized ligands.

  17. 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.

  18. MChip, a low density microarray, differentiates among seasonal human H1N1, North American swine H1N1, and the 2009 pandemic H1N1.

    PubMed

    Heil, Gary L; McCarthy, Troy; Yoon, Kyoung-Jin; Liu, Siyuan; Saad, Magdi D; Smith, Catherine B; Houck, Julie A; Dawson, Erica D; Rowlen, Kathy L; Gray, Gregory C

    2010-11-01

    The MChip uses data from the hybridization of amplified viral RNA to 15 distinct oligonucleotides that target the influenza A matrix (M) gene segment. An artificial neural network (ANN) automates the interpretation of subtle differences in fluorescence intensity patterns from the microarray. The complete process from clinical specimen to identification including amplification of viral RNA can be completed in <8 hours for under US$10. The work presented here represents an effort to expand and test the capabilities of the MChip to differentiate influenza A/H1N1 of various species origin. The MChip ANN was trained to recognize fluorescence image patterns of a variety of known influenza A viruses, including examples of human H1N1, human H3N2, swine H1N1, 2009 pandemic influenza A H1N1, and a wide variety of avian, equine, canine, and swine influenza viruses. Robustness of the MChip ANN was evaluated using 296 blinded isolates. Training of the ANN was expanded by the addition of 71 well-characterized influenza A isolates and yielded relatively high accuracy (little misclassification) in distinguishing unique H1N1 strains: nine human A/H1N1 (88·9% correct), 35 human A/H3N2 (97·1% correct), 31 North American swine A/H1N1 (80·6% correct), 14 2009 pandemic A/H1N1 (87·7% correct), and 23 negative samples (91·3% correct). Genetic diversity among the swine H1N1 isolates may have contributed to the lower success rate for these viruses. The current study demonstrates the MChip has the capability to differentiate the genetic variations among influenza viruses with appropriate ANN training. Further selective enrichment of the ANN will improve its ability to rapidly and reliably characterize influenza viruses of unknown origin. © 2010 Blackwell Publishing Ltd.

  19. Multiple epitope presentation and surface density control enabled by chemoselective immobilization lead to enhanced performance in IgE-binding fingerprinting on peptide microarrays.

    PubMed

    Gori, Alessandro; Cretich, Marina; Vanna, Renzo; Sola, Laura; Gagni, Paola; Bruni, Giulia; Liprino, Marta; Gramatica, Furio; Burastero, Samuele; Chiari, Marcella

    2017-08-29

    Multiple ligand presentation is a powerful strategy to enhance the affinity of a probe for its corresponding target. A promising application of this concept lies in the analytical field, where surface immobilized probes interact with their corresponding targets in the context of complex biological samples. Here we investigate the effect of multiple epitope presentation (MEP) in the challenging context of IgE-detection in serum samples using peptide microarrays, and evaluate the influence of probes surface density on the assay results. Using the milk allergen alpha-lactalbumin as a model, we have synthesized three immunoreactive epitope sequences in a linear, branched and tandem form and exploited a chemoselective click strategy (CuAAC) for their immobilization on the surface of two biosensors, a microarray and an SPR chip both modified with the same clickable polymeric coating. We first demonstrated that a fine tuning of the surface peptide density plays a crucial role to fully exploit the potential of oriented and multiple peptide display. We then compared the three multiple epitope presentations in a microarray assay using sera samples from milk allergic patients, confirming that a multiple presentation, in particular that of the tandem construct, allows for a more efficient characterization of IgE-binding fingerprints at a statistically significant level. To gain insights on the binding parameters that characterize antibody/epitopes affinity, we selected the most reactive epitope of the series (LAC1) and performed a Surface Plasmon Resonance Imaging (SPRi) analysis comparing different epitope architectures (linear versus branched versus tandem). We demonstrated that the tandem peptide provides an approximately twofold increased binding capacity with respect to the linear and branched peptides, that could be attributed to a lower rate of dissociation (Kd). Copyright © 2017 Elsevier B.V. All rights reserved.

  20. The application of high density microarray for analysis of mitogenic signaling and cell-cycle in the adrenal.

    PubMed

    Wang, C; Francis, R; Harirchian, S; Batlle, D; Mayhew, B; Bassett, M; Rainey, W E; Pestell, R G

    2000-11-01

    Angiotensin II (AII) binds to specific G-protein coupled receptors and is mitogenic in adrenal, liver epithelial, and vascular smooth muscle cells. The H295R human adrenocortical cell line, which expresses AII receptors predominantly of the AT1 subclass, proliferates in response to treatment with AII. The induction and maintenance of cellular proliferation involves a precisely coordinated induction of a variety of genes. As the human genome sequencing projects near completion a variety of high throughput technologies have been developed in order to create dynamic displays of genomic responses. One high throughput method, the gridded cDNA microarray has been developed in which immobilised DNA samples are hybridized on glass slides for the identification of global genomic responses. For this purpose high precision robotic microarrayers have been developed at AECOM. The cyclin D1 gene, which encodes the regulatory subunit of the cyclin D1-dependent kinase (CD1K) required for phosphorylation of the retinoblastoma protein (pRB), was induced by AII in H295R cells. Abundance of the cyclin D1 gene is rate-limiting in G1 phase progression of the cell-cycle in a variety of cell types. AII induced cyclin D1 promoter activity through a c-Fos and c-Jun binding sequence at -954 bp. Theabundance of c-Fos within this complex was increased by AII treatment. Analysis of AII signaling in adrenal cells by cDNA microarray demonstrated an induction of the human homologue of Xenopus XPMC2 (HXPMC2). The cDNA for XPMC2 was previously shown to rescue mitotic catastrophe in mutant S. Pombe defective in cdc2 kinase function. Further studies are required to determine the requirement for cyclin D1 and XPMC2H in AII-induced cell-cycle progression and cellular proliferation in the adrenal.

  1. 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

  2. Design, construction and validation of a Plasmodium vivax microarray for the transcriptome profiling of clinical isolates.

    PubMed

    Boopathi, Pon Arunachalam; Subudhi, Amit Kumar; Middha, Sheetal; Acharya, Jyoti; Mugasimangalam, Raja Chinnadurai; Kochar, Sanjay Kumar; Kochar, Dhanpat Kumar; Das, Ashis

    2016-12-01

    High density oligonucleotide microarrays have been used on Plasmodium vivax field isolates to estimate whole genome expression. However, no microarray platform has been experimentally optimized for studying the transcriptome of field isolates. In the present study, we adopted both bioinformatics and experimental testing approaches to select best optimized probes suitable for detecting parasite transcripts from field samples and included them in designing a custom 15K P. vivax microarray. This microarray has long oligonucleotide probes (60mer) that were in-situ synthesized onto glass slides using Agilent SurePrint technology and has been developed into an 8X15K format (8 identical arrays on a single slide). Probes in this array were experimentally validated and represents 4180 P. vivax genes in sense orientation, of which 1219 genes have also probes in antisense orientation. Validation of the 15K array by using field samples (n=14) has shown 99% of parasite transcript detection from any of the samples. Correlation analysis between duplicate probes (n=85) present in the arrays showed perfect correlation (r(2)=0.98) indicating the reproducibility. Multiple probes representing the same gene exhibited similar kind of expression pattern across the samples (positive correlation, r≥0.6). Comparison of hybridization data with the previous studies and quantitative real-time PCR experiments were performed to highlight the microarray validation procedure. This array is unique in its design, and results indicate that the array is sensitive and reproducible. Hence, this microarray could be a valuable functional genomics tool to generate reliable expression data from P. vivax field isolates. Copyright © 2016. Published by Elsevier B.V.

  3. Identification of seroreactive proteins of Leptospira interrogans serovar copenhageni using a high-density protein microarray approach.

    PubMed

    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

    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. 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. 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. 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 diagnosing this neglected tropical disease. Further

  4. 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

  5. Fabrication of DNA microarrays on nanoengineered polymericultrathin film prepared by self-assembly of polyelectrolytemultilayers

    SciTech Connect

    Zhou, X.; Wu Liyou; Zhou, J.-Z.

    2004-08-24

    Microarray-based technology is in need of flexible andcost-effective chemistry for fabrication of oligonucleotide microarrays.We have developed a novel method for the fabrication of oligonucleotidemicroarrays with unmodified oligonucleotide probes on nanoengineeredthree-dimensional thin films that are deposited on glass slides byconsecutive layer-to-layer adsorption of polyelectrolytes. Unmodifiedoligonucleotide probes were spotted and immobilized on these multilayeredpolyelectrolyte thin films (PET) by electrostatic adsorption andentrapment on the porous structure of the PET film. The PET provideshigher probe binding capacity and thus higher hybridization signal thanthat of the traditional two-dimensional aminosilane and poly-L-lysinecoated slides. Immobilized probe densities of 3.4 x 1012/cm2 wereobserved for microarray spots on PET with unmodified 50-meroligonucleotide probes, which is comparable to the immobilized probedensities of alkyamine-modified 50-mer probes end-tethered on analdehyde-functionalized slide. The study of hybridization efficiencyshowed that 90 percent of immobilized probes on PET film are accessibleto target DNA to form duplex format in hybridization. The DNA microarrayfabricated on PET film has wider dynamic range (about 3 orders ofmagnitude) and lower detection limit (0.5 nM) than the conventionalamino- and aldehyde-functionlized slides. Oligonucleotide microarraysfabricated on these PET-coated slides also had consistent spotmorphology. In addition, discrimination of single nucleotide polymorphismof 16S rRNA genes was achieved with the PET-based oligonucleotidemicroarrays. The PET microarrays constructed by our self-assembly processis cost-effective, versatile, and well suited for immobilizing many typesof biological active molecules so that a wide variety of microarrayformats can be developed.

  6. 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.

  7. High-density 16S microarray and clone library-based microbial community composition of the Phoenix spacecraft assembly clean room.

    PubMed

    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.

  8. 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.

  9. Strategy for the design of custom cDNA microarrays.

    PubMed

    Lorenz, Matthias G O; Cortes, Lizette M; Lorenz, Juergen J; Liu, Edison T

    2003-06-01

    DNA microarrays are valuable but expensive tools for expression profiling of cells, tissues, and organs. The design of custom microarrays leads to cost reduction without necessarily compromising their biological value. Here we present a strategy for designing custom cDNA microarrays and constructed a microarray for mouse immunology research (ImmunoChip). The strategy used interrogates expressed sequence tag databases available in the public domain but overcomes many of the problems encountered. Immunologically relevant clusters were selected based on the expression of expressed sequence tags in relevant libraries. Selected clusters were organized in modules, and the best representative clones were identified. When tested, this microarray was found to have minimal clone identity errors or phage contamination and identified molecular signatures of lymphoid cell lines. Our proposed design of custom microarrays avoids probe redundancy, allows the organization of the chip to optimize chip production, and reduces microarray production costs. The strategy described is also useful for the design of oligonucleotide microarrays.

  10. Application of High-Density DNA Resequencing Microarray for Detection and Characterization of Botulinum Neurotoxin-Producing Clostridia

    PubMed Central

    Vanhomwegen, Jessica; Berthet, Nicolas; Mazuet, Christelle; Guigon, Ghislaine; Vallaeys, Tatiana; Stamboliyska, Rayna; Dubois, Philippe; Kennedy, Giulia C.; Cole, Stewart T.; Caro, Valérie; Manuguerra, Jean-Claude; Popoff, Michel-Robert

    2013-01-01

    Background Clostridium botulinum and related clostridia express extremely potent toxins known as botulinum neurotoxins (BoNTs) that cause severe, potentially lethal intoxications in humans. These BoNT-producing bacteria are categorized in seven major toxinotypes (A through G) and several subtypes. The high diversity in nucleotide sequence and genetic organization of the gene cluster encoding the BoNT components poses a great challenge for the screening and characterization of BoNT-producing strains. Methodology/Principal Findings In the present study, we designed and evaluated the performances of a resequencing microarray (RMA), the PathogenId v2.0, combined with an automated data approach for the simultaneous detection and characterization of BoNT-producing clostridia. The unique design of the PathogenID v2.0 array allows the simultaneous detection and characterization of 48 sequences targeting the BoNT gene cluster components. This approach allowed successful identification and typing of representative strains of the different toxinotypes and subtypes, as well as the neurotoxin-producing C. botulinum strain in a naturally contaminated food sample. Moreover, the method allowed fine characterization of the different neurotoxin gene cluster components of all studied strains, including genomic regions exhibiting up to 24.65% divergence with the sequences tiled on the arrays. Conclusions/Significance The severity of the disease demands rapid and accurate means for performing risk assessments of BoNT-producing clostridia and for tracing potentials sources of contamination in outbreak situations. The RMA approach constitutes an essential higher echelon component in a diagnostics and surveillance pipeline. In addition, it is an important asset to characterise potential outbreak related strains, but also environment isolates, in order to obtain a better picture of the molecular epidemiology of BoNT-producing clostridia. PMID:23818983

  11. 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.

  12. Targeting density-enhanced phosphatase-1 (DEP-1) with antisense oligonucleotides improves the metabolic phenotype in high-fat diet-fed mice

    PubMed Central

    2013-01-01

    Background Insulin signaling is tightly controlled by tyrosine dephosphorylation of the insulin receptor through protein-tyrosine-phosphatases (PTPs). DEP-1 is a PTP dephosphorylating tyrosine residues in a variety of receptor tyrosine kinases. Here, we analyzed whether DEP-1 activity is differentially regulated in liver, skeletal muscle and adipose tissue under high-fat diet (HFD), examined the role of DEP-1 in insulin resistance in vivo, and its function in insulin signaling. Results Mice were fed an HFD for 10 weeks to induce obesity-associated insulin resistance. Thereafter, HFD mice were subjected to systemic administration of specific antisense oligonucleotides (ASOs), highly accumulating in hepatic tissue, against DEP-1 or control ASOs. Targeting DEP-1 led to improvement of insulin sensitivity, reduced basal glucose level, and significant reduction of body weight. This was accompanied by lower insulin and leptin serum levels. Suppression of DEP-1 in vivo also induced hyperphosphorylation in the insulin signaling cascade of the liver. Moreover, DEP-1 physically associated with the insulin receptor in situ, and recombinant DEP-1 dephosphorylated the insulin receptor in vitro. Conclusions These results indicate that DEP-1 acts as an endogenous antagonist of the insulin receptor, and downregulation of DEP-1 results in an improvement of insulin sensitivity. DEP-1 may therefore represent a novel target for attenuation of metabolic diseases. PMID:23889985

  13. Universal protein binding microarrays for the comprehensive characterization of the DNA binding specificities of transcription factors

    PubMed Central

    Berger, Michael F.; Bulyk, Martha L.

    2010-01-01

    Protein binding microarray (PBM) technology provides a rapid, high-throughput means of characterizing the in vitro DNA binding specificities of transcription factors (TFs). Using high-density, custom-designed microarrays containing all 10-mer sequence variants, one can obtain comprehensive binding site measurements for any TF, regardless of its structural class or species of origin. Here, we present a protocol for the examination and analysis of TF binding specificities at high resolution using such ‘all 10-mer’ universal PBMs. This procedure involves double-stranding a commercially synthesized DNA oligonucleotide array, binding a TF directly to the double-stranded DNA microarray, and labeling the protein-bound microarray with a fluorophore-conjugated antibody. We describe how to computationally extract the relative binding preferences of the examined TF for all possible contiguous and gapped 8-mers over the full range of affinities, from highest affinity sites to nonspecific sites. Multiple proteins can be tested in parallel in separate chambers on a single microarray, enabling the processing of a dozen or more TFs in a single day. PMID:19265799

  14. Development of a Novel Oligonucleotide Array for Genomic Screening of Chemical Warfare Agent Exposure in Guinea Pigs and Swine

    DTIC Science & Technology

    2004-04-01

    respectively. To address this gap in research tools, we have developed an oligonucleotide microarray that contains representative genes from guinea pig...To address this gap in research tools, we have developed an oligonucleotide microarray that contains representative genes from guinea pig...toxicogenomics. Toxicogenomics is focused on identifying changes in gene expression in response to a toxic chemical and then using that information to

  15. 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.

  16. 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.

  17. 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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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

    PubMed Central

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

    2016-01-01

    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 (p values <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

  19. Genome-wide transcription analyses in rice using tiling microarrays.

    PubMed

    Li, Lei; Wang, Xiangfeng; Stolc, Viktor; Li, Xueyong; Zhang, Dongfen; Su, Ning; Tongprasit, Waraporn; Li, Songgang; Cheng, Zhukuan; Wang, Jun; Deng, Xing Wang

    2006-01-01

    Sequencing and computational annotation revealed several features, including high gene numbers, unusual composition of the predicted genes and a large number of genes lacking homology to known genes, that distinguish the rice (Oryza sativa) genome from that of other fully sequenced model species. We report here a full-genome transcription analysis of the indica rice subspecies using high-density oligonucleotide tiling microarrays. Our results provided expression data support for the existence of 35,970 (81.9%) annotated gene models and identified 5,464 unique transcribed intergenic regions that share similar compositional properties with the annotated exons and have significant homology to other plant proteins. Elucidating and mapping of all transcribed regions revealed an association between global transcription and cytological chromosome features, and an overall similarity of transcriptional activity between duplicated segments of the genome. Collectively, our results provide the first whole-genome transcription map useful for further understanding the rice genome.

  20. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. 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

  2. The delivery of therapeutic oligonucleotides

    PubMed Central

    Juliano, Rudolph L.

    2016-01-01

    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

  3. 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. © The Author 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. 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.

  5. 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.

  6. A note on oligonucleotide expression values not being normally distributed.

    PubMed

    Hardin, Johanna; Wilson, Jason

    2009-07-01

    Novel techniques for analyzing microarray data are constantly being developed. Though many of the methods contribute to biological discoveries, inability to properly evaluate the novel techniques limits their ability to advance science. Because the underlying distribution of microarray data is unknown, novel methods are typically tested against the assumed normal distribution. However, microarray data are not, in fact, normally distributed, and assuming so can have misleading consequences. Using an Affymetrix technical replicate spike-in data set, we show that oligonucleotide expression values are not normally distributed for any of the standard methods for calculating expression values. The resulting data tend to have a large proportion of skew and heavy tailed genes. Additionally, we show that standard methods can give unexpected and misleading results when the data are not well approximated by the normal distribution. Robust methods are therefore recommended when analyzing microarray data. Additionally, new techniques should be evaluated with skewed and/or heavy-tailed data distributions.

  7. Microarray analysis of erythromycin resistance determinants.

    PubMed

    Volokhov, D; Chizhikov, V; Chumakov, K; Rasooly, A

    2003-01-01

    To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.

  8. OligoFaktory: a visual tool for interactive oligonucleotide design.

    PubMed

    Schretter, Colas; Milinkovitch, Michel C

    2006-01-01

    The OligoFaktory is a set of tools for the design, on an arbitrary number of target sequences, of high-quality long oligonucleotide for micro-array, of primer pair for PCR, of siRNA and more. The user-centered interface exists in two flavours: a web portal and a standalone software for Mac OS X Tiger. A unified presentation of results provides overviews with distribution charts and relative location bar graphs, as well as detailed features for each oligonucleotide. Input and output files conform to a common XML interchange file format to allow both automatic generation of input data, archiving, and post-processing of results. The design pipeline can use BLAST servers to evaluate specificity of selected oligonucleotides. The web portal http://ueg.ulb.ac.be/oligofaktory/; the software for Macintosh: http://www.oligofaktory.org/

  9. Allergen diagnosis microarray with high-density immobilization capacity using diamond-like carbon-coated chips for profiling allergen-specific IgE and other immunoglobulins.

    PubMed

    Suzuki, Koichi; Hiyoshi, Mineyoshi; Tada, Hitomi; Bando, Miwa; Ichioka, Takao; Kamemura, Norio; Kido, Hiroshi

    2011-11-14

    The diagnosis of antibody-mediated allergic disorders is based on clinical findings, skin prick tests and detection of allergen-specific IgE in serum. Here, we present a new microarray technique of high-density antigen immobilization using carboxylated arms on the surface of a diamond-like carbon (DLC)-coated chip. High immobilization capacity of antigen on DLC chip at (0.94-7.82)×10(9) molecules mm(-2) allowed the analysis of allergen-specific immunoglobulins against not only purified proteins but also natural allergen extracts with wide assay dynamic range. The higher sensitivity of the allergen-specific IgE detection on DLC chip was observed for comparison with the UniCAP system: the DLC chip allowed lowering the limit of dilution rate in UniCAP system to further dilution at 4-8-fold. High correlations (ρ>0.9-0.85) of allergen-specific IgE values determined by the DLC chip and UniCAP were found in most of 20 different allergens tested. The DLC chip was useful to determine allergen-induced antibodies of IgA, IgG, IgG1, and IgG4 in sera, apart from IgE, as well as secretory IgA in saliva against the same series of allergens on the chip in a minimal amount (1-2 μL) of sample. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. 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.

  11. Stripping custom microRNA microarrays and the lessons learned about probe-slide interactions.

    PubMed

    Zhang, Xiaoxiao; Xu, Wayne; Tan, Jiankang; Zeng, Yan

    2009-03-15

    Microarrays have been used extensively in gene expression profiling and genotyping studies. To reduce the high cost and enhance the consistency of microarray experiments, it is often desirable to strip and reuse microarray slides. Our genome-wide analysis of microRNA expression involves the hybridization of fluorescently labeled nucleic acids to custom-made, spotted DNA microarrays based on GAPSII-coated slides. We describe here a simple and effective method to regenerate such custom microarrays that uses a very low-salt buffer to remove labeled nucleic acids from microarrays. Slides can be stripped and reused multiple times without significantly compromising data quality. Moreover, our analyses of the performance of regenerated slides identifies parameters that influence the attachment of oligonucleotide probes to GAPSII slides, shedding light on the interactions between DNA and the microarray surface and suggesting ways in which to improve the design of oligonucleotide probes.

  12. 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.

  13. 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

  14. Oligonucleotide Fingerprint Identification for Microarray-Based Pathogen Diagnostic Assays

    DTIC Science & Technology

    2006-10-01

    Yersinia species), or a set of organisms that may or may not have any phylogenetic relationship (e.g. to detect a viral or a bacterial family) and (2) the... phylogenetic tree or published data. The target and neighbor will contain common DNA sequences, which, obviously, cannot be used as DNA fingerprints. DNA... Yersinia pestis and 250 fingerprints for Francisella tularensis are presented. Availability: The implemented algorithm is available upon request

  15. An oligonucleotide microarray to characterize multidrug resistant plasmids

    USDA-ARS?s Scientific Manuscript database

    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...

  16. 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.

  17. Development of a microarray for identification of pathogenic Clostridium species

    PubMed Central

    Janvilisri, Tavan; Scaria, Joy; Gleed, Robin; Fubini, Susan; Bonkosky, Michelle M.; Gröhn, Yrjö T.; Chang, Yung-Fu

    2009-01-01

    In recent years, Clostridium species have rapidly reemerged as human and animal pathogens. The detection and identification of pathogenic Clostridium species is therefore critical for clinical diagnosis and antimicrobial therapy. Traditional diagnostic techniques for clostridia are laborious, time-consuming and may adversely affect the therapeutic outcome. In this study, we developed an oligonucleotide diagnostic microarray for pathogenic Clostridium species. The microarray specificity was tested against 65 Clostridium isolates. The applicability of this microarray in a clinical setting was assessed with the use of mock stool samples. The microarray was successful in discriminating at least four species with the limit of detection as low as 104 CFU/ml. In addition, the pattern of virulence and antibiotic resistance genes of tested strains were determined through the microarrays. This approach demonstrates the high-throughput detection and identification of Clostridium species and provides advantages over traditional methods. Microarray-based techniques are promising applications for clinical diagnosis and epidemiological investigations. PMID:19879710

  18. Application of DNA microarray technology to gerontological studies.

    PubMed

    Masuda, Kiyoshi; Kuwano, Yuki; Nishida, Kensei; Rokutan, Kazuhito

    2013-01-01

    Gene expression patterns change dramatically in aging and age-related events. The DNA microarray is now recognized as a useful device in molecular biology and widely used to identify the molecular mechanisms of aging and the biological effects of drugs for therapeutic purpose in age-related diseases. Recently, numerous technological advantages have led to the evolution of DNA microarrays and microarray-based techniques, revealing the genomic modification and all transcriptional activity. Here, we show the step-by-step methods currently used in our lab to handling the oligonucleotide microarray and miRNA microarray. Moreover, we introduce the protocols of ribonucleoprotein [RNP] immunoprecipitation followed by microarray analysis (RIP-chip) which reveal the target mRNA of age-related RNA-binding proteins.

  19. Microarray analysis of ox-LDL (oxidized low-density lipoprotein)-regulated genes in human coronary artery smooth muscle cells.

    PubMed

    Minta, Joe; Jungwon Yun, James; St Bernard, Rosanne

    2010-01-01

    Recent studies suggest that circulating LDL (low-density lipoproteins) play a central role in the pathogenesis of atherosclerosis, and the oxidized form (ox-LDL) is highly atherogenic. Deposits of ox-LDL have been found in atherosclerotic plaques, and ox-LDL has been shown to promote monocyte recruitment, foam cell formation and the transition of quiescent and contractile vascular SMCs (smooth muscle cells) to the migratory and proliferative phenotype. SMC phenotype transition and hyperplasia are the pivotal events in the pathogenesis of atherosclerosis. To comprehend the complex molecular mechanisms involved in ox-LDL-mediated SMC phenotype transition, we have compared the differential gene expression profiles of cultured quiescent human coronary artery SMCs with cells induced with ox-LDL for 3 and 21 h using Affymetrix HG-133UA cDNA microarray chips. Assignment of the regulated genes into functional groups indicated that several genes involved in metabolism, membrane transport, cell-cell interactions, signal transduction, transcription, translation, cell migration, proliferation and apoptosis were differentially expressed. Our data suggests that the interaction of ox-LDL with its cognate receptors on SMCs modulates the induction of several growth factors and cytokines, which activate a variety of intracellular signalling mechanisms (including PI3K, MAPK, Jak/STAT, sphingosine, Rho kinase pathways) that contribute to SMC transition from the quiescent and contractile phenotype to the proliferative and migratory phenotype. Our study has also identified several genes (including CDC27, cyclin A1, cyclin G2, glypican 1, MINOR, p15 and apolipoprotein) not previously implicated in ox-LDL-induced SMC phenotype transition and substantially extends the list of potential candidate genes involved in atherogenesis.

  20. Genotypic assessment of drug-resistant tuberculosis in Baghdad and other Iraqi provinces using low-cost and low-density DNA microarrays.

    PubMed

    Al-Rubaye, Dalal Saleh; Henihan, Grace; Al-Abasly, Ameraa K Abas; Seagar, Amie-Louise; Al-Attraqchi, Azhar Ab F; Schulze, Holger; Hashim, Dhafer Salman; Kamil, Jinan Khalid; Laurenson, Ian F; Bachmann, Till T

    2016-02-01

    We report on a molecular investigation carried out to ascertain the prevalence of drug-resistant tuberculosis (TB) and the specific gene mutations responsible for resistance to rifampicin (RIF) and/or isoniazid (INH) in Iraq. In total, 110 clinical isolates from category II TB cases from Baghdad (58%) and several Iraqi provinces (42%) were analysed using colorimetric, low-cost and low-density (LCD) microarrays (MYCO-Direct and MYCO-Resist LCD array kits) to identify the point mutations responsible for resistance in Mycobacterium tuberculosis isolates. We found 76 patients (69.1%) had resistant strains, of which 40 (36%) were multidrug-resistant (MDR)-TB. Where mono-resistance was identified, it was found to be predominantly to RIF (83%). The most common mutations were rpoB S531L (50%), inhA C15T (25%) and katG S315T (15%). The most common MDR-TB genotypes were rpoB S531L with inhA C15T (60%) and rpoB S531L with katG S315T (20%). Where phenotypic analysis of clinical isolates was also performed, genotypic data were found to show excellent correlation with phenotypic results. Correlation was found between the MYCO-Resist LCD array and GenoType MTBDRplus for detection of resistance to RIF. Our study shows MDR-TB in 36% of category II TB cases in Baghdad and surrounding Iraqi provinces, which reflects the World Health Organization findings based on phenotypic studies. Diagnosis of TB and MDR-TB using culture-based tests is a significant impediment to global TB control. The LCD arrays investigated herein are easy to use, sensitive and specific molecular tools for TB resistance profiling in resource-limited laboratory settings.

  1. Reproducible and inexpensive probe preparation for oligonucleotide arrays.

    PubMed

    Zhang, Y; Price, B D; Tetradis, S; Chakrabarti, S; Maulik, G; Makrigiorgos, G M

    2001-07-01

    We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

  2. 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

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

    PubMed

    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; Scanlan, David J; Paerl, Ryan W; Stuart, Rhona; Zehr, Jonathan P

    2014-07-01

    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.

  4. 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...

  5. 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...

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

    PubMed

    Bilgic, Tugba; Klok, Harm-Anton

    2015-11-09

    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.

  7. EvOligo: A Novel Software to Design and Group Libraries of Oligonucleotides Applicable for Nucleic Acid-Based Experiments.

    PubMed

    Milewski, Marek C; Kamel, Karol; Kurzynska-Kokorniak, Anna; Chmielewski, Marcin K; Figlerowicz, Marek

    2017-03-15

    Experimental methods based on DNA and RNA hybridization, such as multiplex polymerase chain reaction, multiplex ligation-dependent probe amplification, or microarray analysis, require the use of mixtures of multiple oligonucleotides (primers or probes) in a single test tube. To provide an optimal reaction environment, minimal self- and cross-hybridization must be achieved among these oligonucleotides. To address this problem, we developed EvOligo, which is a software package that provides the means to design and group DNA and RNA molecules with defined lengths. EvOligo combines two modules. The first module performs oligonucleotide design, and the second module performs oligonucleotide grouping. The software applies a nearest-neighbor model of nucleic acid interactions coupled with a parallel evolutionary algorithm to construct individual oligonucleotides, and to group the molecules that are characterized by the weakest possible cross-interactions. To provide optimal solutions, the evolutionary algorithm sorts oligonucleotides into sets, preserves preselected parts of the oligonucleotides, and shapes their remaining parts. In addition, the oligonucleotide sets can be designed and grouped based on their melting temperatures. For the user's convenience, EvOligo is provided with a user-friendly graphical interface. EvOligo was used to design individual oligonucleotides, oligonucleotide pairs, and groups of oligonucleotide pairs that are characterized by the following parameters: (1) weaker cross-interactions between the non-complementary oligonucleotides and (2) more uniform ranges of the oligonucleotide pair melting temperatures than other available software products. In addition, in contrast to other grouping algorithms, EvOligo offers time-efficient sorting of paired and unpaired oligonucleotides based on various parameters defined by the user.

  8. Evaluation of high-resolution microarray platforms for genomic profiling of bone tumours

    PubMed Central

    2010-01-01

    Background Several high-density oligonucleotide microarray platforms are available for genome-wide single nucleotide polymorphism (SNP) detection and microarray-based comparative genomic hybridisation (array CGH), which may be used to detect copy number aberrations in human tumours. As part of the EuroBoNeT network of excellence for research on bone tumours (eurobonet.eu), we have evaluated four different commercial high-resolution microarray platforms in order to identify the most appropriate technology for mapping DNA copy number aberrations in such tumours. Findings DNA from two different cytogenetically well-characterized bone sarcoma cell lines, representing a simple and a complex karyotype, respectively, was tested in duplicate on four high-resolution microarray platforms; Affymetrix Genome-Wide Human SNP Array 6.0, Agilent Human Genome CGH 244A, Illumina HumanExon510s-duo and Nimblegen HG18 CGH 385 k WG tiling v1.0. The data was analysed using the platform-specific analysis software, as well as a platform-independent analysis algorithm. DNA copy number was measured at six specific chromosomes or chromosomal regions, and compared with the expected ratio based on available cytogenetic information. All platforms performed well in terms of reproducibility and were able to delimit and score small amplifications and deletions at similar resolution, but Agilent microarrays showed better linearity and dynamic range. The platform-specific analysis software provided with each platform identified in general correct copy numbers, whereas using a platform-independent analysis algorithm, correct copy numbers were determined mainly for Agilent and Affymetrix microarrays. Conclusions All platforms performed reasonably well, but Agilent microarrays showed better dynamic range, and like Affymetrix microarrays performed well with the platform-independent analysis software, implying more robust data. Bone tumours like osteosarcomas are heterogeneous tumours with complex

  9. 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. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. An undergraduate laboratory exercise to study the effect of darkness on plant gene expression using DNA microarray.

    PubMed

    Chang, Ming-Mei; Briggs, George M

    2007-11-01

    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 exercise using an Arabidopsis DNA microarray to study the gene expression of Brassica rapa, Wisconsin Fast Plant. Genes involved in senescence, cell wall loosening/degradation, and sugar transport were the most upregulated, while those involved in photosynthesis, the elimination of reactive oxygen intermediates associated with photooxidative stress and auxin synthesis, were the most downregulated. Students were able to complete the experiment successfully. Throughout the exercise, they learned various important molecular techniques including RNA isolation, quantification, reverse transcription, cRNA synthesis, labeling and purification, and microarray hybridization, washing, scanning, and feature extraction. The exercise can be integrated into a college-level molecular biology laboratory. The procedure used can be adapted to examine other effects on other organisms. Copyright © 2007 International Union of Biochemistry and Molecular Biology, Inc.

  11. DNA microarrays on silicon nanostructures: optimization of the multilayer stack for fluorescence detection.

    PubMed

    Oillic, C; Mur, P; Blanquet, E; Delapierre, G; Vinet, F; Billon, T

    2007-04-15

    To improve the sensitivity of fluorescence detection in DNA microarrays, the use of silicon nanostructures based on chemical vapor deposition (CVD) processes adopted for the growth of rough polycrystalline silicon was investigated. These substrates present advantages of two main properties which could lead to an enhancement of the fluorescence detection, i.e. (i) the increase of the available surface area in order to achieve a high loading capacity of biomolecules and (ii) the optimization of the stack of silicon nanostructures support. Indeed, the structures were elaborated on an initial thermal oxide layer and then covered with a silicon oxide layer, obtained by oxidation and allowing the functionalization for the subsequent grafting of DNA probes. Moreover, these oxide layers play a part in the fluorescence detection. The influence of the silicon oxide layer thickness above and below the silicon grains in close relation with the density of nanostructures on the emitted fluorescence was emphasized. This paper presents an experimental characterization of the fluorescence intensity and the optimization of the different layers that composed the substrate used for DNA microarrays. The performances of the microarrays were investigated by means of hybridization experiments using complementary fluorescent labeled-oligonucleotides targets. Our results indicate that an optimized substrate can be designed and that the use of oxidized silicon nanostructures for support of biochip could be a strategy for improving the sensitivity of fluorescence detection.

  12. Multiplex detection and identification of viral, bacterial, and protozoan pathogens in human blood and plasma using a high-density resequencing pathogen microarray platform.

    PubMed

    Kourout, Moussa; Fisher, Carolyn; Purkayastha, Anjan; Tibbetts, Clark; Winkelman, Valerie; Williamson, Phillip; Nakhasi, Hira L; Duncan, Robert

    2016-06-01

    The implementation of nucleic acid-based tests for blood donor screening has improved the safety of the blood supply; however, the increasing number of emerging pathogen tests is burdensome. Development of multiplex testing platforms that allow simultaneous screening for different pathogens is a potential solution. The TessArray resequencing microarray is a platform that allows multiplex detection and identification of 97 different blood-borne pathogens in one single test. The objective was to evaluate the lowest concentration detected in blood or plasma, species discrimination, and applicability of the TessArray microarray platform for testing blood donors. Human blood or plasma spiked with selected pathogens (10,000, 1000, or 100 cells or copies/mL), including three viral, four bacterial, and four protozoan pathogens were each tested on this platform. The nucleic acids were extracted, amplified using multiplexed sets of pooled specific primers, fragmented, labeled, and hybridized to a microarray. Finally, the detected sequences were identified using an automated genomic database alignment algorithm. The performance of this platform demonstrated detection for spiked bacterial and protozoan pathogens of 100 cells/mL and viral pathogens as low as 100 copies/mL. Coded specimens, including spiked and negative controls, were identified correctly for blood specimens (31/32, 97%) and for plasma specimens (20/22, 91%) demonstrating the effectiveness of the platform. These results indicated that the TessArray microarray platform could be employed for multiplex detection and identification, with a high level of discriminatory power for numerous blood-borne pathogen targets with potential for use in blood safety. © 2016 AABB.

  13. Transcriptome-Wide High-Density Microarray Analysis Reveals Differential Gene Transcription in Periprosthetic Tissue From Hips With Chronic Periprosthetic Joint Infection vs Aseptic Loosening.

    PubMed

    Omar, Mohamed; Klawonn, Frank; Brand, Stephan; Stiesch, Meike; Krettek, Christian; Eberhard, Jörg

    2017-01-01

    Differentiating between periprosthetic hip infection and aseptic hip prosthesis loosening can be challenging, especially in patients with chronic infections. This study used whole-genome microarray analysis to investigate the transcriptomes of periprosthetic hip tissues to identify genes that are differentially transcripted between chronic periprosthetic hip infection and aseptic hip prosthesis loosening. In this pilot study, a total of 24 patients with either chronic periprosthetic hip infection (n = 12) or aseptic hip prosthesis loosening (n = 12) were analyzed. Periprosthetic hip infection was diagnosed based on modified criteria of the Musculoskeletal Infection Society. To evaluate differences in gene transcription, whole-genome microarray analysis was performed on the mRNA of periprosthetic tissue. Microarray analysis revealed differential gene transcription in periprosthetic hip tissue affected by chronic hip infection vs aseptic hip prosthesis loosening. A total of 39 genes had area under the curve values greater than 0.9 for diagnosing chronic periprosthetic hip infection; 5 genes had annotations relevant to infection and metabolism. The 39 genes also included 7 genes that were differentially transcribed but that have no apparent connection to immune response processes plus 27 genes with unknown function. Differences in gene transcription profiles might represent novel diagnostic targets that can be used to differentiate between chronic periprosthetic hip infections and aseptic hip prosthesis loosening. Secondary metabolites of differentially transcripted genes might serve as easily accessible markers for detecting chronic periprosthetic joint infection in future. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Radiolabeled oligonucleotides for antisense imaging

    PubMed Central

    Iyer, Arun K; He, Jiang

    2011-01-01

    Oligonucleotides radiolabeled with isotopes emitting γ-rays (for SPECT imaging) or positrons (for PET imaging) can be useful for targeting messenger RNA (mRNA) thereby serving as non-invasive imaging tools for detection of gene expression in vivo (antisense imaging). Radiolabeled oligonucleotides may also be used for monitoring their in vivo fate, thereby helping us better understand the barriers to its delivery for antisense targeting. These developments have led to a new area of molecular imaging and targeting, utilizing radiolabeled antisense oligonucleotides. However, the success of antisense imaging relies heavily on overcoming the barriers for its targeted delivery in vivo. Furthermore, the low ability of the radiolabeled antisense oligonucleotide to subsequently internalize into the cell and hybridize with its target mRNA poses additional challenges in realizing its potentials. This review covers the advances in the antisense imaging probe development for PET and SPECT, with an emphasis on radiolabeling strategies, stability, delivery and in vivo targeting. PMID:21822406

  15. 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

  16. 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

  17. 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

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

    PubMed

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

    2015-06-01

    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.

  19. DNA microarrays: an introduction to the technology.

    PubMed

    Bilitewski, Ursula

    2009-01-01

    DNA microarrays allow the comprehensive genetic analysis of an organism or a sample. They are based on probes, which are immobilized in an ordered two-dimensional pattern on substrates, such as nylon membranes or glass slides. Probes are either spotted cDNAs or oligonucleotides and are designed to be specific for an organism, a gene, a genetic variant (mutation or polymorphism), or intergenic regions. Thus, they can be used for example for genotyping, expression analysis, or studies of protein-DNA interactions, and in the biomedical field they allow the detection of pathogens, antibiotic resistances, gene mutations and polymorphisms, and pathogenic states and can guide therapy. Microarrays, which cover the whole genome of an organism, are as well available as those which are focussed on genes related to a certain diagnostic application.

  20. DNA microarray technology for the microbiologist: an overview.

    PubMed

    Ehrenreich, Armin

    2006-11-01

    DNA microarrays have found widespread use as a flexible tool to investigate bacterial metabolism. Their main advantage is the comprehensive data they produce on the transcriptional response of the whole genome to an environmental or genetic stimulus. This allows the microbiologist to monitor metabolism and to define stimulons and regulons. Other fields of application are the identification of microorganisms or the comparison of genomes. The importance of this technology increases with the number of sequenced genomes and the falling prices for equipment and oligonucleotides. Knowledge of DNA microarrays is of rising relevance for many areas in microbiological research. Much literature has been published on various specific aspects of this technique that can be daunting to the casual user and beginner. This article offers a comprehensive outline of microarray technology for transcription analysis in microbiology. It shortly discusses the types of DNA microarrays available, the printing of custom arrays, common labeling strategies for targets, hybridization, scanning, normalization, and clustering of expression data.

  1. The ultimate chip shot: can microarray technology deliver for neuroscience?

    PubMed

    Nisenbaum, L K

    2002-01-01

    The use of cDNA and oligonucleotide microarrays, or 'chips', is emerging as a powerful, new technology in the field of neuroscience for examining gene expression in a high-throughput fashion. The application of microarray technology to the study of brain and behavior has lagged behind other areas of biology such as cancer and yeast genetics due to the challenges presented by the heterogeneous and complex organization of the nervous system. This review provides a brief overview of available microarray technology as well as a description of experimental considerations in planning and implementing a neuroscience-based array study. Successful implementation of microarray technology within the field of neuroscience will provide a molecular approach to studying systems neurobiology, leading to insights into areas ranging from fundamental questions of developmental neurobiology to neurological and psychiatric disorders.

  2. Oligonucleotide conjugates for therapeutic applications.

    PubMed

    Winkler, Johannes

    2013-07-01

    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 mechanisms and pharmacokinetic properties.

  3. 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

  4. 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.

  5. Living-cell microarrays.

    PubMed

    Yarmush, Martin L; King, Kevin R

    2009-01-01

    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.

  6. Hole hopping rates in single strand oligonucleotides

    NASA Astrophysics Data System (ADS)

    Borrelli, Raffaele; Capobianco, Amedeo; Peluso, Andrea

    2014-08-01

    The rates of hole transfer between guanine and adenine in single strand DNA have been evaluated by using Fermi’s golden rule and Kubo’s generating function approach for the Franck-Condon weighted density of states. The whole sets of the normal modes and vibrational frequencies of the two nucleobases, obtained at DFT/B3LYP level of calculation, have been considered in computations. The results show that in single strand the pyramidalization/planarization mode of the amino groups of both nucleobases plays the major role. At room temperature, the Franck-Condon density of states extends over a wide range of hole site energy difference, 0-1 eV, giving some hints about the design of oligonucleotides of potential technological interest.

  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. Thermodynamics of Oligonucleotide Duplex Melting

    NASA Astrophysics Data System (ADS)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-05-01

    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 rigorous thermodynamic analysis to an important biochemical problem. Because the stacking of base pairs on top of one another is a significant factor in the energetics of oligonucleotide melting, several investigators have applied van't Hoff analysis to melting temperature data using a nearest-neighbor model and have obtained entropies and enthalpies for the stacking of bases. The present article explains how the equilibrium constant for the dissociation of strands from double-stranded oligonucleotides can be expressed in terms of the total strand concentration and thus how the total strand concentration influences the melting temperature. It also presents a simplified analysis based on the entropies and enthalpies of stacking that is manually tractable so that students can work examples to help them understand the thermodynamics of oligonucleotide melting.

  9. Molecular Mechanisms of Antisense Oligonucleotides.

    PubMed

    Crooke, Stanley T

    2017-04-01

    In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

  10. Molecular Mechanisms of Antisense Oligonucleotides

    PubMed Central

    2017-01-01

    In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs. PMID:28080221

  11. 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…

  12. Impact of surface chemistry and blocking strategies on DNA microarrays.

    PubMed

    Taylor, Scott; Smith, Stephanie; Windle, Brad; Guiseppi-Elie, Anthony

    2003-08-15

    The surfaces and immobilization chemistries of DNA microarrays are the foundation for high quality gene expression data. Four surface modification chemistries, poly-L-lysine (PLL), 3-glycidoxypropyltrimethoxysilane (GPS), DAB-AM-poly(propyleminime hexadecaamine) dendrimer (DAB) and 3-aminopropyltrimethoxysilane (APS), were evaluated using cDNA and oligonucleotide sub-arrays. Two un-silanized glass surfaces, RCA-cleaned and immersed in Tris-EDTA buffer were also studied. DNA on amine-modified surfaces was fixed by UV (90 mJ/cm(2)), while DNA on GPS-modified surfaces was immobilized by covalent coupling. Arrays were blocked with either succinic anhydride (SA), bovine serum albumin (BSA) or left unblocked prior to hybridization with labeled PCR product. Quality factors evaluated were surface affinity for cDNA versus oligonucleotides, spot and background intensity, spotting concentration and blocking chemistry. Contact angle measurements and atomic force microscopy were preformed to characterize surface wettability and morphology. The GPS surface exhibited the lowest background intensity regardless of blocking method. Blocking the arrays did not affect raw spot intensity, but affected background intensity on amine surfaces, BSA blocking being the lowest. Oligonucleotides and cDNA on unblocked GPS-modified slides gave the best signal (spot-to-background intensity ratio). Under the conditions evaluated, the unblocked GPS surface along with amine covalent coupling was the most appropriate for both cDNA and oligonucleotide microarrays.

  13. Performing custom microRNA microarray experiments.

    PubMed

    Zhang, Xiaoxiao; Zeng, Yan

    2011-10-28

    microRNAs (miRNAs) are a large family of ˜ 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes (1). Complex genomes encode at least hundreds of miRNAs, which primarily inhibit the expression of a vast number of target genes post-transcriptionally (2, 3). miRNAs control a broad range of biological processes (1). In addition, altered miRNA expression has been associated with human diseases such as cancers, and miRNAs may serve as biomarkers for diseases and prognosis (4, 5). It is important, therefore, to understand the expression and functions of miRNAs under many different conditions. Three major approaches have been employed to profile miRNA expression: real-time PCR, microarray, and deep sequencing. The technique of miRNA microarray has the advantage of being high-throughput, generally less expensive, and most of the experimental and analysis steps can be carried out in a molecular biology laboratory at most universities, medical schools and associated hospitals. Here, we describe a method for performing custom miRNA microarray experiments. A miRNA probe set will be printed on glass slides to produce miRNA microarrays. RNA is isolated using a method or reagent that preserves small RNA species, and then labeled with a fluorescence dye. As a control, reference DNA oligonucleotides corresponding to a subset of miRNAs are also labeled with a different fluorescence dye. The reference DNA will serve to demonstrate the quality of the slide and hybridization and will also be used for data normalization. The RNA and DNA are mixed and hybridized to a microarray slide containing probes for most of the miRNAs in the database. After washing, the slide is scanned to obtain images, and intensities of the individual spots quantified. These raw signals will be further processed and analyzed as the expression data of the corresponding miRNAs. Microarray slides can be stripped and regenerated to reduce the cost of microarrays and to enhance the

  14. Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

    PubMed Central

    LeProust, Emily M.; Peck, Bill J.; Spirin, Konstantin; McCuen, Heather Brummel; Moore, Bridget; Namsaraev, Eugeni; Caruthers, Marvin H.

    2010-01-01

    We have achieved the ability to synthesize thousands of unique, long oligonucleotides (150mers) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used. While there has been significant demand for libraries of long oligos (150mer and more), the yields in conventional DNA synthesis and the associated side reactions have previously limited the availability of oligonucleotide pools to lengths <100 nt. Using novel array based depurination assays, we show that the depurination side reaction is the limiting factor for the synthesis of libraries of long oligonucleotides on Agilent Technologies’ SurePrint® DNA microarray platform. We also demonstrate how depurination can be controlled and reduced by a novel detritylation process to enable the synthesis of high quality, long (150mer) oligonucleotide libraries and we report the characterization of synthesis efficiency for such libraries. Oligonucleotide libraries prepared with this method have changed the economics and availability of several existing applications (e.g. targeted resequencing, preparation of shRNA libraries, site-directed mutagenesis), and have the potential to enable even more novel applications (e.g. high-complexity synthetic biology). PMID:20308161

  15. 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

  16. The Role Radius of Curvature Plays in Thiolated Oligonucleotide Loading on Gold Nanoparticles

    PubMed Central

    Hill, Haley D.; Millstone, Jill E.; Banholzer, Matthew J.; Mirkin, Chad A.

    2011-01-01

    We show that by correlating the radius of curvature of spherical gold nanoparticles of varying sizes with their respective thiol terminated oligonucleotide loading densities that a mathematical relationship can be derived for predicting the loading of oligonucleotides on anisotropic gold nanomaterials. This mathematical relationship was tested with gold nanorods (radius 17.5 nm, length 475 nm) where the measured number of oligonucleotides per rod (3330 ±110) was within experimental error of the predicted loading of 3244 oligonucleotides from the derivation. Additionally, we show that once gold nanoparticles reach a diameter of approximately 60 nm (and certainly 100 nm) the local surface experienced by the oligonucleotide is highly similar to that of a planar surface. PMID:19236080

  17. Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide

    PubMed Central

    Hwang, Byungjin; Bang, Duhee

    2016-01-01

    All synthetic DNA materials require prior programming of the building blocks of the oligonucleotide sequences. The development of a programmable microarray platform provides cost-effective and time-efficient solutions in the field of data storage using DNA. However, the scalability of the synthesis is not on par with the accelerating sequencing capacity. Here, we report on a new paradigm of generating genetic material (writing) using a degenerate oligonucleotide and optomechanical retrieval method that leverages sequencing (reading) throughput to generate the desired number of oligonucleotides. As a proof of concept, we demonstrate the feasibility of our concept in digital information storage in DNA. In simulation, the ability to store data is expected to exponentially increase with increase in degenerate space. The present study highlights the major framework change in conventional DNA writing paradigm as a sequencer itself can become a potential source of making genetic materials. PMID:27876825

  18. 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

  19. Chaotic mixer improves microarray hybridization.

    PubMed

    McQuain, Mark K; Seale, Kevin; Peek, Joel; Fisher, Timothy S; Levy, Shawn; Stremler, Mark A; Haselton, Frederick R

    2004-02-15

    Hybridization is an important aspect of microarray experimental design which influences array signal levels and the repeatability of data within an array and across different arrays. Current methods typically require 24h and use target inefficiently. In these studies, we compare hybridization signals obtained in conventional static hybridization, which depends on diffusional target delivery, with signals obtained in a dynamic hybridization chamber, which employs a fluid mixer based on chaotic advection theory to deliver targets across a conventional glass slide array. Microarrays were printed with a pattern of 102 identical probe spots containing a 65-mer oligonucleotide capture probe. Hybridization of a 725-bp fluorescently labeled target was used to measure average target hybridization levels, local signal-to-noise ratios, and array hybridization uniformity. Dynamic hybridization for 1h with 1 or 10ng of target DNA increased hybridization signal intensities approximately threefold over a 24-h static hybridization. Similarly, a 10- or 60-min dynamic hybridization of 10ng of target DNA increased hybridization signal intensities fourfold over a 24h static hybridization. In time course studies, static hybridization reached a maximum within 8 to 12h using either 1 or 10ng of target. In time course studies using the dynamic hybridization chamber, hybridization using 1ng of target increased to a maximum at 4h and that using 10ng of target did not vary over the time points tested. In comparison to static hybridization, dynamic hybridization reduced the signal-to-noise ratios threefold and reduced spot-to-spot variation twofold. Therefore, we conclude that dynamic hybridization based on a chaotic mixer design improves both the speed of hybridization and the maximum level of hybridization while increasing signal-to-noise ratios and reducing spot-to-spot variation.

  20. Identification of candidate lung cancer susceptibility genes in mouse using oligonucleotide arrays

    PubMed Central

    Lemon, W; Bernert, H; Sun, H; Wang, Y; You, M

    2002-01-01

    We applied microarray gene expression profiling to lungs from mouse strains having variable susceptibility to lung tumour development as a means to identify, within known quantitative trait loci (QTLs), candidate genes responsible for susceptibility or resistance to lung cancer. At least eight chromosomal regions of mice have been mapped and verified to be linked with lung tumour susceptibility or resistance. In this study, high density oligonucleotide arrays were used to measure the relative expression levels of >36 000 genes and ESTs in lung tissues of A/J, BALB/cJ, SM/J, C3H/HeJ, and C57BL/6J mice. A number of differentially expressed genes were found in each of the lung cancer susceptibility QTLs. Bioinformatic analysis of the differentially expressed genes located within QTLs produced 28 susceptibility candidates and 22 resistance candidates. These candidates may be extremely helpful in the ultimate identification of the precise genes responsible for lung tumour susceptibility or resistance in mice and, through follow up, humans. Complete data sets are available at http://thinker.med.ohio-state.edu. PMID:12205107

  1. Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

    PubMed

    Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

    2014-01-01

    Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

  2. Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

    PubMed Central

    Murgha, Yusuf E.; Rouillard, Jean-Marie; Gulari, Erdogan

    2014-01-01

    Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification. PMID:24733454

  3. 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

  4. Overview of Protein Microarrays

    PubMed Central

    Reymond Sutandy, FX; Qian, Jiang; Chen, Chien-Sheng; Zhu, Heng

    2013-01-01

    Protein microarray is an emerging technology that provides a versatile platform for characterization of hundreds of thousands of proteins in a highly parallel and high-throughput way. Two major classes of protein microarrays are defined to describe their applications: analytical and functional protein microarrays. In addition, tissue or cell lysates can also be fractionated and spotted on a slide to form a reverse-phase protein microarray. While the fabrication technology is maturing, applications of protein microarrays, especially functional protein microarrays, have flourished during the past decade. Here, we will first review recent advances in the protein microarray technologies, and then present a series of examples to illustrate the applications of analytical and functional protein microarrays in both basic and clinical research. The research areas will include detection of various binding properties of proteins, study of protein posttranslational modifications, analysis of host-microbe interactions, profiling antibody specificity, and identification of biomarkers in autoimmune diseases. As a powerful technology platform, it would not be surprising if protein microarrays will become one of the leading technologies in proteomic and diagnostic fields in the next decade. PMID:23546620

  5. Oligonucleotide-based antiviral strategies.

    PubMed

    Schubert, S; Kurreck, J

    2006-01-01

    In the age of extensive global traffic systems, the close neighborhood of man and livestock in some regions of the world, as well as inadequate prevention measures and medical care in poorer countries, greatly facilitates the emergence and dissemination of new virus strains. The appearance of avian influenza viruses that can infect humans, the spread of the severe acute respiratory syndrome (SARS) virus, and the unprecedented raging of human immunodeficiency virus (HIV) illustrate the threat of a global virus pandemic. In addition, viruses like hepatitis B and C claim more than one million lives every year for want of efficient therapy. Thus, new approaches to prevent virus propagation are urgently needed. Antisense strategies are considered a very attractive means of inhibiting viral replication, as oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The ensuing targeted destruction of viral RNA should interfere with viral replication without entailing negative effects on ongoing cellular processes. In this review, we will give some examples of the employment of antisense oligonucleotides, ribozymes, and RNA interference strategies for antiviral purposes. Currently, in spite of encouraging results in preclinical studies, only a few antisense oligonucleotides and ribozymes have turned out to be efficient antiviral compounds in clinical trials. The advent of RNA interference now seems to be refueling hopes for decisive progress in the field of therapeutic employment of antisense strategies.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  7. Microarray-Based Detection of 90 Antibiotic Resistance Genes of Gram-Positive Bacteria

    PubMed Central

    Perreten, Vincent; Vorlet-Fawer, Lorianne; Slickers, Peter; Ehricht, Ralf; Kuhnert, Peter; Frey, Joachim

    2005-01-01

    A disposable microarray was developed for detection of up to 90 antibiotic resistance genes in gram-positive bacteria by hybridization. Each antibiotic resistance gene is represented by two specific oligonucleotides chosen from consensus sequences of gene families, except for nine genes for which only one specific oligonucleotide could be developed. A total of 137 oligonucleotides (26 to 33 nucleotides in length with similar physicochemical parameters) were spotted onto the microarray. The microarrays (ArrayTubes) were hybridized with 36 strains carrying specific antibiotic resistance genes that allowed testing of the sensitivity and specificity of 125 oligonucleotides. Among these were well-characterized multidrug-resistant strains of Enterococcus faecalis, Enterococcus faecium, and Lactococcus lactis and an avirulent strain of Bacillus anthracis harboring the broad-host-range resistance plasmid pRE25. Analysis of two multidrug-resistant field strains allowed the detection of 12 different antibiotic resistance genes in a Staphylococcus haemolyticus strain isolated from mastitis milk and 6 resistance genes in a Clostridium perfringens strain isolated from a calf. In both cases, the microarray genotyping corresponded to the phenotype of the strains. The ArrayTube platform presents the advantage of rapidly screening bacteria for the presence of antibiotic resistance genes known in gram-positive bacteria. This technology has a large potential for applications in basic research, food safety, and surveillance programs for antimicrobial resistance. PMID:15872258

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

    USDA-ARS?s Scientific Manuscript database

    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...

  9. 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

  10. 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.

  11. 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

  12. Novel photocleavable universal support for oligonucleotide synthesis.

    PubMed

    Anderson, Emma; Brown, Tom; Picken, Douglas

    2003-01-01

    A novel photocleavable universal support for the automated solid phase synthesis of oligonucleotides is described. The linker between the growing oligonucleotide chain and CPG support contains a nucleophilic amine protected with a photocleavable group. On exposure to UV light, this group is detached and the free amine affords cleavage of the oligonucleotide from the support. The use of long wavelength UV light avoids damage to the DNA.

  13. 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.

  14. SOAP: short oligonucleotide alignment program.

    PubMed

    Li, Ruiqiang; Li, Yingrui; Kristiansen, Karsten; Wang, Jun

    2008-03-01

    We have developed a program SOAP for efficient gapped and ungapped alignment of short oligonucleotides onto reference sequences. The program is designed to handle the huge amounts of short reads generated by parallel sequencing using the new generation Illumina-Solexa sequencing technology. SOAP is compatible with numerous applications, including single-read or pair-end resequencing, small RNA discovery and mRNA tag sequence mapping. SOAP is a command-driven program, which supports multi-threaded parallel computing, and has a batch module for multiple query sets. http://soap.genomics.org.cn.

  15. Microarray in parasitic infections

    PubMed Central

    Sehgal, Rakesh; Misra, Shubham; Anand, Namrata; Sharma, Monika

    2012-01-01

    Modern biology and genomic sciences are rooted in parasitic disease research. Genome sequencing efforts have provided a wealth of new biological information that promises to have a major impact on our understanding of parasites. Microarrays provide one of the major high-throughput platforms by which this information can be exploited in the laboratory. Many excellent reviews and technique articles have recently been published on applying microarrays to organisms for which fully annotated genomes are at hand. However, many parasitologists work on organisms whose genomes have been only partially sequenced. This review is mainly focused on how to use microarray in these situations. PMID:23508469

  16. Wavelet-based detection of transcriptional activity on a novel Staphylococcus aureus tiling microarray

    PubMed Central

    2012-01-01

    Background High-density oligonucleotide microarray is an appropriate technology for genomic analysis, and is particulary useful in the generation of transcriptional maps, ChIP-on-chip studies and re-sequencing of the genome.Transcriptome analysis of tiling microarray data facilitates the discovery of novel transcripts and the assessment of differential expression in diverse experimental conditions. Although new technologies such as next-generation sequencing have appeared, microarrays might still be useful for the study of small genomes or for the analysis of genomic regions with custom microarrays due to their lower price and good accuracy in expression quantification. Results Here, we propose a novel wavelet-based method, named ZCL (zero-crossing lines), for the combined denoising and segmentation of tiling signals. The denoising is performed with the classical SUREshrink method and the detection of transcriptionally active regions is based on the computation of the Continuous Wavelet Transform (CWT). In particular, the detection of the transitions is implemented as the thresholding of the zero-crossing lines. The algorithm described has been applied to the public Saccharomyces cerevisiae dataset and it has been compared with two well-known algorithms: pseudo-median sliding window (PMSW) and the structural change model (SCM). As a proof-of-principle, we applied the ZCL algorithm to the analysis of the custom tiling microarray hybridization results of a S. aureus mutant deficient in the sigma B transcription factor. The challenge was to identify those transcripts whose expression decreases in the absence of sigma B. Conclusions The proposed method archives the best performance in terms of positive predictive value (PPV) while its sensitivity is similar to the other algorithms used for the comparison. The computation time needed to process the transcriptional signals is low as compared with model-based methods and in the same range to those based on the use of

  17. Calibration and assessment of channel-specific biases in microarray data with extended dynamical range

    PubMed Central

    Bengtsson, Henrik; Jönsson, Göran; Vallon-Christersson, Johan

    2004-01-01

    Background Non-linearities in observed log-ratios of gene expressions, also known as intensity dependent log-ratios, can often be accounted for by global biases in the two channels being compared. Any step in a microarray process may introduce such offsets and in this article we study the biases introduced by the microarray scanner and the image analysis software. Results By scanning the same spotted oligonucleotide microarray at different photomultiplier tube (PMT) gains, we have identified a channel-specific bias present in two-channel microarray data. For the scanners analyzed it was in the range of 15–25 (out of 65,535). The observed bias was very stable between subsequent scans of the same array although the PMT gain was greatly adjusted. This indicates that the bias does not originate from a step preceding the scanner detector parts. The bias varies slightly between arrays. When comparing estimates based on data from the same array, but from different scanners, we have found that different scanners introduce different amounts of bias. So do various image analysis methods. We propose a scanning protocol and a constrained affine model that allows us to identify and estimate the bias in each channel. Backward transformation removes the bias and brings the channels to the same scale. The result is that systematic effects such as intensity dependent log-ratios are removed, but also that signal densities become much more similar. The average scan, which has a larger dynamical range and greater signal-to-noise ratio than individual scans, can then be obtained. Conclusions The study shows that microarray scanners may introduce a significant bias in each channel. Such biases have to be calibrated for, otherwise systematic effects such as intensity dependent log-ratios will be observed. The proposed scanning protocol and calibration method is simple to use and is useful for evaluating scanner biases or for obtaining calibrated measurements with extended dynamical

  18. Oligonucleotide therapeutics for human leukaemia.

    PubMed

    Gewirtz, A M

    1997-01-01

    The concept of antisense oligonucleotide 'therapeutics' has generated a great deal of controversy. Questions abound regarding the mechanism of action of these compounds, their reliability and their ultimate utility. These problems are compounded by the 'hype', which has attended their development, and the inability of workers in this area to meet the expectations raised by its most zealous proponents. Nevertheless, it is worth pointing out that there have been some notable gene disruption successes with this technique that have stood up to rigorous scrutiny. Our own work with c-myb as a target is perhaps a reasonable example. Though much remains to be accomplished before antisense drugs are commonly, and usefully, employed in the clinic, it is important to remember what motivates their development. Gene-targeted drugs have the promise of exquisite specificity and the potential to do much good with little toxicity. Accordingly, antisense oligonucleotides can serve as a paradigm of rational drug development. For all these reasons then, we believe that efforts should be increased to decipher the mechanism of action of antisense oligodeoxynucleotides, and to learn how they may be successfully employed in the clinic.

  19. Detection of fungal DNA in peritoneal fluids by a PCR DNA low-density microarray system and quantitation of serum (1-3)-β-D-glucan in the diagnosis of peritoneal candidiasis.

    PubMed

    Corrales, Isabel; Giménez, Estela; Aguilar, Gerardo; Delgado, Carlos; Puig, Jaime; Izquierdo, Ana; Belda, Javier; Navarro, David

    2015-02-01

    Microbiological documentation of peritoneal candidiasis (PC) is hampered by the low numbers of yeasts observable by direct microscopic examination and recoverable by culture methods. The performance of a polymerase chain reaction (PCR) DNA Low-Density Microarray System (CLART STIs B) was compared to that of BACTEC FX automated culture method for the detection of Candida spp. in 161 peritoneal fluids (PF) from patients with peritonitis. The clinical utility of (1-3)-β-d-glucan (BDG) antigenemia in the diagnosis of PC was evaluated in 42 of these patients. The overall agreement between the PCR assay and the culture method was good (κ = 0.790), and their sensitivities were 93.5% and 74.19%, respectively. Serum BDG levels in patients with Candida spp. in PFs (median, 200.3 pg/mL; Range, 22.0-523.4 pg/mL) was significantly higher (P = 0.002) than those found in patients without the yeast (median, 25.3 pg/mL; Range, 0-523.4 pg/mL). Our study demonstrates the potential clinical utility of molecular methods and the measurement of serum BDG levels for the diagnosis of PC. © The Author 2014. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. 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

  1. Emerging Use of Gene Expression Microarrays in Plant Physiology

    DOE PAGES

    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

  2. Plant-pathogen interactions: what microarray tells about it?

    PubMed

    Lodha, T D; Basak, J

    2012-01-01

    Plant defense responses are mediated by elementary regulatory proteins that affect expression of thousands of genes. Over the last decade, microarray technology has played a key role in deciphering the underlying networks of gene regulation in plants that lead to a wide variety of defence responses. Microarray is an important tool to quantify and profile the expression of thousands of genes simultaneously, with two main aims: (1) gene discovery and (2) global expression profiling. Several microarray technologies are currently in use; most include a glass slide platform with spotted cDNA or oligonucleotides. Till date, microarray technology has been used in the identification of regulatory genes, end-point defence genes, to understand the signal transduction processes underlying disease resistance and its intimate links to other physiological pathways. Microarray technology can be used for in-depth, simultaneous profiling of host/pathogen genes as the disease progresses from infection to resistance/susceptibility at different developmental stages of the host, which can be done in different environments, for clearer understanding of the processes involved. A thorough knowledge of plant disease resistance using successful combination of microarray and other high throughput techniques, as well as biochemical, genetic, and cell biological experiments is needed for practical application to secure and stabilize yield of many crop plants. This review starts with a brief introduction to microarray technology, followed by the basics of plant-pathogen interaction, the use of DNA microarrays over the last decade to unravel the mysteries of plant-pathogen interaction, and ends with the future prospects of this technology.

  3. 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…

  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. Bromodeoxyuridine-labeled oligonucleotides as tools for oligonucleotide uptake studies.

    PubMed

    Maszewska, Maria; Kobylańska, Anna; Gendaszewska-Darmach, Edyta; Koziołkiewicz, Maria

    2002-12-01

    The mechanisms by which various oligonucleotides (ODNs) and their analogs enter cells are not fully understood. A common technique used in studies on cellular uptake of ODNs is their conjugation with fluorochromes. However, fluorescently labeled ODNs may vary from the parent compounds in charge and hydrophilicity, and they may interact differently with some components of cellular membranes. In this report, we present an alternative method based on the immunofluorescent detection of ODNs with incorporated 5-bromo-2'-deoxyuridine (BrdUrd). Localization of BrdUrd-modified ODNs has been achieved using FITC-labeled anti-BrdUrd antibodies. This technique allowed determination of the differences in cellular uptake of phosphodiester (PO) and phosphorothioate (PS) ODNs and their derivatives conjugated with cholesterol and menthol. The immunocytochemical method also has shown that the cellular uptake of some ODNs may be influenced by specific sequences that are responsible for the formation of higher-order structures.

  6. Influence of the length of target DNA overhang proximal to the array surface on discrimination of single-base mismatches on a 25-mer oligonucleotide array

    PubMed Central

    2014-01-01

    Background The performance of probes on an oligonucleotide microarray can be characterised in terms of hybridisation signal strength and the ability to discriminate sequence mismatches between the probe and the hybridising target strand, such as those resulting from SNPs. Various properties of the probe affect mismatch discrimination, such as probe length and the position of mismatched bases, and the effects of these factors have been well characterised in a variety of array formats. Results A low-density microarray was developed to systematically investigate the effect of a probe’s position within hybridised target PCR products on the tolerance and discrimination of single-nucleotide mismatches between the probe and target. In line with previous reports, hybridisation signals were attenuated by different degrees depending on the identity of the mismatch, the position of the mismatch within the probe, and the length of the PCR product. However, the same mismatch caused different degrees of attenuation depending on the position of the probe within the hybridising product, such that improved mismatch discrimination was observed for PCR products where a greater proportion of the total length was proximal to the array surface. Conclusions These results suggest that the degree of mismatch discrimination can be influenced by the choice of PCR primers, providing a means by which array performance could be fine-tuned in addition to manipulation of the properties of the probes themselves. PMID:24742004

  7. [Recent progress and prospect in oligonucleotide therapeutics].

    PubMed

    Yoshioka, Kotaro; Kuwahara, Hiroya; Nishina, Kazutaka; Nagata, Tetsuya; Yokota, Takanori

    2015-06-01

    Remarkable progress has been made in chemical modification and nonviral delivery systems that improve the properties and efficacy of therapeutics oligonucleotides therapeutics, such as antisense oligonucleotide (ASO) and small interfering RNA(siRNA). ASOs act through various mechanisms including the degradation of mRNA by RNase H (gapmer-type ASO) and the modulation alternative splicing patterns(splice switching oligonucleotide). Recent favorable outcomes in clinical trials for cancers and genetic diseases such as familial amyloid polyneuropathy and Duchenne muscular dystrophy indicate high clinical potency of oligonucleotide therapeutics. Here we reviewed recent advances in basic properties and clinical applications of ASO and siRNA, and provide future perspective on oligonucleotide therapeutics.

  8. DNA Microarray Technology

    SciTech Connect

    WERNER-WASHBURNE, MARGARET; DAVIDSON, GEORGE S.

    2002-01-01

    Collaboration between Sandia National Laboratories and the University of New Mexico Biology Department resulted in the capability to train students in microarray techniques and the interpretation of data from microarray experiments. These studies provide for a better understanding of the role of stationary phase and the gene regulation involved in exit from stationary phase, which may eventually have important clinical implications. Importantly, this research trained numerous students and is the basis for three new Ph.D. projects.

  9. 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.

  10. Adaptive resolution simulation of oligonucleotides.

    PubMed

    Netz, Paulo A; Potestio, Raffaello; Kremer, Kurt

    2016-12-21

    Nucleic acids are characterized by a complex hierarchical structure and a variety of interaction mechanisms with other molecules. These features suggest the need of multiscale simulation methods in order to grasp the relevant physical properties of deoxyribonucleic acid (DNA) and RNA using in silico experiments. Here we report an implementation of a dual-resolution modeling of a DNA oligonucleotide in physiological conditions; in the presented setup only the nucleotide molecule and the solvent and ions in its proximity are described at the atomistic level; in contrast, the water molecules and ions far from the DNA are represented as computationally less expensive coarse-grained particles. Through the analysis of several structural and dynamical parameters, we show that this setup reliably reproduces the physical properties of the DNA molecule as observed in reference atomistic simulations. These results represent a first step towards a realistic multiscale modeling of nucleic acids and provide a quantitatively solid ground for their simulation using dual-resolution methods.

  11. Adaptive resolution simulation of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Netz, Paulo A.; Potestio, Raffaello; Kremer, Kurt

    2016-12-01

    Nucleic acids are characterized by a complex hierarchical structure and a variety of interaction mechanisms with other molecules. These features suggest the need of multiscale simulation methods in order to grasp the relevant physical properties of deoxyribonucleic acid (DNA) and RNA using in silico experiments. Here we report an implementation of a dual-resolution modeling of a DNA oligonucleotide in physiological conditions; in the presented setup only the nucleotide molecule and the solvent and ions in its proximity are described at the atomistic level; in contrast, the water molecules and ions far from the DNA are represented as computationally less expensive coarse-grained particles. Through the analysis of several structural and dynamical parameters, we show that this setup reliably reproduces the physical properties of the DNA molecule as observed in reference atomistic simulations. These results represent a first step towards a realistic multiscale modeling of nucleic acids and provide a quantitatively solid ground for their simulation using dual-resolution methods.

  12. A model of base-call resolution on broad-spectrum pathogen detection resequencing DNA microarrays.

    PubMed

    Malanoski, Anthony P; Lin, Baochuan; Stenger, David A

    2008-06-01

    Oligonucleotide microarrays offer the potential to efficiently test for multiple organisms, an excellent feature for surveillance applications. Among these, resequencing microarrays are of particular interest, as they possess additional unique capabilities to track pathogens' genetic variations and perform detailed discrimination of closely related organisms. However, this potential can only be realized if the costs of developing the detection microarray are kept at a manageable level. Selection and verification of the probes are key factors affecting microarray design costs that can be reduced through the development and use of in silico modeling. Models created for other types of microarrays do not meet all the required criteria for this type of microarray. We describe here in silico methods for designing resequencing microarrays targeted for multiple organism detection. The model development presented here has focused on accurate base-call prediction in regions that are applicable to resequencing microarrays designed for multiple organism detection, a variation from other uses of a predictive model in which perfect prediction of all hybridization events is necessary. The model will assist in simplifying the design of resequencing microarrays and in reduction of the time and costs required for their development for new applications.

  13. 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. © 2010 Blackwell Verlag GmbH.

  14. D-MaPs - DNA-microarray projects: Web-based software for multi-platform microarray analysis

    PubMed Central

    2009-01-01

    The web application D-Maps provides a user-friendly interface to researchers performing studies based on microarrays. The program was developed to manage and process one- or two-color microarray data obtained from several platforms (currently, GeneTAC, ScanArray, CodeLink, NimbleGen and Affymetrix). Despite the availability of many algorithms and many software programs designed to perform microarray analysis on the internet, these usually require sophisticated knowledge of mathematics, statistics and computation. D-maps was developed to overcome the requirement of high performance computers or programming experience. D-Maps performs raw data processing, normalization and statistical analysis, allowing access to the analyzed data in text or graphical format. An original feature presented by D-Maps is GEO (Gene Expression Omnibus) submission format service. The D-MaPs application was already used for analysis of oligonucleotide microarrays and PCR-spotted arrays (one- and two-color, laser and light scanner). In conclusion, D-Maps is a valuable tool for microarray research community, especially in the case of groups without a bioinformatic core. PMID:21637530

  15. D-MaPs - DNA-microarray projects: Web-based software for multi-platform microarray analysis.

    PubMed

    Carazzolle, Marcelo F; Herig, Taís S; Deckmann, Ana C; Pereira, Gonçalo A G

    2009-07-01

    The web application D-Maps provides a user-friendly interface to researchers performing studies based on microarrays. The program was developed to manage and process one- or two-color microarray data obtained from several platforms (currently, GeneTAC, ScanArray, CodeLink, NimbleGen and Affymetrix). Despite the availability of many algorithms and many software programs designed to perform microarray analysis on the internet, these usually require sophisticated knowledge of mathematics, statistics and computation. D-maps was developed to overcome the requirement of high performance computers or programming experience. D-Maps performs raw data processing, normalization and statistical analysis, allowing access to the analyzed data in text or graphical format. An original feature presented by D-Maps is GEO (Gene Expression Omnibus) submission format service. The D-MaPs application was already used for analysis of oligonucleotide microarrays and PCR-spotted arrays (one- and two-color, laser and light scanner). In conclusion, D-Maps is a valuable tool for microarray research community, especially in the case of groups without a bioinformatic core.

  16. A Novel Pan-Flavivirus Detection and Identification Assay Based on RT-qPCR and Microarray

    PubMed Central

    Sachse, Konrad; Ziegler, Ute; Keller, Markus

    2017-01-01

    The genus Flavivirus includes arthropod-borne viruses responsible for a large number of infections in humans and economically important animals. While RT-PCR protocols for specific detection of most Flavivirus species are available, there has been also a demand for a broad-range Flavivirus assay covering all members of the genus. It is particularly challenging to balance specificity at genus level with equal sensitivity towards each target species. In the present study, a novel assay combining a SYBR Green-based RT-qPCR with a low-density DNA microarray has been developed. Validation experiments confirmed that the RT-qPCR exhibited roughly equal sensitivity of detection and quantification for all flaviviruses tested. These PCR products are subjected to hybridization on a microarray carrying 84 different oligonucleotide probes that represent all known Flavivirus species. This assay has been used as a screening and confirmation tool for Flavivirus presence in laboratory and field samples, and it performed successfully in international External Quality Assessment of NAT studies. Twenty-six Flavivirus strains were tested with the assay, showing equivalent or superior characteristics compared with the original or even with species-specific RT-PCRs. As an example, test results on West Nile virus detection in a panel of 340 mosquito pool samples from Greece are presented. PMID:28626758

  17. Ultrathin oligonucleotide layers for fluorescence-based DNA sensors

    NASA Astrophysics Data System (ADS)

    Furch, M.; Ueberfeld, J.; Hartmann, Andreas; Bock, Daniel; Seeger, Stefan

    1996-11-01

    Preliminary investigations into the design of an affinity sensor using evanescent wave technology concentrate upon the means of immobilization of the receptor molecules. In this work DNA served as the selective recognition element. The molecular principle of a sequence-selective biosensor for DNA is based on a sandwich-hybridization assay wherein the analyte, a single-stranded (ss)DNA, bound specifically to both an immobilized capture probe and a dye-labeled oligonucleotide in free solution. The efficiency of the capture array depends on the density of highly organized oligonucleotides on the waveguide surface and correlates therefore directly with the specificity and the sensitivity of the sensor. In the present approach using the Langmuir- Blodgett technique cinnamoylbutylether-cellulose monolayers were transferred onto optical fibers or planar waveguides. These films served as matrices for the immobilization of biotinylated oligonucleotides via streptavidin. For the first time streptavidin was immobilized by that manner. The specificity of the streptavidin layer or the following bounded nucleic acid molecules were controlled by an enzyme- linked immunosorbent assay (ELISA). Finally, this application has also shown to be suitable for the detection of Salmonella, which is an important pathogen associated with acute gastroenteritidis and food borne diseases.

  18. Functional GPCR microarrays.

    PubMed

    Hong, Yulong; Webb, Brian L; Su, Hui; Mozdy, Eric J; Fang, Ye; Wu, Qi; Liu, Li; Beck, Jonathan; Ferrie, Ann M; Raghavan, Srikanth; Mauro, John; Carre, Alain; Müeller, Dirk; Lai, Fang; Rasnow, Brian; Johnson, Michael; Min, Hosung; Salon, John; Lahiri, Joydeep

    2005-11-09

    This paper describes G-protein-coupled receptor (GPCR) microarrays on porous glass substrates and functional assays based on the binding of a europium-labeled GTP analogue. The porous glass slides were made by casting a glass frit on impermeable glass slides and then coating with gamma-aminopropyl silane (GAPS). The emitted fluorescence was captured on an imager with a time-gated intensified CCD detector. Microarrays of the neurotensin receptor 1, the cholinergic receptor muscarinic 2, the opioid receptor mu, and the cannabinoid receptor 1 were fabricated by pin printing. The selective agonism of each of the receptors was observed. The screening of potential antagonists was demonstrated using a cocktail of agonists. The amount of activation observed was sufficient to permit determinations of EC50 and IC50. Such microarrays could potentially streamline drug discovery by helping integrate primary screening with selectivity and safety screening without compromising the essential functional information obtainable from cellular assays.

  19. 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.

  20. Multievidence microarray mining.

    PubMed

    Seifert, Martin; Scherf, Matthias; Epple, Anton; Werner, Thomas

    2005-10-01

    Microarray mining is a challenging task because of the superposition of several processes in the data. We believe that the combination of microarray data-based analyses (statistical significance analysis of gene expression) with array-independent analyses (literature-mining and promoter analysis) enables some of the problems of traditional array analysis to be overcome. As a proof-of-principle, we revisited publicly available microarray data derived from an experiment with platelet-derived growth factor (PDGF)-stimulated fibroblasts. Our strategy revealed results beyond the detection of the major metabolic pathway known to be linked to the PDGF response: we were able to identify the crosstalking regulatory networks underlying the metabolic pathway without using a priori knowledge about the experiment.

  1. Complex DNA nanostructures from oligonucleotide ensembles.

    PubMed

    Mathur, Divita; Henderson, Eric R

    2013-04-19

    The first synthetic DNA nanostructures were created by self-assembly of a small number of oligonucleotides. Introduction of the DNA origami method provided a new paradigm for designing and creating two- and three-dimensional DNA nanostructures by folding a large single-stranded DNA and 'stapling' it together with a library of oligonucleotides. Despite its power and wide-ranging implementation, the DNA origami technique suffers from some limitations. Foremost among these is the limited number of useful single-stranded scaffolds of biological origin. This report describes a new approach to creating large DNA nanostructures exclusively from synthetic oligonucleotides. The essence of this approach is to replace the single-stranded scaffold in DNA origami with a library of oligonucleotides termed "scaples" (scaffold staples). Scaples eliminate the need for scaffolds of biological origin and create new opportunities for producing larger and more diverse DNA nanostructures as well as simultaneous assembly of distinct structures in a "single-pot" reaction.

  2. Microfabricated Fountain Pens for High-Density DNA Arrays

    PubMed Central

    Reese, Matthew O.; van Dam, R. Michae; Scherer, Axel; Quake, Stephen R.

    2003-01-01

    We used photolithographic microfabrication techniques to create very small stainless steel fountain pens that were installed in place of conventional pens on a microarray spotter. Because of the small feature size produced by the microfabricated pens, we were able to print arrays with up to 25,000 spots/cm2, significantly higher than can be achieved by other deposition methods. This feature density is sufficiently large that a standard microscope slide can contain multiple replicates of every gene in a complex organism such as a mouse or human. We tested carryover during array printing with dye solution, labeled DNA, and hybridized DNA, and we found it to be indistinguishable from background. Hybridization also showed good sequence specificity to printed oligonucleotides. In addition to improved slide capacity, the microfabrication process offers the possibility of low-cost mass-produced pens and the flexibility to include novel pen features that cannot be machined with conventional techniques. PMID:12975313

  3. 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

  4. Incorporation of an aldehyde function in oligonucleotides.

    PubMed

    Tilquin, J M; Dechamps, M; Sonveaux, E

    2001-01-01

    A nucleotide-like phosphoramidite building block that has the nucleic base replaced by the tert-butyldimethylsilyl-protected styrene glycol was synthesized. After the automatic synthesis of an oligonucleotide incorporating this synthon, the benzaldehyde function was generated by fluoride deprotection and oxidation by sodium periodate. In a similar manner, an oligonucleotide where a nucleic base was replaced by the (CH2)8CH=O chain was synthesized and conjugated with biotin derivatives.

  5. DNA microarray technology in toxicogenomics of aquatic models: methods and applications.

    PubMed

    Ju, Zhenlin; Wells, Melissa C; Walter, Ronald B

    2007-02-01

    Toxicogenomics represents the merging of toxicology with genomics and bioinformatics to investigate biological functions of genome in response to environmental contaminants. Aquatic species have traditionally been used as models in toxicology to characterize the actions of environmental stresses. Recent completion of the DNA sequencing for several fish species has spurred the development of DNA microarrays allowing investigators access to toxicogenomic approaches. However, since microarray technology is thus far limited to only a few aquatic species and derivation of biological meaning from microarray data is highly dependent on statistical arguments, the full potential of microarray in aquatic species research has yet to be realized. Herein we review some of the issues related to construction, probe design, statistical and bioinformatical data analyses, and current applications of DNA microarrays. As a model a recently developed medaka (Oryzias latipes) oligonucleotide microarray was described to highlight some of the issues related to array technology and its application in aquatic species exposed to hypoxia. Although there are known non-biological variations present in microarray data, it remains unquestionable that array technology will have a great impact on aquatic toxicology. Microarray applications in aquatic toxicogenomics will range from the discovery of diagnostic biomarkers, to establishment of stress-specific signatures and molecular pathways hallmarking the adaptation to new environmental conditions.

  6. Microarray analysis of p-anisaldehyde-induced transcriptome of Saccharomyces cerevisiae.

    PubMed

    Yu, Lu; Guo, Na; Yang, Yi; Wu, Xiuping; Meng, Rizeng; Fan, Junwen; Ge, Fa; Wang, Xuelin; Liu, Jingbo; Deng, Xuming

    2010-03-01

    p-Anisaldehyde (4-methoxybenzaldehyde), an extract from Pimpinella anisum L. seeds, is a potential novel preservative. To reveal the possible action mechanism of p-anisaldehyde against microorganisms, yeast-based commercial oligonucleotide microarrays were used to analyze the genome-wide transcriptional changes in response to p-anisaldehyde. Quantitative real-time RT-PCR was performed for selected genes to verify the microarray results. We interpreted our microarray data with the clustering tool, T-profiler. Analysis of microarray data revealed that p-anisaldehyde induced the expression of genes related to sulphur assimilation, aromatic aldehydes metabolism, and secondary metabolism, which demonstrated that the addition of p-anisaldehyde may influence the normal metabolism of aromatic aldehydes. This genome-wide transcriptomics approach revealed first insights into the response of Saccharomyces cerevisiae (S. cerevisiae) to p-anisaldehyde challenge.

  7. 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…

  8. Protein Microarray Technology

    PubMed Central

    Hall, David A.; Ptacek, Jason

    2007-01-01

    Protein chips have emerged as a promising approach for a wide variety of applications including the identification of protein-protein interactions, protein-phospholipid interactions, small molecule targets, and substrates of proteins kinases. They can also be used for clinical diagnostics and monitoring disease states. This article reviews current methods in the generation and applications of protein microarrays. PMID:17126887

  9. 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…

  10. On-chip synthesis of RNA aptamer microarrays for multiplexed protein biosensing with SPR imaging measurements.

    PubMed

    Chen, Yulin; Nakamoto, Kohei; Niwa, Osamu; Corn, Robert M

    2012-06-05

    Microarrays of RNA aptamers are fabricated in a one-step, multiplexed enzymatic synthesis on gold thin films in a microfluidic format and then employed in the detection of protein biomarkers with surface plasmon resonance imaging (SPRI) measurements. Single-stranded RNA (ssRNA) oligonucleotides are transcribed on-chip from double-stranded DNA (dsDNA) templates attached to microarray elements (denoted as generator elements) by the surface transcription reaction of T7 RNA polymerase. As they are synthesized, the ssRNA oligonucleotides diffuse in the microfluidic channel and are quickly captured by hybridization adsorption onto adjacent single-stranded DNA (ssDNA) microarray elements (denoted as detector elements) that contain a sequence complementary to 5'-end of the ssRNA. The RNA aptamers attached to these detector elements are subsequently used in SPRI measurements for the bioaffinity detection of protein biomarkers. The microfluidic generator-detector element format permits the simultaneous fabrication of multiple ssRNA oligonucleotides with different capture sequences that can hybridize simultaneously to distinct detector elements and thus create a multiplexed aptamer microarray. In an initial set of demonstration experiments, SPRI measurements are used to monitor the bioaffinity adsorption of human thrombin (hTh) and vascular endothelial growth factor (VEGF) proteins onto RNA aptamer microarrays fabricated in situ with this on-chip RNA polymerase synthesis methodology. Additional SPRI measurements of the hydrolysis and desorption of the surface-bound ssRNA aptamers with a surface RNase H are used to verify the capture of ssRNA with RNA-DNA surface hybridization onto the detector elements. The on-chip RNA synthesis described here is an elegant, one-step multiplexed methodology for the rapid and contamination-free fabrication of RNA aptamer microarrays for protein biosensing with SPRI.

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

    PubMed

    Biyani, Manish; Ichiki, Takanori

    2015-07-14

    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.

  12. 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. PMID:27600226

  13. Genotyping and detection of common avian and human origin-influenza viruses using a portable chemiluminescence imaging microarray.

    PubMed

    Zhang, Yingjie; Liu, Qiqi; Wang, Dou; Chen, Suhong; Wang, Xiaobo; Wang, Shengqi

    2016-01-01

    Influenza viruses are divided into three types, A, B, and C. Human influenza A and B viruses can cause seasonal epidemics, but influenza C causes only a mild respiratory illness. Influenza A virus can infect various host species. In 2013, human-infectious avian influenza A (H7N9) was first reported in China. By the second week of 2014, there were 210 laboratory-confirmed human cases in the country, and the mortality rate eventually reached 22 %. Rapid and accurate diagnosis of influenza viruses is important for clinical management and epidemiology. In this assay, a cost-effective chemiluminescence (CL) detection oligonucleotide microarray was developed to genotype and detect avian influenza A (H7N9), avian influenza A (H5N1), 2009 influenza A (H1N1), seasonal influenza A (H1N1), and seasonal influenza A (H3N2). Influenza A viruses and influenza B viruses were also generally detected using this microarray. The results of detection of 40 cultivated influenza virus strains showed that the microarray was able to distinguish the subtypes of these influenza viruses very well. The microarray possessed similar or 10 fold higher limit of detection than the real-time RT-PCR method. Sixty-six clinical swab samples were detected using this microarray and verified with real time RT-PCR to evaluate the efficiency of this microarray for clinical testing. A reliable CL detection oligonucleotide microarray had been developed to genotype and detected these influenza viruses.

  14. Assessing the Detection Capacity of Microarrays as Bio/Nanosensing Platforms

    PubMed Central

    Lee, Ju Seok; Song, Joon Jin; Kim, Jin-Woo

    2013-01-01

    Microarray is one of the most powerful detection systems with multiplexing and high throughput capability. It has significant potential as a versatile biosensing platform for environmental monitoring, pathogen detection, medical therapeutics, and drug screening to name a few. To date, however, microarray applications are still limited to preliminary screening of genome-scale transcription profiling or gene ontology analysis. Expanding the utility of microarrays as a detection tool for various biological and biomedical applications requires information about performance such as the limits of detection and quantification, which are considered as an essential information to decide the detection sensitivity of sensing devices. Here we present a calibration design that integrates detection limit theory and linear dynamic range to obtain a performance index of microarray detection platform using oligonucleotide arrays as a model system. Two different types of limits of detection and quantification are proposed by the prediction or tolerance interval for two common cyanine fluorescence dyes, Cy3 and Cy5. Besides oligonucleotide, the proposed method can be generalized to other microarray formats with various biomolecules such as complementary DNA, protein, peptide, carbohydrate, tissue, or other small biomolecules. Also, it can be easily applied to other fluorescence dyes for further dye chemistry improvement. PMID:24324959

  15. Enhanced oligonucleotide-nanoparticle conjugate stability using thioctic acid modified oligonucleotides.

    PubMed

    Dougan, Jennifer A; Karlsson, Camilla; Smith, W Ewen; Graham, Duncan

    2007-01-01

    Metallic nanoparticles of gold functionalized with oligonucleotides conventionally use a terminal thiol modification and have been used in a wide range of applications. Although readily available, the oligonucleotide-nanoparticle conjugates prepared in this way suffer from a lack of stability when exposed to a variety of small molecules or elevated temperatures. If silver is used in place of gold then this lack of stability is even more pronounced. In this study we report the synthesis of highly stabilized oligonucleotide-nanoparticle conjugates using a simple oligonucleotide modification. A modified solid support was used to generate 3'-thioctic acid modified oligonucleotides by treatment with an N-hydroxysuccimidyl ester of thioctic acid. Unusually, both gold and silver nanoparticles have been investigated in this study and show that these disulphide-modified oligonucleotide probes offer significant improvements in nanoparticle stability when treated with dithiothreitol (DTT) compared with monothiol analogues. This is a significant advance in oligonucleotide-nanoparticle conjugate stability and for the first time allows silver nanoparticles to be prepared that are more stable than standard gold-thiol functionalized nanoparticles. This opens up the possibility of using silver nanoparticles functionalized with oligonucleotides as an alternative to gold.

  16. A novel design of whole-genome microarray probes for Saccharomyces cerevisiae which minimizes cross-hybridization

    PubMed Central

    Talla, Emmanuel; Tekaia, Fredj; Brino, Laurent; Dujon, Bernard

    2003-01-01

    Background Numerous DNA microarray hybridization experiments have been performed in yeast over the last years using either synthetic oligonucleotides or PCR-amplified coding sequences as probes. The design and quality of the microarray probes are of critical importance for hybridization experiments as well as subsequent analysis of the data. Results We present here a novel design of Saccharomyces cerevisiae microarrays based on a refined annotation of the genome and with the aim of reducing cross-hybridization between related sequences. An effort was made to design probes of similar lengths, preferably located in the 3'-end of reading frames. The sequence of each gene was compared against the entire yeast genome and optimal sub-segments giving no predicted cross-hybridization were selected. A total of 5660 novel probes (more than 97% of the yeast genes) were designed. For the remaining 143 genes, cross-hybridization was unavoidable. Using a set of 18 deletant strains, we have experimentally validated our cross-hybridization procedure. Sensitivity, reproducibility and dynamic range of these new microarrays have been measured. Based on this experience, we have written a novel program to design long oligonucleotides for microarray hybridizations of complete genome sequences. Conclusions A validated procedure to predict cross-hybridization in microarray probe design was defined in this work. Subsequently, a novel Saccharomyces cerevisiae microarray (which minimizes cross-hybridization) was designed and constructed. Arrays are available at Eurogentec S. A. Finally, we propose a novel design program, OliD, which allows automatic oligonucleotide design for microarrays. The OliD program is available from authors. PMID:14499002

  17. Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides.

    PubMed

    Graham, Mark J; Lee, Richard G; Brandt, Teresa A; Tai, Li-Jung; Fu, Wuxia; Peralta, Raechel; Yu, Rosie; Hurh, Eunju; Paz, Erika; McEvoy, Bradley W; Baker, Brenda F; Pham, Nguyen C; Digenio, Andres; Hughes, Steven G; Geary, Richard S; Witztum, Joseph L; Crooke, Rosanne M; Tsimikas, Sotirios

    2017-07-20

    Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins. We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, P<0.01 for all doses vs. placebo) and in levels of triglycerides (reductions of 33.2 to 63.1%), LDL cholesterol (1.3 to 32.9%), very-low-density lipoprotein cholesterol (27.9 to 60.0%), non-high-density lipoprotein cholesterol (10.0 to 36.6%), apolipoprotein B (3.4 to 25.7%), and apolipoprotein C-III (18.9 to 58.8%). Three participants who received the antisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in

  18. Cross-platform analysis of cancer microarray data improves gene expression based classification of phenotypes

    PubMed Central

    Warnat, Patrick; Eils, Roland; Brors, Benedikt

    2005-01-01

    Background The extensive use of DNA microarray technology in the characterization of the cell transcriptome is leading to an ever increasing amount of microarray data from cancer studies. Although similar questions for the same type of cancer are addressed in these different studies, a comparative analysis of their results is hampered by the use of heterogeneous microarray platforms and analysis methods. Results In contrast to a meta-analysis approach where results of different studies are combined on an interpretative level, we investigate here how to directly integrate raw microarray data from different studies for the purpose of supervised classification analysis. We use median rank scores and quantile discretization to derive numerically comparable measures of gene expression from different platforms. These transformed data are then used for training of classifiers based on support vector machines. We apply this approach to six publicly available cancer microarray gene expression data sets, which consist of three pairs of studies, each examining the same type of cancer, i.e. breast cancer, prostate cancer or acute myeloid leukemia. For each pair, one study was performed by means of cDNA microarrays and the other by means of oligonucleotide microarrays. In each pair, high classification accuracies (> 85%) were achieved with training and testing on data instances randomly chosen from both data sets in a cross-validation analysis. To exemplify the potential of this cross-platform classification analysis, we use two leukemia microarray data sets to show that important genes with regard to the biology of leukemia are selected in an integrated analysis, which are missed in either single-set analysis. Conclusion Cross-platform classification of multiple cancer microarray data sets yields discriminative gene expression signatures that are found and validated on a large number of microarray samples, generated by different laboratories and microarray technologies

  19. 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

  20. [Design and realization of a microarray data analysis platform].

    PubMed

    Sun, Xian-He; Guo, Yun-Bo; Liu, Na; Ma, Li; Deng, Qin-Kai

    2011-04-01

    To design a platform for microarray data analysis and processing in the browser/server mode running in Linux operating system. Based on the Apache HTTP server, the platform, programmed with Perl language, integrated R language and Bioconductor packages for processing and analysis of the input data of oligonucleotide arrays and two-color spotted arrays. Users were allowed to submit data and parameter configurations to the platform via the web page, and the results of analysis were also returned via the web page. With an easy operation and high performance, the platform fulfilled the functions of processing, quality assessment, biological annotation and statistical analysis of the data from oligonucleotide arrays and two-color spotted arrays. Using the platform, we analyzed the gene expression profiles in Mtb-stimulated macrophages of three clinical phenotypes, namely latent TB (LTB), pulmonary (PTB) and meningeal (TBM), and obtained valuable clues for identifying tuberculosis susceptibility genes. We also analyzed the effect of INH treatment on Mycobacterium tuberculosis gene expression in various dormancy models, such as hypoxia and KatG mutant, and found that a set of genes responded to INH treatment during exponential growth but not in dormancy, and that the overall number of differentially regulated genes was reduced in the cells in low metabolic state. The platform we have constructed integrates comprehensive resources, and with a user-friendly interface, allows direct result visualization to facilitate microarray data analysis.

  1. Biolog phenotype microarrays.

    PubMed

    Shea, April; Wolcott, Mark; Daefler, Simon; Rozak, David A

    2012-01-01

    Phenotype microarrays nicely complement traditional genomic, transcriptomic, and proteomic analysis by offering opportunities for researchers to ground microbial systems analysis and modeling in a broad yet quantitative assessment of the organism's physiological response to different metabolites and environments. Biolog phenotype assays achieve this by coupling tetrazolium dyes with minimally defined nutrients to measure the impact of hundreds of carbon, nitrogen, phosphorous, and sulfur sources on redox reactions that result from compound-induced effects on the electron transport chain. Over the years, we have used Biolog's reproducible and highly sensitive assays to distinguish closely related bacterial isolates, to understand their metabolic differences, and to model their metabolic behavior using flux balance analysis. This chapter describes Biolog phenotype microarray system components, reagents, and methods, particularly as they apply to bacterial identification, characterization, and metabolic analysis.

  2. 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

  3. Demonstrating a multi-drug resistant Mycobacterium tuberculosis amplification microarray.

    PubMed

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

    2014-04-25

    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.

  4. An oligonucleotide hybridization approach to DNA sequencing.

    PubMed

    Khrapko, K R; Lysov YuP; Khorlyn, A A; Shick, V V; Florentiev, V L; Mirzabekov, A D

    1989-10-09

    We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

  5. Analyzing Microarray Data.

    PubMed

    Hung, Jui-Hung; Weng, Zhiping

    2017-03-01

    Because there is no widely used software for analyzing RNA-seq data that has a graphical user interface, this protocol provides an example of analyzing microarray data using Babelomics. This analysis entails performing quantile normalization and then detecting differentially expressed genes associated with the transgenesis of a human oncogene c-Myc in mice. Finally, hierarchical clustering is performed on the differentially expressed genes using the Cluster program, and the results are visualized using TreeView.

  6. 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.

  7. 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.

  8. EvoOligo: oligonucleotide probe design with multiobjective evolutionary algorithms.

    PubMed

    Shin, Soo-Yong; Lee, In-Hee; Cho, Young-Min; Yang, Kyung-Ae; Zhang, Byoung-Tak

    2009-12-01

    Probe design is one of the most important tasks in successful deoxyribonucleic acid microarray experiments. We propose a multiobjective evolutionary optimization method for oligonucleotide probe design based on the multiobjective nature of the probe design problem. The proposed multiobjective evolutionary approach has several distinguished features, compared with previous methods. First, the evolutionary approach can find better probe sets than existing simple filtering methods with fixed threshold values. Second, the multiobjective approach can easily incorporate the user's custom criteria or change the existing criteria. Third, our approach tries to optimize the combination of probes for the given set of genes, in contrast to other tools that independently search each gene for qualifying probes. Lastly, the multiobjective optimization method provides various sets of probe combinations, among which the user can choose, depending on the target application. The proposed method is implemented as a platform called EvoOligo and is available for service on the web. We test the performance of EvoOligo by designing probe sets for 19 types of Human Papillomavirus and 52 genes in the Arabidopsis Calmodulin multigene family. The design results from EvoOligo are proven to be superior to those from well-known existing probe design tools, such as OligoArray and OligoWiz.

  9. Spatially Defined Oligonucleotide Arrays. Technical Report for Phase II

    SciTech Connect

    2000-06-15

    The goal of the Human Genome Project is to sequence all 3 billion base pairs of the human genome. Progress in this has been rapid; GenBank{reg_sign} finished 1994 with 286 million bases of sequence and grew by 2470 in the first quarter of 1995. The challenge to the scientific community is to understand the biological relevance of this genetic information. In most cases the sequence being generated for any single region of the genome represents the genotype of a single individual. A complete understanding of the function of specific genes and other regions of the genome and their role in human disease and development will only become apparent when the sequence of many more individuals is known. Access to genetic information is ultimately limited by the ability to screen DNA sequence. Although the pioneering sequencing methods of Sanger et al. (15) and Maxam and Gilbert (11) have become standard in virtually all molecular biology laboratories, the basic protocols remain largely unchanged. The throughput of this sequencing technology is now becoming the rate-limiting step in both large-scale sequencing projects such as the Human Genome Project and the subsequent efforts to understand genetic diversity. This has inspired the development of advanced DNA sequencing technologies (9), Incremental improvements to Sanger sequencing have been made in DNA labeling and detection. High-speed electrophoresis methods using ultrathin gels or capillary arrays are now being more widely employed. However, these methods are throughput-limited by their sequential nature and the speed and resolution of separations. This limitation will become more pronounced as the need to rapidly screen newly discovered genes for biologically relevant polymorphisms increases. An alternative to gel-based sequencing is to use high-density oligonucleotide probe arrays. Oligonucleotide probe arrays display specific oligonucleotide probes at precise locations in a high density, information-rich format (5

  10. Three microarray platforms: an analysis of their concordance in profiling gene expression

    PubMed Central

    Petersen, David; Chandramouli, GVR; Geoghegan, Joel; Hilburn, Joanne; Paarlberg, Jonathon; Kim, Chang Hee; Munroe, David; Gangi, Lisa; Han, Jing; Puri, Raj; Staudt, Lou; Weinstein, John; Barrett, J Carl; Green, Jeffrey; Kawasaki, Ernest S

    2005-01-01

    Background Microarrays for the analysis of gene expression are of three different types: short oligonucleotide (25–30 base), long oligonucleotide (50–80 base), and cDNA (highly variable in length). The short oligonucleotide and cDNA arrays have been the mainstay of expression analysis to date, but long oligonucleotide platforms are gaining in popularity and will probably replace cDNA arrays. As part of a validation study for the long oligonucleotide arrays, we compared and contrasted expression profiles from the three formats, testing RNA from six different cell lines against a universal reference standard. Results The three platforms had 6430 genes in common. In general, correlation of gene expression levels across the platforms was good when defined by concordance in the direction of expression difference (upregulation or downregulation), scatter plot analysis, principal component analysis, cell line correlation or quantitative RT-PCR. The overall correlations (r values) between platforms were in the range 0.7 to 0.8, as determined by analysis of scatter plots. When concordance was measured for expression ratios significant at p-values of <0.05 and at expression threshold levels of 1.5 and 2-fold, the agreement among the platforms was very high, ranging from 93% to 100%. Conclusion Our results indicate that the long oligonucleotide platform is highly suitable for expression analysis and compares favorably with the cDNA and short oligonucleotide varieties. All three platforms can give similar and reproducible results if the criterion is the direction of change in gene expression and minimal emphasis is placed on the magnitude of change. PMID:15876355

  11. Surface chemistries for antibody microarrays

    SciTech Connect

    Seurynck-Servoss, Shannon L.; Baird, Cheryl L.; Rodland, Karin D.; Zangar, Richard C.

    2007-05-01

    Enzyme-linked immunosorbent assay (ELISA) microarrays promise to be a powerful tool for the detection of disease biomarkers. The original technology for printing ELISA microarray chips and capturing antibodies on slides was derived from the DNA microarray field. However, due to the need to maintain antibody structure and function when immobilized, surface chemistries used for DNA microarrays are not always appropriate for ELISA microarrays. In order to identify better surface chemistries for antibody capture, a number of commercial companies and academic research groups have developed new slide types that could improve antibody function in microarray applications. In this review we compare and contrast the commercially available slide chemistries, as well as highlight some promising recent advances in the field.

  12. DNA sequence analysis by hybridization with oligonucleotide microchips : MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides.

    SciTech Connect

    Stomakhin, A. A.; Vasiliskov, V. A.; Timofeev, E.; Schulga, D.; Cotter, R. J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Moscow Inst. of Physics and Technology; Middle Atlantic Mass Spectrometry Lab.; Johns Hopkins Univ. School of Medicine

    2000-01-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA-8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10-15{sup o}C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA-8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.

  13. DNA sequence analysis by hybridization with oligonucleotide microchips: MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides

    PubMed Central

    Stomakhin, Andrey A.; Vasiliskov, Vadim A.; Timofeev, Edward; Schulga, Dennis; Cotter, Richard J.; Mirzabekov, Andrei D.

    2000-01-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA–8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10–15°C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA–8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed. PMID:10666462

  14. Oligonucleotide recombination in gram negative bacteria

    USDA-ARS?s Scientific Manuscript database

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  15. Liver as a target for oligonucleotide therapeutics.

    PubMed

    Sehgal, Alfica; Vaishnaw, Akshay; Fitzgerald, Kevin

    2013-12-01

    Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be more akin to small molecule therapeutics because they are small,completely synthetic in origin, do not integrate into the host genome,and have a defined duration of therapeutic activity after which effects recover to baseline. They offer a high degree of specificity at the genetic level, thereby reducing off-target effects.At the same time, they provide a strategy for targeting any gene in the genome, including transcripts that produce mutated proteins.Oligonucleotide-based therapeutics include short interfering RNA (siRNA), that degrade target mRNA through RISC mediated RNAi; anti-miRs, that target miRNAs; miRNA mimics, that regulate target mRNA; antisense oligonucleotides, that may be working through RNAseH mediated mRNA decay; mRNA upregulation,by targeting long non-coding RNAs; and oligonucleotides induced alternative splicing [1]. All these approaches require some minimal degree of homology at the nucleic acid sequence level for them to be functional. The different mechanisms of action and their relevant activity are outlined in Fig. 1. Besides homology,RNA secondary structure has also been exploited in the case of ribozymes and aptamers, which act by binding to nucleic acids or proteins, respectively. While there have been many reports of gene knockdown and gene modulation in cell lines and mice with all these methods, very few have advanced to clinical stages.The main obstacle to date has been the safe and effective intracellular delivery of these compounds in higher species, including humans. Indeed, their action requires direct interaction with DNA/RNA within the target cell so even when one solves the issues of tissue and cellular access, intracellular/intranuclear location represents yet another barrier to

  16. Formation of N-branched oligonucleotides as by-products in solid-phase oligonucleotide synthesis.

    PubMed

    Cazenave, Christian; Bathany, Katell; Rayner, Bernard

    2006-01-01

    During the synthesis of oligonucleotides by the standard phosphoramidite method using 2'-deoxycytidine- derivatized solid support, a side reaction was observed that gave rise to the formation of high molecular weight N-branched oligomers having two identical chains linked to the 3'-terminal 2'-deoxycytidine. Postsynthesis treatment with neat triethylamine trihydrofluoride selectively cleaved the phosphoramidate linkage and converted the N-branched oligomers back to the expected oligonucleotides.

  17. Ecotoxicogenomics: Microarray interlaboratory comparability.

    PubMed

    Vidal-Dorsch, Doris E; Bay, Steven M; Moore, Shelly; Layton, Blythe; Mehinto, Alvine C; Vulpe, Chris D; Brown-Augustine, Marianna; Loguinov, Alex; Poynton, Helen; Garcia-Reyero, Natàlia; Perkins, Edward J; Escalon, Lynn; Denslow, Nancy D; Cristina, Colli-Dula R; Doan, Tri; Shukradas, Shweta; Bruno, Joy; Brown, Lorraine; Van Agglen, Graham; Jackman, Paula; Bauer, Megan

    2016-02-01

    Transcriptomic analysis can complement traditional ecotoxicology data by providing mechanistic insight, and by identifying sub-lethal organismal responses and contaminant classes underlying observed toxicity. Before transcriptomic information can be used in monitoring and risk assessment, it is necessary to determine its reproducibility and detect key steps impacting the reliable identification of differentially expressed genes. A custom 15K-probe microarray was used to conduct transcriptomics analyses across six laboratories with estuarine amphipods exposed to cyfluthrin-spiked or control sediments (10 days). Two sample types were generated, one consisted of total RNA extracts (Ex) from exposed and control samples (extracted by one laboratory) and the other consisted of exposed and control whole body amphipods (WB) from which each laboratory extracted RNA. Our findings indicate that gene expression microarray results are repeatable. Differentially expressed data had a higher degree of repeatability across all laboratories in samples with similar RNA quality (Ex) when compared to WB samples with more variable RNA quality. Despite such variability a subset of genes were consistently identified as differentially expressed across all laboratories and sample types. We found that the differences among the individual laboratory results can be attributed to several factors including RNA quality and technical expertise, but the overall results can be improved by following consistent protocols and with appropriate training. Published by Elsevier Ltd.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  19. Fecal source tracking in water using a mitochondrial DNA microarray.

    PubMed

    Vuong, Nguyet-Minh; Villemur, Richard; Payment, Pierre; Brousseau, Roland; Topp, Edward; Masson, Luke

    2013-01-01

    A mitochondrial-based microarray (mitoArray) was developed for rapid identification of the presence of 28 animals and one family (cervidae) potentially implicated in fecal pollution in mixed activity watersheds. Oligonucleotide probes for genus or subfamily-level identification were targeted within the 12S rRNA - Val tRNA - 16S rRNA region in the mitochondrial genome. This region, called MI-50, was selected based on three criteria: 1) the ability to be amplified by universal primers 2) these universal primer sequences are present in most commercial and domestic animals of interest in source tracking, and 3) that sufficient sequence variation exists within this region to meet the minimal requirements for microarray probe discrimination. To quantify the overall level of mitochondrial DNA (mtDNA) in samples, a quantitative-PCR (Q-PCR) universal primer pair was also developed. Probe validation was performed using DNA extracted from animal tissues and, for many cases, animal-specific fecal samples. To reduce the amplification of potentially interfering fish mtDNA sequences during the MI-50 enrichment step, a clamping PCR method was designed using a fish-specific peptide nucleic acid. DNA extracted from 19 water samples were subjected to both array and independent PCR analyses. Our results confirm that the mitochondrial microarray approach method could accurately detect the dominant animals present in water samples emphasizing the potential for this methodology in the parallel scanning of a large variety of animals normally monitored in fecal source tracking.

  20. Statistical designs for two-color spotted microarray experiments.

    PubMed

    Chai, Feng-Shun; Liao, Chen-Tuo; Tsai, Shin-Fu

    2007-04-01

    Two-color cDNA or oligonucleotide-based spotted microarrays have been commonly used in measuring the expression levels of thousands of genes simultaneously. To realize the immense potential of this powerful new technology, budgeted within limited resources or other constraints, practical designs with high efficiencies are in demand. In this study, we address the design issue concerning the arrangement of the mRNA samples labeled with fluorescent dyes and hybridized on the slides. A normalization model is proposed to characterize major sources of systematic variation in a two-color microarray experiment. This normalization model establishes a connection between designs for two-color microarray experiments with a particular class of classical row-column designs. A heuristic algorithm for constructing A-optimal or highly efficient designs is provided. Statistical optimality results are found for some of the designs generated from the algorithm. It is believed that the constructed designs are the best or very close to the best possible for estimating the relative gene expression levels among the mRNA samples of interest.

  1. Detection and Identification of Mycobacterium Species Isolates by DNA Microarray

    PubMed Central

    Fukushima, Masao; Kakinuma, Kenichi; Hayashi, Hiroshi; Nagai, Hiroko; Ito, Kunihiko; Kawaguchi, Ryuji

    2003-01-01

    Rapid identification of Mycobacterium species isolates is necessary for the effective management of tuberculosis. Recently, analysis of DNA gyrase B subunit (gyrB) genes has been identified as a suitable means for the identification of bacterial species. We describe a microarray assay based on gyrB gene sequences that can be used for the identification of Mycobacteria species. Primers specific for a gyrB gene region common to all mycobacteria were synthesized and used for PCR amplification of DNA purified from clinical samples. A set of oligonucleotide probes for specific gyrB gene regions was developed for the identification of 14 Mycobacterium species. Each probe was spotted onto a silylated glass slide with an arrayer and used for hybridization with fluorescently labeled RNA derived from amplified sample DNA to yield a pattern of positive spots. This microarray produced unique hybridization patterns for each species of mycobacteria and could differentiate closely related bacterial species. Moreover, the results corresponded well with those obtained by the conventional culture method for the detection of mycobacteria. We conclude that a gyrB-based microarray can rapidly detect and identify closely related mycobacterial species and may be useful in the diagnosis and effective management of tuberculosis. PMID:12791887

  2. Microbial diagnostic microarray for food‐ and water‐borne pathogens

    PubMed Central

    Kostić, Tanja; Stessl, Beatrix; Wagner, Martin; Sessitsch, Angela; Bodrossy, Levente

    2010-01-01

    Summary A microbial diagnostic microarray for the detection of the most relevant bacterial food‐ and water‐borne pathogens and indicator organisms was developed and thoroughly validated. The microarray platform based on sequence‐specific end labelling of oligonucleotides and the pyhylogenetically robust gyrB marker gene allowed a highly specific (resolution on genus/species level) and sensitive (0.1% relative and 104 cfu absolute detection sensitivity) detection of the target pathogens. Validation was performed using a set of reference strains and a set of spiked environmental samples. Reliability of the obtained data was additionally verified by independent analysis of the samples via fluorescence in situ hybridization (FISH) and conventional microbiological reference methods. The applicability of this diagnostic system for food analysis was demonstrated through extensive validation using artificially and naturally contaminated spiked food samples. The microarray‐based pathogen detection was compared with the corresponding microbiological reference methods (performed according to the ISO norm). Microarray results revealed high consistency with the reference microbiological data. PMID:21255342

  3. Effects of stray light on the fidelity of photodirected oligonucleotide array synthesis.

    PubMed

    Garland, Peter B; Serafinowski, Pawel J

    2002-10-01

    Fabrication of high density oligonucleotide arrays using metal on glass photolithographic masks is inflexible and expensive. Maskless methods using computer-controlled projection have been proposed and implemented, but associated stray light effects on photodirected oligonucleotide synthesis and their analysis have not been reported. We have developed a theoretical approach: it predicts that the stray light content of the output of digital micromirror devices and other spatial light modulators of similar performance (contrast ratio approximately 400) will cause extensive random base insertions. For example, use of a digital micromirror device for synthesis of a 20mer array will result in the majority of oligonucleotide chains being 21mers or 22mers. This chain lengthening effect of stray light would not be preventable when synthesis involves a directly photosensitive 5'-blocking group. If the 5'-blocking group is acid labile and released with photogenerated acid, the presence of low concentrations of weak base will prevent the effect of stray light. We have demonstrated experimentally the anticipated chain lengthening effect of stray light on photoacid-dependent synthesis of oligonucleotides and prevention of the effect by low concentrations of n-octylamine. The application of these findings should facilitate the development of maskless fabrication and availability of high density and high fidelity user-designed arrays for research applications.

  4. The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

    PubMed

    Dubrovin, E V; Presnova, G V; Rubtsova, M Yu; Egorov, A M; Grigorenko, V G; Yaminsky, I V

    2015-01-01

    Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

  5. 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

  6. Chemical ligation of oligonucleotides using an electrophilic phosphorothioester

    PubMed Central

    Oikawa, Ryota; Hayakawa, Mayu; Takamori, Shono; Kimura, Yasuaki; Abe, Naoko; Tsuji, Genichiro; Matsuda, Akira; Shuto, Satoshi; Ito, Yoshihiro

    2017-01-01

    Abstract We developed a new approach for chemical ligation of oligonucleotides using the electrophilic phosphorothioester (EPT) group. A nucleophilic phosphorothioate group on oligonucleotides was converted into the EPT group by treatment with Sanger's reagent (1-fluoro-2,4-dinitrobenzene). EPT oligonucleotides can be isolated, stored frozen, and used for the ligation reaction. The reaction of the EPT oligonucleotide and an amino-modified oligonucleotide took place without any extra reagents at pH 7.0–8.0 at room temperature, and resulted in a ligation product with a phosphoramidate bond with a 39–85% yield. This method has potential uses in biotechnology and chemical biology. PMID:28520986

  7. Transcriptomic analysis of floral organs from Phalaenopsis orchid by using oligonucleotide microarray.

    PubMed

    Hsiao, Yu-Yun; Huang, Tian-Hsiang; Fu, Chih-Hsiung; Huang, Shi-Ching; Chen, Yi-Jun; Huang, Yueh-Min; Chen, Wen-Huei; Tsai, Wen-Chieh; Chen, Hong-Hwa

    2013-04-10

    Orchids are one of the most species rich of all angiosperm families. Their extraordinary floral diversity, especially conspicuous labellum morphology, makes them the successful species during evolution process. Because of the fine and delicate development of the perianth, orchid provides a rich subject for studying developmental biology. However, study on molecular mechanism underling orchid floral development is still in its infancy. In this study, we developed an oligomicroarray containing 14,732 unigenes based on the information of expressed sequence tags derived from Phalaenopsis orchids. We applied the oligomicroarray to compare transcriptome among different types of floral organs including sepal, petal and labellum. We discovered that 173, 11, and 285 unigenes were highly differentially expressed in sepal, petal, and labellum, respectively. These unigenes were annotated with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and transcription factor family. Unigenes involved in energy metabolism, lipid metabolism, and terpenoid metabolism are significantly differentially distributed between labellum and two types of tepal (sepal and petal). Labellum-dominant unigenes encoding MADS-box and sepal-dominant unigenes encoding WRKY transcription factors were also identified. Further studies are required but data suggest that it will be possible to identify genes better adapted to sepal, petal and labellum function. The developed functional genomic tool will narrow the gap between approaches based on model organisms with plenty genomic resources and species that are important for developmental and evolutionary studies. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

    PubMed

    Hatters, D M; Wilson, L; Atcliffe, B W; Mulhern, T D; Guzzo-Pernell, N; Howlett, G J

    2001-07-01

    Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways.

  9. Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

    PubMed Central

    Hatters, D M; Wilson, L; Atcliffe, B W; Mulhern, T D; Guzzo-Pernell, N; Howlett, G J

    2001-01-01

    Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways. PMID:11423421

  10. Microarrays in cancer research.

    PubMed

    Grant, Geraldine M; Fortney, Amanda; Gorreta, Francesco; Estep, Michael; Del Giacco, Luca; Van Meter, Amy; Christensen, Alan; Appalla, Lakshmi; Naouar, Chahla; Jamison, Curtis; Al-Timimi, Ali; Donovan, Jean; Cooper, James; Garrett, Carleton; Chandhoke, Vikas

    2004-01-01

    Microarray technology has presented the scientific community with a compelling approach that allows for simultaneous evaluation of all cellular processes at once. Cancer, being one of the most challenging diseases due to its polygenic nature, presents itself as a perfect candidate for evaluation by this approach. Several recent articles have provided significant insight into the strengths and limitations of microarrays. Nevertheless, there are strong indications that this approach will provide new molecular markers that could be used in diagnosis and prognosis of cancers. To achieve these goals it is essential that there is a seamless integration of clinical and molecular biological data that allows us to elucidate genes and pathways involved in various cancers. To this effect we are currently evaluating gene expression profiles in human brain, ovarian, breast and hematopoetic, lung, colorectal, head and neck and biliary tract cancers. To address the issues we have a joint team of scientists, doctors and computer scientists from two Virginia Universities and a major healthcare provider. The study has been divided into several focus groups that include; Tissue Bank Clinical & Pathology Laboratory Data, Chip Fabrication, QA/QC, Tissue Devitalization, Database Design and Data Analysis, using multiple microarray platforms. Currently over 300 consenting patients have been enrolled in the study with the largest number being that of breast cancer patients. Clinical data on each patient is being compiled into a secure and interactive relational database and integration of these data elements will be accomplished by a common programming interface. This clinical database contains several key parameters on each patient including demographic (risk factors, nutrition, co-morbidity, familial history), histopathology (non genetic predictors), tumor, treatment and follow-up information. Gene expression data derived from the tissue samples will be linked to this database, which

  11. 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

  12. Dysbiosis of fecal microbiota in Crohn's disease patients as revealed by a custom phylogenetic microarray.

    PubMed

    Kang, Seungha; Denman, Stuart E; Morrison, Mark; Yu, Zhongtang; Dore, Joel; Leclerc, Marion; McSweeney, Chris S

    2010-12-01

    A custom phylogenetic microarray composed of small subunit ribosomal RNA probes, representing ≈500 bacterial species from the human and animal gut, was developed and evaluated for analysis of gut microbial diversity using fecal samples from healthy subjects and Crohn's disease (CD) patients. Oligonucleotide probes (≈40 mer) used on the microarray were selected from published articles or designed with the "GoArray" microarray probe design program using selected bacterial 16S rRNA sequences. Fecal 16S rDNA from individual samples of six healthy subjects and six CD patients were used as template to generate fluorescently labeled cRNA that was hybridized to the microarray. Differences revealed by the microarray in relative abundance of microbial populations between healthy and diseased patients were verified using quantitative real-time polymerase chain reaction (PCR) with species-specific primer sets. The microarray analyses showed that Eubacterium rectale, Bacteroides fragilis group, B. vulgatus, Ruminococcus albus, R. callidus, R. bromii, and Faecalibacterium prausnitzii were 5-10-fold more abundant in the healthy subjects than in the CD patients, while Enterococcus sp., Clostridium difficile, Escherichia coli, Shigella flexneri, and Listeria sp. were more abundant in the CD group. The microarray detected differences in abundance of bacterial populations within the phylum Firmicutes that had been reported previously for the same samples based on phylogenetic analysis of metagenomic clone libraries. In addition, the microarray showed that Enterococcus sp. was in higher abundance in the CD patients. This microarray should be another useful tool to examine the diversity and abundance of human intestinal microbiota. Copyright © 2010 Crohn's & Colitis Foundation of America, Inc.

  13. Striptease on glass: validation of an improved stripping procedure for in situ microarrays.

    PubMed

    Hahnke, Karin; Jacobsen, Marc; Gruetzkau, Andreas; Gruen, Joachim R; Koch, Markus; Emoto, Masashi; Meyer, Thomas F; Walduck, Anna; Kaufmann, Stefan H E; Mollenkopf, Hans-Joachim

    2007-01-30

    Microarrays have rapidly become an indispensable tool for gene analysis. Microarray experiments can be cost prohibitive, however, largely due to the price of the arrays themselves. Whilst different methods for stripping filter arrays on membranes have been established, only very few protocols are published for thermal and chemical stripping of microarrays on glass. Most of these protocols for stripping microarrays on glass were developed in combination with specific surface chemistry and different coatings for covalently immobilizing presynthesized DNA in a deposition process. We have developed a method for stripping commercial in situ microarrays using a multi-step procedure. We present a method that uses mild chemical degradation complemented by enzymatic treatment. We took advantage of the differences in biochemical properties of covalently linked DNA oligonucleotides on in situ synthesized microarrays and the antisense cRNA hybridization probes. The success of stripping protocols for microarrays on glass was critically dependent on the type of arrays, the nature of sample used for hybridization, as well as hybridization and washing conditions. The protocol employs alkali hydrolysis of the cRNA, several enzymatic degradation steps using RNAses and Proteinase K, combined with appropriate washing steps. Stripped arrays were rehybridized using the same protocols as for new microarrays. The stripping method was validated with microarrays from different suppliers and rehybridization of stripped in situ arrays yielded comparable results to hybridizations done on unused, new arrays with no significant loss in precision or accuracy. We show that stripping of commercial in situ arrays is feasible and that reuse of stripped arrays gave similar results compared to unused ones. This was true even for biological samples that show only slight differences in their expression profiles. Our analyses indicate that the stripping procedure does not significantly influence data

  14. 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.

  15. 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.

  16. In situ synthesis of DNA microarray on functionalized cyclic olefin copolymer substrate.

    PubMed

    Saaem, Ishtiaq; Ma, Kuo-Sheng; Marchi, Alexandria N; LaBean, Thomas H; Tian, Jingdong

    2010-02-01

    Thermoplastic materials such as cyclic-olefin copolymers (COC) provide a versatile and cost-effective alternative to the traditional glass or silicon substrate for rapid prototyping and industrial scale fabrication of microdevices. To extend the utility of COC as an effective microarray substrate, we developed a new method that enabled for the first time in situ synthesis of DNA oligonucleotide microarrays on the COC substrate. To achieve high-quality DNA synthesis, a SiO(2) thin film array was prepatterned on the inert and hydrophobic COC surface using RF sputtering technique. The subsequent in situ DNA synthesis was confined to the surface of the prepatterned hydrophilic SiO(2) thin film features by precision delivery of the phosphoramidite chemistry using an inkjet DNA synthesizer. The in situ SiO(2)-COC DNA microarray demonstrated superior quality and stability in hybridization assays and thermal cycling reactions. Furthermore, we demonstrate that pools of high-quality mixed-oligos could be cleaved off the SiO(2)-COC microarrays and used directly for construction of DNA origami nanostructures. It is believed that this method will not only enable synthesis of high-quality and low-cost COC DNA microarrays but also provide a basis for further development of integrated microfluidics microarrays for a broad range of bioanalytical and biofabrication applications.

  17. Systematic review of accuracy of prenatal diagnosis for abnormal chromosome diseases by microarray technology.

    PubMed

    Xu, H B; Yang, H; Liu, G; Chen, H

    2014-10-31

    The accuracy of prenatal diagnosis for abnormal chromosome diseases by chromosome microarray technology and karyotyping were compared. A literature search was carried out in the MEDLINE database with the keywords "chromosome" and "karyotype" and "genetic testing" and "prenatal diagnosis" and "oligonucleotide array sequence". The studies obtained were filtered by using the QUADAS tool, and studies conforming to the quality standard were fully analyzed. There was one paper conforming to the QUADAS standards including 4406 gravidas with adaptability syndromes of prenatal diagnosis including elderly parturient women, abnormal structure by type-B ultrasound, and other abnormalities. Microarray technology yielded successful diagnoses in 4340 cases (98.8%), and there was no need for tissue culture in 87.9% of the samples. All aneuploids and non-parallel translocations in 4282 cases of non-chimera identified by karyotyping could be detected using microarray analysis technology, whereas parallel translocations and fetal triploids could not be detected by microarray analysis technology. In the samples with normal karyotyping results, type-B ultrasound showed that 6% of chromosomal deficiencies or chromosome duplications could be detected by microarray technology, and the same abnormal chromosomes were detected in 1.7% of elderly parturient women and samples with positive serology screening results. In the prenatal diagnosis test, compared with karyotyping, microarray technology could identify the extra cell genetic information with clinical significance, aneuploids, and non-parallel translocations; however, its disadvantage is that it could not identify parallel translocations and triploids.

  18. Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates.

    PubMed Central

    Bonfils, E; Depierreux, C; Midoux, P; Thuong, N T; Monsigny, M; Roche, A C

    1992-01-01

    Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides--substituted on their 5'-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3'-end, on the other hand,--were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides. Images PMID:1408764

  19. A novel 4p16.3 microduplication distal to WHSC1 and WHSC2 characterized by oligonucleotide array with new phenotypic features.

    PubMed

    Cyr, Andrew B; Nimmakayalu, Manjunath; Longmuir, Susannah Q; Patil, Shivanand R; Keppler-Noreuil, Kim M; Shchelochkov, Oleg A

    2011-09-01

    Larger imbalances on chromosome 4p in the form of deletions associated with Wolf-Hirschhorn syndrome (WHS) and duplications of chromosome 4p have a defined clinical phenotype. The critical region for both these clinical disorders has been narrowed based on the genotype-phenotype correlations. However, cryptic rearrangements in this region have been reported infrequently. We report on a male patient with a microduplication of chromosome 4p, who presents with findings of macrocephaly, irregular iris pigmentation-heterochromia, and preserved linear growth in addition to overlapping features of trisomy 4p such as seizures, delayed psychomotor development, and dysmorphic features including prominent glabella, low-set ears, and short neck. Using a high-density oligonucleotide microarray, we have identified a novel submicroscopic duplication involving dosage sensitive genes TACC3, FGFR3, and LETM1. The microduplication did not involve WHSC1 and WHSC2 which are considered in the critical region for WHS and trisomy 4p. This patient's presentation and genomic findings help further delineate clinical significance of re-arrangements in the 4p16 region without the involvement of WHS critical region.

  20. Development of multiplex DNA electronic microarrays using a universal adaptor system for detection of single nucleotide polymorphisms.

    PubMed

    Tsang, Shirley; Sun, Zhonghe; Stewart, Claudia; Lum, Nicole; Frankenberger, Casey; Subleski, Marianne; Rasmussen, Lynn; Munroe, David J

    2004-04-01

    The NanoChip electronic microarray is designed for the rapid detection of genetic variation in research and clinical diagnosis. We have developed a multiplex electronic microarray assay, specific for single nucleotide polymorphism (SNP) genotyping and mutation detection, using universal adaptor sequences tailed to the 5' end of PCR primers specific to each target. PCR products, amplified by primers directed to the universal adaptor sequence, are immobilized on the microarray either directly or via capture oligonucleotides complementary to the universal adaptor sequence. This simple modification results in a significant increase in fidelity with improved specificity and accuracy. In addition, the multiplexing of genetic variant detection allows increased throughput and significantly reduced cost per assay. This general schema can also be applied to other microarray and macroarray formats.

  1. Development and evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in Acid Mine Drainages and bioleaching systems.

    PubMed

    Yin, Huaqun; Cao, Linhui; Qiu, Guanzhou; Wang, Dianzuo; Kellogg, Laurie; Zhou, Jizhong; Dai, Zhimin; Liu, Xueduan

    2007-07-01

    To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. The functional genes in the microarray were involved in carbon metabolism (158), nitrogen metabolism (72), sulfur metabolism (39), iron metabolism (68), DNA replication and repair (97), metal-resistance (27), membrane-relate gene (16), transposon (13) and IST sequence (11). Based on the results of microarray hybridizations, 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 ng of genomic DNA in the absence of background DNA. Strong linear relationships between the signal intensity and the target DNA were observed (r(2) approximately 0.98). Application of this type of the microarray to analyze the acidic environments and bioleaching systems demonstrated that the developed microarray appeared to be useful for profiling differences in microbial community structures of acidic environments and bioleaching systems. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of genes related with acidophilic microorganism and the microbial community in acidic environments and bioleaching systems, although more work is needed to improve.

  2. Antisense oligonucleotides in therapy for neurodegenerative disorders.

    PubMed

    Evers, Melvin M; Toonen, Lodewijk J A; van Roon-Mom, Willeke M C

    2015-06-29

    Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation.

  3. 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. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. AMOD: a morpholino oligonucleotide selection tool

    PubMed Central

    Klee, Eric W.; Shim, Kyong Jin; Pickart, Michael A.; Ekker, Stephen C.; Ellis, Lynda B. M.

    2005-01-01

    AMOD is a web-based program that aids in the functional evaluation of nucleotide sequences through sequence characterization and antisense morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative morpholino oligonucleotides. Using these properties, morpholino candidates are selected and compared with genomic and transcriptome databases with the goal to identify target-specific enriched morpholinos. AMOD has been used at the University of Minnesota to design ∼200 morpholinos for a functional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at . PMID:15980523

  5. The prebiotic synthesis of deoxythymidine oligonucleotides

    NASA Technical Reports Server (NTRS)

    Stephen-Sherwood, E.; Odom, D. G.; Oro, J.

    1974-01-01

    Deoxythymidine 5 prime-triphosphate in the presence of deoxythymidine 5 prime-phosphate, cyanamide and 4-amino-5-imidazole carboxamide polymerizes under drying conditions at moderate temperatures (60 to 90 C) to yield oligonucleotides of up to four units in length. Enzymatic analysis indicated that the majority of these oligomers contained natural 3 prime-5 prime phosphodiester bonds. This reaction offers a possible method for the formation of deoxyoligonucleotides under primitive earth conditions.

  6. A New Distribution Family for Microarray Data.

    PubMed

    Kelmansky, Diana Mabel; Ricci, Lila

    2017-02-10

    The traditional approach with microarray data has been to apply transformations that approximately normalize them, with the drawback of losing the original scale. The alternative stand point taken here is to search for models that fit the data, characterized by the presence of negative values, preserving their scale; one advantage of this strategy is that it facilitates a direct interpretation of the results. A new family of distributions named gpower-normal indexed by p∈R is introduced and it is proven that these variables become normal or truncated normal when a suitable gpower transformation is applied. Expressions are given for moments and quantiles, in terms of the truncated normal density. This new family can be used to model asymmetric data that include non-positive values, as required for microarray analysis. Moreover, it has been proven that the gpower-normal family is a special case of pseudo-dispersion models, inheriting all the good properties of these models, such as asymptotic normality for small variances. A combined maximum likelihood method is proposed to estimate the model parameters, and it is applied to microarray and contamination data. Rcodes are available from the authors upon request.

  7. Cationic Nanohydrogel Particles for Therapeutic Oligonucleotide Delivery.

    PubMed

    Leber, Nadine; Nuhn, Lutz; Zentel, Rudolf

    2017-06-12

    Short pharmaceutical active oligonucleotides such as small interfering RNA (siRNA) or cytidine-phosphate-guanosine (CpG) are considered as powerful therapeutic alternatives, especially to medicate hard-to-treat diseases (e.g., liver fibrosis or cancer). Unfortunately, these molecules are equipped with poor pharmacokinetic properties that prevent them from translation. Well-defined nanosized carriers can provide opportunities to optimize their delivery and guide them to their site of action. Among several concepts, this Feature Article focuses on cationic nanohydrogel particles as a universal delivery system for small anionic molecules including siRNA and CpG. Cationic nanohydrogels are derived from preaggregated precursor block copolymers, which are further cross-linked to obtain well-defined nanoparticles of tunable sizes and with (degradable) cationic cores. Novel opportunities for oligonucleotide delivery in vitro and in vivo with respect to liver fibrosis therapies will be highlighted as well as perspectives toward modulating the immune system. In general, the approach of covalently stabilized cationic carrier systems can contribute to find advanced oligonucleotide therapeutics. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Development of a high-throughput microfluidic integrated microarray for the detection of chimeric bioweapons.

    SciTech Connect

    Sheppod, Timothy; Satterfield, Brent; Hukari, Kyle W.; West, Jason A. A.; Hux, Gary A.

    2006-10-01

    The advancement of DNA cloning has significantly augmented the potential threat of a focused bioweapon assault, such as a terrorist attack. With current DNA cloning techniques, toxin genes from the most dangerous (but environmentally labile) bacterial or viral organism can now be selected and inserted into robust organism to produce an infinite number of deadly chimeric bioweapons. In order to neutralize such a threat, accurate detection of the expressed toxin genes, rather than classification on strain or genealogical decent of these organisms, is critical. The development of a high-throughput microarray approach will enable the detection of unknowns chimeric bioweapons. The development of a high-throughput microarray approach will enable the detection of unknown bioweapons. We have developed a unique microfluidic approach to capture and concentrate these threat genes (mRNA's) upto a 30 fold concentration. These captured oligonucleotides can then be used to synthesize in situ oligonucleotide copies (cDNA probes) of the captured genes. An integrated microfluidic architecture will enable us to control flows of reagents, perform clean-up steps and finally elute nanoliter volumes of synthesized oligonucleotides probes. The integrated approach has enabled a process where chimeric or conventional bioweapons can rapidly be identified based on their toxic function, rather than being restricted to information that may not identify the critical nature of the threat.

  9. Discovery of RNA Binding Small Molecules Using Small Molecule Microarrays.

    PubMed

    Connelly, Colleen M; Abulwerdi, Fardokht A; Schneekloth, John S

    2017-01-01

    New methods to identify RNA-binding small molecules open yet unexplored opportunities for the pharmacological modulation of RNA-driven biology and disease states. One such approach is the use of small molecule microarrays (SMMs). Typically, SMMs are generated by spatially arraying and covalently linking a library of small molecules to a glass surface. Next, incubation of the arrays with a fluorescently labeled RNA reveals binding interactions that are detected upon slide imaging. The relative ease with which SMMs are manufactured enables the screening of multiple oligonucleotides in parallel against tens of thousands of small molecules, providing information about both binding and selectivity of identified RNA-small molecule interactions. This approach is useful for screening a broad variety of structurally and functionally diverse RNAs. Here, we present a general method for the preparation and use of SMMs to rapidly identify small molecules that selectively bind to an RNA of interest.

  10. Microarray simulator as educational tool.

    PubMed

    Ruusuvuori, Pekka; Nykter, Matti; Mäkiraatikka, Eeva; Lehmussola, Antti; Korpelainen, Tomi; Erkkilä, Timo; Yli-Harja, Olli

    2007-01-01

    As many real-world applications, microarray measurements are inapplicable for large-scale teaching purposes due to their laborious preparation process and expense. Fortunately, many phases of the array preparation process can be efficiently demonstrated by using a software simulator tool. Here we propose the use of microarray simulator as an aiding tool in teaching of computational biology. Three case studies on educational use of the simulator are presented, which demonstrate the effect of gene knock-out, synthetic time series, and effect of noise sources. We conclude that the simulator, used for teaching the principles of microarray measurement technology, proved to be a useful tool in education.

  11. A novel universal DNA labeling and amplification system for rapid microarray-based detection of 117 antibiotic resistance genes in Gram-positive bacteria.

    PubMed

    Strauss, Christian; Endimiani, Andrea; Perreten, Vincent

    2015-01-01

    A rapid and simple DNA labeling system has been developed for disposable microarrays and has been validated for the detection of 117 antibiotic resistance genes abundant in Gram-positive bacteria. The DNA was fragmented and amplified using phi-29 polymerase and random primers with linkers. Labeling and further amplification were then performed by classic PCR amplification using biotinylated primers specific for the linkers. The microarray developed by Perreten et al. (Perreten, V., Vorlet-Fawer, L., Slickers, P., Ehricht, R., Kuhnert, P., Frey, J., 2005. Microarray-based detection of 90 antibiotic resistance genes of gram-positive bacteria. J.Clin.Microbiol. 43, 2291-2302.) was improved by additional oligonucleotides. A total of 244 oligonucleotides (26 to 37 nucleotide length and with similar melting temperatures) were spotted on the microarray, including genes conferring resistance to clinically important antibiotic classes like β-lactams, macrolides, aminoglycosides, glycopeptides and tetracyclines. Each antibiotic resistance gene is represented by at least 2 oligonucleotides designed from consensus sequences of gene families. The specificity of the oligonucleotides and the quality of the amplification and labeling were verified by analysis of a collection of 65 strains belonging to 24 species. Association between genotype and phenotype was verified for 6 antibiotics using 77 Staphylococcus strains belonging to different species and revealed 95% test specificity and a 93% predictive value of a positive test. The DNA labeling and amplification is independent of the species and of the target genes and could be used for different types of microarrays. This system has also the advantage to detect several genes within one bacterium at once, like in Staphylococcus aureus strain BM3318, in which up to 15 genes were detected. This new microarray-based detection system offers a large potential for applications in clinical diagnostic, basic research, food safety and

  12. Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

    PubMed Central

    2013-01-01

    Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA

  13. Challenges for MicroRNA Microarray Data Analysis

    PubMed Central

    Wang, Bin; Xi, Yaguang

    2013-01-01

    Microarray is a high throughput discovery tool that has been broadly used for genomic research. Probe-target hybridization is the central concept of this technology to determine the relative abundance of nucleic acid sequences through fluorescence-based detection. In microarray experiments, variations of expression measurements can be attributed to many different sources that influence the stability and reproducibility of microarray platforms. Normalization is an essential step to reduce non-biological errors and to convert raw image data from multiple arrays (channels) to quality data for further analysis. In general, for the traditional microarray analysis, most established normalization methods are based on two assumptions: (1) the total number of target genes is large enough (>10,000); and (2) the expression level of the majority of genes is kept constant. However, microRNA (miRNA) arrays are usually spotted in low density, due to the fact that the total number of miRNAs is less than 2,000 and the majority of miRNAs are weakly or not expressed. As a result, normalization methods based on the above two assumptions are not applicable to miRNA profiling studies. In this review, we discuss a few representative microarray platforms on the market for miRNA profiling and compare the traditional methods with a few novel strategies specific for miRNA microarrays. PMID:24163754

  14. Reverse Phase Protein Microarrays.

    PubMed

    Baldelli, Elisa; Calvert, Valerie; Hodge, Alex; VanMeter, Amy; Petricoin, Emanuel F; Pierobon, Mariaelena

    2017-01-01

    While genes and RNA encode information about cellular status, proteins are considered the engine of the cellular machine, as they are the effective elements that drive all cellular functions including proliferation, migration, differentiation, and apoptosis. Consequently, investigations of the cellular protein network are considered a fundamental tool for understanding cellular functions.Alteration of the cellular homeostasis driven by elaborate intra- and extracellular interactions has become one of the most studied fields in the era of personalized medicine and targeted therapy. Increasing interest has been focused on developing and improving proteomic technologies that are suitable for analysis of clinical samples. In this context, reverse-phase protein microarrays (RPPA) is a sensitive, quantitative, high-throughput immunoassay for protein analyses of tissue samples, cells, and body fluids.RPPA is well suited for broad proteomic profiling and is capable of capturing protein activation as well as biochemical reactions such as phosphorylation, glycosylation, ubiquitination, protein cleavage, and conformational alterations across hundreds of samples using a limited amount of biological material. For these reasons, RPPA represents a valid tool for protein analyses and generates data that help elucidate the functional signaling architecture through protein-protein interaction and protein activation mapping for the identification of critical nodes for individualized or combinatorial targeted therapy.

  15. DNA nanostructure-based universal microarray platform for high-efficiency multiplex bioanalysis in biofluids.

    PubMed

    Li, Zhenhua; Zhao, Bin; Wang, Dongfang; Wen, Yanli; Liu, Gang; Dong, Haoqing; Song, Shiping; Fan, Chunhai

    2014-10-22

    Microarrays of biomolecules have greatly promoted the development of the fields of genomics, proteomics, and clinical assays because of their remarkably parallel and high-throughput assay capability. Immobilization strategies for biomolecules on a solid support surface play a crucial role in the fabrication of high-performance biological microarrays. In this study, rationally designed DNA tetrahedra carrying three amino groups and one single-stranded DNA extension were synthesized by the self-assembly of four oligonucleotides, followed by high-performance liquid chromatography purification. We fabricated DNA tetrahedron-based microarrays by covalently coupling the DNA tetrahedron onto glass substrates. After their biorecognition capability was evaluated, DNA tetrahedron microarrays were utilized for the analysis of different types of bioactive molecules. The gap hybridization strategy, the sandwich configuration, and the engineering aptamer strategy were employed for the assay of miRNA biomarkers, protein cancer biomarkers, and small molecules, respectively. The arrays showed good capability to anchor capture biomolecules for improving biorecognition. Addressable and high-throughput analysis with improved sensitivity and specificity had been achieved. The limit of detection for let-7a miRNA, prostate specific antigen, and cocaine were 10 fM, 40 pg/mL, and 100 nM, respectively. More importantly, we demonstrated that the microarray platform worked well with clinical serum samples and showed good relativity with conventional chemical luminescent immunoassay. We have developed a novel approach for the fabrication of DNA tetrahedron-based microarrays and a universal DNA tetrahedron-based microarray platform for the detection of different types of bioactive molecules. The microarray platform shows great potential for clinical diagnosis.

  16. Chemistry of Natural Glycan Microarray

    PubMed Central

    Song, Xuezheng; Heimburg-Molinaro, Jamie; Cummings, Richard D.; Smith, David F.

    2014-01-01

    Glycan microarrays have become indispensable tools for studying protein-glycan interactions. Along with chemo-enzymatic synthesis, glycans isolated from natural sources have played important roles in array development and will continue to be a major source of glycans. N- and O-glycans from glycoproteins, and glycans from glycosphingolipids can be released from corresponding glycoconjugates with relatively mature methods, although isolation of large numbers and quantities of glycans are still very challenging. Glycosylphosphatidylinositol (GPI)-anchors and glycosaminoglycans (GAGs) are less represented on current glycan microarrays. Glycan microarray development has been greatly facilitated by bifunctional fluorescent linkers, which can be applied in a “Shotgun Glycomics” approach to incorporate isolated natural glycans. Glycan presentation on microarrays may affect glycan binding by GBPs, often through multivalent recognition by the GBP. PMID:24487062

  17. Template-Directed Ligation of Peptides to Oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  18. Detection of SPO11-oligonucleotide complexes from mouse testes.

    PubMed

    Pan, Jing; Keeney, Scott

    2009-01-01

    The SPO11 protein generates programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination. Endonucleolytic cleavage 3' to the DSB sites releases SPO11 from DNA, leaving SPO11 covalently associated with an oligonucleotide. This chapter describes detection of the release product, SPO11-oligonucleotide complexes, from mouse testis lysates. The method for determining the size of SPO11-associated oligonucleotides is also provided.

  19. microDuMIP: target-enrichment technique for microarray-based duplex molecular inversion probes

    PubMed Central

    Yoon, Jung-Ki; Ahn, Jinwoo; Kim, Han Sang; Han, Soo Min; Jang, Hoon; Lee, Min Goo; Lee, Ji Hyun; Bang, Duhee

    2015-01-01

    Molecular inversion probe (MIP)-based capture is a scalable and effective target-enrichment technology that can use synthetic single-stranded oligonucleotides as probes. Unlike the straightforward use of synthetic oligonucleotides for low-throughput target capture, high-throughput MIP capture has required laborious protocols to generate thousands of single-stranded probes from DNA microarray because of multiple enzymatic steps, gel purifications and extensive PCR amplifications. Here, we developed a simple and efficient microarray-based MIP preparation protocol using only one enzyme with double-stranded probes and improved target capture yields by designing probes with overlapping targets and unique barcodes. To test our strategy, we produced 11 510 microarray-based duplex MIPs (microDuMIPs) and captured 3554 exons of 228 genes in a HapMap genomic DNA sample (NA12878). Under our protocol, capture performance and precision of calling were compatible to conventional MIP capture methods, yet overlapping targets and unique barcodes allowed us to precisely genotype with as little as 50 ng of input genomic DNA without library preparation. microDuMIP method is simpler and cheaper, allowing broader applications and accurate target sequencing with a scalable number of targets. PMID:25414325

  20. In Situ-Synthesized Novel Microarray Optimized for Mouse Stem Cell and Early Developmental Expression Profiling

    PubMed Central

    Carter, Mark G.; Hamatani, Toshio; Sharov, Alexei A.; Carmack, Condie E.; Qian, Yong; Aiba, Kazuhiro; Ko, Naomi T.; Dudekula, Dawood B.; Brzoska, Pius M.; Hwang, S. Stuart; Ko, Minoru S.H.

    2003-01-01

    Applications of microarray technologies to mouse embryology/genetics have been limited, due to the nonavailability of microarrays containing large numbers of embryonic genes and the gap between microgram quantities of RNA required by typical microarray methods and the miniscule amounts of tissue available to researchers. To overcome these problems, we have developed a microarray platform containing in situ-synthesized 60-mer oligonucleotide probes representing approximately 22,000 unique mouse transcripts, assembled primarily from sequences of stem cell and embryo cDNA libraries. We have optimized RNA labeling protocols and experimental designs to use as little as 2 ng total RNA reliably and reproducibly. At least 98% of the probes contained in the microarray correspond to clones in our publicly available collections, making cDNAs readily available for further experimentation on genes of interest. These characteristics, combined with the ability to profile very small samples, make this system a resource for stem cell and embryogenomics research. [Supplemental material is available online at www.genome.org and at the NIA Mouse cDNA Project Web site, http://lgsun.grc.nia.nih.gov/cDNA/cDNA.html.] PMID:12727912

  1. Fabrication of DNA Microarrays on Polydopamine-Modified Gold Thin Films for SPR Imaging Measurements

    PubMed Central

    Wood, Jennifer B.; Szyndler, Megan W.; Halpern, Aaron R.; Cho, Kyunghee

    2013-01-01

    Polydopamine (PDA) films were fabricated on thin film gold substrates in a single-step polymerization-deposition process from dopamine solutions and then employed in the construction of robust DNA microarrays for the ultra-sensitive detection of biomolecules with nanoparticle-enhanced surface plasmon resonance (SPR) imaging. PDA multilayers with thicknesses varying from 1 to 5 nm were characterized with a combination of scanning angle SPR and AFM experiments, and 1.3 ± 0.2 nm PDA multilayers were chosen as an optimal thickness for the SPR imaging measurements. DNA microarrays were then fabricated by the reaction of amine-functionalized single-stranded DNA (ssDNA) oligonucleotides with PDA-modified gold thin film microarray elements, and were subsequently employed in SPR imaging measurements of DNA hybridization adsorption and protein-DNA binding. Concurrent control experiments with noncomplementary ssDNA sequences demonstrated that the adhesive PDA multilayer was also able to provide good resistance to the nonspecific binding of biomolecules. Finally, a series of SPR imaging measurements of the hybridization adsorption of DNA-modified gold nanoparticles onto mixed sequence DNA microarrays were used to confirm that the use of PDA multilayer films is a simple, rapid and versatile method for fabricating DNA microarrays for ultrasensitive nanoparticle-enhanced SPR imaging biosensing. PMID:23902428

  2. Simultaneous discrimination between 15 fish pathogens by using 16S ribosomal DNA PCR and DNA microarrays.

    PubMed

    Warsen, Adelaide E; Krug, Melissa J; LaFrentz, Stacey; Stanek, Danielle R; Loge, Frank J; Call, Douglas R

    2004-07-01

    We developed a DNA microarray suitable for simultaneous detection and discrimination between multiple bacterial species based on 16S ribosomal DNA (rDNA) polymorphisms using glass slides. Microarray probes (22- to 31-mer oligonucleotides) were spotted onto Teflon-masked, epoxy-silane-derivatized glass slides using a robotic arrayer. PCR products (ca. 199 bp) were generated using biotinylated, universal primer sequences, and these products were hybridized overnight (55 degrees C) to the microarray. Targets that annealed to microarray probes were detected using a combination of Tyramide Signal Amplification and Alexa Fluor 546. This methodology permitted 100% specificity for detection of 18 microbes, 15 of which were fish pathogens. With universal 16S rDNA PCR (limited to 28 cycles), detection sensitivity for purified control DNA was equivalent to <150 genomes (675 fg), and this sensitivity was not adversely impacted either by the presence of competing bacterial DNA (1.1 x 10(6) genomes; 5 ng) or by the addition of up to 500 ng of fish DNA. Consequently, coupling 16S rDNA PCR with a microarray detector appears suitable for diagnostic detection and surveillance for commercially important fish pathogens.

  3. Fabrication of DNA microarrays on polydopamine-modified gold thin films for SPR imaging measurements.

    PubMed

    Wood, Jennifer B; Szyndler, Megan W; Halpern, Aaron R; Cho, Kyunghee; Corn, Robert M

    2013-08-27

    Polydopamine (PDA) films were fabricated on thin film gold substrates in a single-step polymerization-deposition process from dopamine solutions and then employed in the construction of robust DNA microarrays for the ultrasensitive detection of biomolecules with nanoparticle-enhanced surface plasmon resonance (SPR) imaging. PDA multilayers with thicknesses varying from 1 to 5 nm were characterized with a combination of scanning angle SPR and AFM experiments, and 1.3 ± 0.2 nm PDA multilayers were chosen as an optimal thickness for the SPR imaging measurements. DNA microarrays were then fabricated by the reaction of amine-functionalized single-stranded DNA (ssDNA) oligonucleotides with PDA-modified gold thin film microarray elements, and were subsequently employed in SPR imaging measurements of DNA hybridization adsorption and protein-DNA binding. Concurrent control experiments with non-complementary ssDNA sequences demonstrated that the adhesive PDA multilayer was also able to provide good resistance to the nonspecific binding of biomolecules. Finally, a series of SPR imaging measurements of the hybridization adsorption of DNA-modified gold nanoparticles onto mixed sequence DNA microarrays were used to confirm that the use of PDA multilayer films is a simple, rapid, and versatile method for fabricating DNA microarrays for ultrasensitive nanoparticle-enhanced SPR imaging biosensing.

  4. An event-specific DNA microarray to identify genetically modified organisms in processed foods.

    PubMed

    Kim, Jae-Hwan; Kim, Su-Youn; Lee, Hyungjae; Kim, Young-Rok; Kim, Hae-Yeong

    2010-05-26

    We developed an event-specific DNA microarray system to identify 19 genetically modified organisms (GMOs), including two GM soybeans (GTS-40-3-2 and A2704-12), thirteen GM maizes (Bt176, Bt11, MON810, MON863, NK603, GA21, T25, TC1507, Bt10, DAS59122-7, TC6275, MIR604, and LY038), three GM canolas (GT73, MS8xRF3, and T45), and one GM cotton (LLcotton25). The microarray included 27 oligonucleotide probes optimized to identify endogenous reference targets, event-specific targets, screening targets (35S promoter and nos terminator), and an internal target (18S rRNA gene). Thirty-seven maize-containing food products purchased from South Korean and US markets were tested for the presence of GM maize using this microarray system. Thirteen GM maize events were simultaneously detected using multiplex PCR coupled with microarray on a single chip, at a limit of detection of approximately 0.5%. Using the system described here, we detected GM maize in 11 of the 37 food samples tested. These results suggest that an event-specific DNA microarray system can reliably detect GMOs in processed foods.

  5. Development and application of the active surveillance of pathogens microarray to monitor bacterial gene flux

    PubMed Central

    Stabler, Richard A; Dawson, Lisa F; Oyston, Petra CF; Titball, Richard W; Wade, Jim; Hinds, Jason; Witney, Adam A; Wren, Brendan W

    2008-01-01

    Background Human and animal health is constantly under threat by emerging pathogens that have recently acquired genetic determinants that enhance their survival, transmissibility and virulence. We describe the construction and development of an Active Surveillance of Pathogens (ASP) oligonucleotide microarray, designed to 'actively survey' the genome of a given bacterial pathogen for virulence-associated genes. Results The microarray consists of 4958 reporters from 151 bacterial species and include genes for the identification of individual bacterial species as well as mobile genetic elements (transposons, plasmid and phage), virulence genes and antibiotic resistance genes. The ASP microarray was validated with nineteen bacterial pathogens species, including Francisella tularensis, Clostridium difficile, Staphylococcus aureus, Enterococcus faecium and Stenotrophomonas maltophilia. The ASP microarray identified these bacteria, and provided information on potential antibiotic resistance (eg sufamethoxazole resistance and sulfonamide resistance) and virulence determinants including genes likely to be acquired by horizontal gene transfer (e.g. an alpha-haemolysin). Conclusion The ASP microarray has potential in the clinic as a diagnostic tool, as a research tool for both known and emerging pathogens, and as an early warning system for pathogenic bacteria that have been recently modified either naturally or deliberately. PMID:18844996

  6. Fast large scale oligonucleotide selection using the longest common factor approach.

    PubMed

    Rahmann, Sven

    2003-07-01

    We present a fast method that selects oligonucleotide probes (such as DNA 25-mers) for microarray experiments on a truly large scale. For example, reliable oligos for human genes can be found within four days, a speedup of one to two orders of magnitude compared to previous approaches. This speed is attained by using the longest common substring as a specificity measure for candidate oligos. We present a space- and time-efficient algorithm, based on a suffix array with additional information, to compute matching statistics (lengths of longest matches) between all candidate oligos and all remaining sequences. With the matching statistics available, we show how to incorporate constraints such as oligo length, melting temperature, and self-complementarity into the selection process at a postprocessing stage. As a result, we can now design custom oligos for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.

  7. Synthesis of oligonucleotides on a soluble support

    PubMed Central

    2017-01-01

    Oligonucleotides are usually prepared in lab scale on a solid support with the aid of a fully automated synthesizer. Scaling up of the equipment has allowed industrial synthesis up to kilogram scale. In spite of this, solution-phase synthesis has received continuous interest, on one hand as a technique that could enable synthesis of even larger amounts and, on the other hand, as a gram scale laboratory synthesis without any special equipment. The synthesis on a soluble support has been regarded as an approach that could combine the advantageous features of both the solution and solid-phase syntheses. The critical step of this approach is the separation of the support-anchored oligonucleotide chain from the monomeric building block and other small molecular reagents and byproducts after each coupling, oxidation and deprotection step. The techniques applied so far include precipitation, extraction, chromatography and nanofiltration. As regards coupling, all conventional chemistries, viz. phosphoramidite, H-phosphonate and phosphotriester strategies, have been attempted. While P(III)-based phosphoramidite and H-phosphonate chemistries are almost exclusively used on a solid support, the “outdated” P(V)-based phosphotriester chemistry still offers one major advantage for the synthesis on a soluble support; the omission of the oxidation step simplifies the coupling cycle. Several of protocols developed for the soluble-supported synthesis allow the preparation of both DNA and RNA oligomers of limited length in gram scale without any special equipment, being evidently of interest for research groups that need oligonucleotides in large amounts for research purposes. However, none of them has really tested at such a scale that the feasibility of their industrial use could be critically judged. PMID:28781703

  8. Identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis

    SciTech Connect

    Bao, X.; Sinha, M. |; Liu, T.; Hong, C.; Luxon, B.A. |; Garofalo, R.P. ||; Casola, A. ||

    2008-04-25

    Human metapneumovirus (hMPV) is a major cause of lower respiratory tract infections in infants, elderly and immunocompromised patients. Little is known about the response to hMPV infection of airway epithelial cells, which play a pivotal role in initiating and shaping innate and adaptive immune responses. In this study, we analyzed the transcriptional profiles of airway epithelial cells infected with hMPV using high-density oligonucleotide microarrays. Of the 47,400 transcripts and variants represented on the Affimetrix GeneChip Human Genome HG-U133 plus 2 array, 1601 genes were significantly altered following hMPV infection. Altered genes were then assigned to functional categories and mapped to signaling pathways. Many up-regulated genes are involved in the initiation of pro-inflammatory and antiviral immune responses, including chemokines, cytokines, type I interferon and interferon-inducible proteins. Other important functional classes up-regulated by hMPV infection include cellular signaling, gene transcription and apoptosis. Notably, genes associated with antioxidant and membrane transport activity, several metabolic pathways and cell proliferation were down-regulated in response to hMPV infection. Real-time PCR and Western blot assays were used to confirm the expression of genes related to several of these functional groups. The overall result of this study provides novel information on host gene expression upon infection with hMPV and also serves as a foundation for future investigations of genes and pathways involved in the pathogenesis of this important viral infection. Furthermore, it can facilitate a comparative analysis of other paramyxoviral infections to determine the transcriptional changes that are conserved versus the one that are specific to individual pathogens.

  9. Antisense delivery using protamine–oligonucleotide particles

    PubMed Central

    Junghans, Monika; Kreuter, Jörg; Zimmer, Andreas

    2000-01-01

    Protamine, a polycationic peptide (mol. wt 4000–4500), was evaluated as a potential penetration enhancer for phosphodiester antisense oligonucleotides (ODNs). Unique complexes in the form of nanoparticles were spontaneously formed, which we call ‘proticles’. The stability of the particles and the ODNs bound into the proticles was examined in foetal calf serum and cell culture medium. FITC-labelled ODNs bound to protamine showed an increased cellular uptake into human histiocytic lymphoma U 937 cells compared to free ODNs. Proticles significantly decreased cellular growth in a cell proliferation assay using ODNs against the c-myc proto-oncogene. PMID:10773093

  10. Analysis of oligonucleotide array experiments with repeated measures using mixed models.

    PubMed

    Li, Hao; Wood, Constance L; Getchell, Thomas V; Getchell, Marilyn L; Stromberg, Arnold J

    2004-12-30

    Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease) or absence (Control) of the disease, and brain regions including olfactory bulb (OB) or cerebellum (CER). In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH) procedure of controlling false discovery rate (FDR) at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the alpha-level (alphanew = 0.0033) determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD) procedure at the level of alphanew to control the family-wise error rate (FWER) for each gene examined. A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

  11. Analysis of oligonucleotide array experiments with repeated measures using mixed models

    PubMed Central

    Li, Hao; Wood, Constance L; Getchell, Thomas V; Getchell, Marilyn L; Stromberg, Arnold J

    2004-01-01

    Background Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease) or absence (Control) of the disease, and brain regions including olfactory bulb (OB) or cerebellum (CER). In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. Results In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH) procedure of controlling false discovery rate (FDR) at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the α-level (αnew = 0.0033) determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD) procedure at the level of αnew to control the family-wise error rate (FWER) for each gene examined. Conclusions A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER. PMID:15626348

  12. Microarray analysis of R-gene-mediated resistance to viruses.

    PubMed

    Ishihara, Takeaki; Sato, Yukiyo; Takahashi, Hideki

    2015-01-01

    The complex process for host-plant resistance to viruses is precisely regulated by a number of genes and signaling compounds. Thus, global gene expression analysis can provide a powerful tool to grasp the complex molecular network for resistance to viruses. The procedures for comparative global gene expression profiling of virus-resistant and control plants by microarray analysis include RNA extraction, cDNA synthesis, cRNA labeling, hybridization, array scanning, and data mining steps. There are several platforms for the microarray analysis. Commercial services for the steps from cDNA synthesis to array scanning are now widely available; however, the data manipulation step is highly dependent on the experimental design and research focus. The protocols presented here are optimized for analyzing global gene expression during the R gene-conferred defense response using commercial oligonucleotide-based arrays. We also demonstrate a technique to screen for differentially expressed genes using Excel software and a simple Internet tool-based data mining approach for characterizing the identified genes.

  13. Novel microarray design strategy to study complex bacterial communities.

    PubMed

    Huyghe, Antoine; Francois, Patrice; Charbonnier, Yvan; Tangomo-Bento, Manuela; Bonetti, Eve-Julie; Paster, Bruce J; Bolivar, Ignacio; Baratti-Mayer, Denise; Pittet, Didier; Schrenzel, Jacques

    2008-03-01

    Assessing bacterial flora composition appears to be of increasing importance to fields as diverse as physiology, development, medicine, epidemiology, the environment, and the food industry. We report here the development and validation of an original microarray strategy that allows analysis of the phylogenic composition of complex bacterial mixtures. The microarray contains approximately 9,500 feature elements targeting 16S rRNA gene-specific regions. Probe design was performed by selecting oligonucleotide sequences specific to each node of the seven levels of the bacterial phylogenetic tree (domain, phylum, class, order, family, genus, and species). This approach, based on sequence information, allows analysis of the bacterial contents of complex bacterial mixtures to detect both known and unknown microorganisms. The presence of unknown organisms can be suspected and mapped on the phylogenetic tree, indicating where to refine analysis. Initial proof-of-concept experiments were performed on oral bacterial communities. Our results show that this hierarchical approach can reveal minor changes (

  14. Antisense Oligonucleotide Therapy in Diabetic Retinopathy

    PubMed Central

    Hnik, Peter; Boyer, David S.; Grillone, Lisa R.; Clement, John G.; Henry, Scott P.; Green, Ellen A.

    2009-01-01

    Diabetic retinopathy is one of the leading causes of blindness in the United States and other parts of the world. Historically, laser photocoagulation and vitrectomy surgery have been used for the treatment of diabetic retinopathy, including diabetic macular edema. Both procedures have proven to be useful under certain conditions but have their limitations. New pathways and processes that promote diabetic retinopathy have been identified, and several new therapeutic approaches are under investigation. These new therapies may be beneficial in the treatment of diabetic retinopathy and include antivascular endothelial growth factor agents, corticosteroids, and therapies that may potentially target a number of additional diabetic retinopathy-related factors and processes, including antisense oligonucleotides. Second-generation antisense oligonucleotides, such as iCo-007, may offer a significant advantage in the treatment of diabetic retinopathy by downregulating the signal pathways of multiple growth factors that seem to play a critical role in the process of ocular angiogenesis and vascular leakage. Benefits of such molecules are expected to include the specificity of the kinase target and an extended half-life, resulting in less frequent intravitreal drug administration, resistance to molecule degradation, and a good safety profile. PMID:20144342

  15. Gene cloning based on long oligonucleotide probes

    SciTech Connect

    Wood, W.I.

    1987-01-01

    The most commonly used technique for gene cloning has been to utilize oligonucleotide probe based on protein sequence data. Of course this approach requires characterized and purified protein so that at least a portion of amino acid sequence can be determined and used to infer the corresponding DNA sequence. Based on the amino acid sequence information, either short or long oligonucleotide probes can be synthesized chemically. Long probes are typically 30-100 nucleotides long and are a single sequence based on a best guess for each codon. The long probe approach was first used to screen for three different genes: bovine trypsin inhibitor, human insulin-like growth factor I, and human factor IX. There are three advantages of long probes. (1) Any stretch of amino acid sequence 10 or longer can be used. (2) The amino acid sequence need not be absolutely correct. (3) These probes can be used to screen high-complexity libraries with fewer false positives. In spite of the uncertainties over codon selection, the long probe approach is currently the method of choice in screening for genes based on protein sequence data.

  16. BIOCONJUGATION OF OLIGONUCLEOTIDES FOR TREATING LIVER FIBROSIS

    PubMed Central

    Ye, Zhaoyang; Hajj Houssein, Houssam S.; Mahato, Ram I.

    2009-01-01

    Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is in urgent need to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remains the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of α1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed. PMID:18154454

  17. Template switching between PNA and RNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1995-01-01

    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  18. Template switching between PNA and RNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1995-01-01

    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  19. Microarray Technologies in Fungal Diagnostics.

    PubMed

    Rupp, Steffen

    2017-01-01

    Microarray technologies have been a major research tool in the last decades. In addition they have been introduced into several fields of diagnostics including diagnostics of infectious diseases. Microarrays are highly parallelized assay systems that initially were developed for multiparametric nucleic acid detection. From there on they rapidly developed towards a tool for the detection of all kind of biological compounds (DNA, RNA, proteins, cells, nucleic acids, carbohydrates, etc.) or their modifications (methylation, phosphorylation, etc.). The combination of closed-tube systems and lab on chip devices with microarrays further enabled a higher automation degree with a reduced contamination risk. Microarray-based diagnostic applications currently complement and may in the future replace classical methods in clinical microbiology like blood cultures, resistance determination, microscopic and metabolic analyses as well as biochemical or immunohistochemical assays. In addition, novel diagnostic markers appear, like noncoding RNAs and miRNAs providing additional room for novel nucleic acid based biomarkers. Here I focus an microarray technologies in diagnostics and as research tools, based on nucleic acid-based arrays.

  20. [First experiences with preimplantation genetic screening of chromosomal aberrations using oligonucleotide-based array comparative genomic hybridization].

    PubMed

    Kuglík, Petr; Smetana, Jan; Němcová, Darja; Vallová, Vladimíra; Mikulášová, Aneta; Gaillyová, Renata; Hubinka, Vít; Koudelka, Marek

    2015-01-01

    Preimplantation genetic diagnosis (PGD) is a complex approach for detecting genetic abnormalities in early-stage embryos using genetic or molecular cytogenetic methods. Recently, single cell genomic methods based on DNA microarrays have been used for PGD. In the presented paper, we discuss and demonstrate the possibility to detect copy number variation (CNVs) in trophectoderm cells biopsied from 5-day embryos using 60-mer oligonucleotide-based array-CGH with CytoSure 8 × 15K Aneuploidy Array. Whereas this microarray platform was originally designed for analysis of unamplified DNA derived from many cells, the new methods, developed for single-cell genomics, allow the application of oligo arrays technology in preimplanation genetic diagnosis. Preclinical validation of single cell array-CGH was made by analysis of 30 positive and negative controls. Validation process included whole genome amplification of DNA from 5-10 cells with normal karyotype and from samples with known aneuploidies and structural aberrations. Subsequently, we analyzed the whole genome profiles in 118 embryos; aneuploidies of chromosomes were observed in 26.7%; segmental imbalances were proved in 6.8% of embryos. Our first experience confirmed that this oligonucleotide-based array technique enables high-resolution preimplantation aneuploidy screening of all the 23 chromosome pairs and sensitive preimplantation diagnosis of segmental imbalances such as deletions, duplications and amplifications.

  1. Disulfide-linked oligonucleotide phosphorothioates - Novel analogues of nucleic acids

    NASA Technical Reports Server (NTRS)

    Wu, Taifeng; Orgel, Leslie E.

    1991-01-01

    The synthesis of phosphorothioate analogs of oligonucleotides by the oxidation of deoxyadenosine 3',5'-bisphosphorothioate (3) was attempted. Cyclization of 3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5'-terminal phosphorotioate group undergoes efficient chain-extension when oxidized in the presence of 3.

  2. Oligonucleotide therapies for disorders of the nervous system.

    PubMed

    Khorkova, Olga; Wahlestedt, Claes

    2017-03-01

    Oligonucleotide therapies are currently experiencing a resurgence driven by advances in backbone chemistry and discoveries of novel therapeutic pathways that can be uniquely and efficiently modulated by the oligonucleotide drugs. A quarter of a century has passed since oligonucleotides were first applied in living mammalian brain to modulate gene expression. Despite challenges in delivery to the brain, multiple oligonucleotide-based compounds are now being developed for treatment of human brain disorders by direct delivery inside the blood brain barrier (BBB). Notably, the first new central nervous system (CNS)-targeted oligonucleotide-based drug (nusinersen/Spinraza) was approved by US Food and Drug Administration (FDA) in late 2016 and several other compounds are in advanced clinical trials. Human testing of brain-targeted oligonucleotides has highlighted unusual pharmacokinetic and pharmacodynamic properties of these compounds, including complex active uptake mechanisms, low systemic exposure, extremely long half-lives, accumulation and gradual release from subcellular depots. Further work on oligonucleotide uptake, development of formulations for delivery across the BBB and relevant disease biology studies are required for further optimization of the oligonucleotide drug development process for brain applications.

  3. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  4. The role of antisense oligonucleotide therapy in patients with familial hypercholesterolemia: risks, benefits, and management recommendations.