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Sample records for efficient dna microarray

  1. Robust and efficient synthetic method for forming DNA microarrays.

    PubMed

    Dolan, P L; Wu, Y; Ista, L K; Metzenberg, R L; Nelson, M A; Lopez, G P

    2001-11-01

    The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

  2. Model selection and efficiency testing for normalization of cDNA microarray data

    PubMed Central

    Futschik, Matthias; Crompton, Toni

    2004-01-01

    In this study we present two novel normalization schemes for cDNA microarrays. They are based on iterative local regression and optimization of model parameters by generalized cross-validation. Permutation tests assessing the efficiency of normalization demonstrated that the proposed schemes have an improved ability to remove systematic errors and to reduce variability in microarray data. The analysis also reveals that without parameter optimization local regression is frequently insufficient to remove systematic errors in microarray data. PMID:15287982

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

    NASA Astrophysics Data System (ADS)

    Qamhieh, K.; Pettitt, B. Montgomery

    2015-03-01

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

  4. 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.

  5. 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.

  6. 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

  7. Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

    PubMed

    Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

    2013-04-01

    The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

  8. Luminescent dye-doped or rare-earth-doped monodisperse silica nanospheres as efficient labels in DNA microarrays

    NASA Astrophysics Data System (ADS)

    Enrichi, F.; Riccò, R.; Meneghello, A.; Pierobon, R.; Marinello, F.; Schiavuta, P.

    2009-08-01

    Luminescent nanoparticles are gaining more and more interest in bio-labeling and bio-imaging applications, like for example DNA microarray. This is a high-throughput technology used for detection and quantification of nucleic acid molecules and other ones of biological interest. The analysis is resulting by specific hybridization between probe sequences deposited in array and a target ss-DNA usually expressed by PCR and functionalized by a fluorescent dye. These organic labels have well known disadvantages like photobleaching and limited sensitivity. Quantum dots may be used as alternatives, but they present troubles like blinking, toxicity and excitation wavelengths out of the usual range of commercial instruments, lowering their efficiency. Therefore in this work we investigate a different strategy, based on the use of inorganic silica nanospheres incorporating standard luminescent dyes or rare earth doped nanocrystals. In the first case it is possible to obtain a high luminescence emission signal, due to the high number of dye molecules that can be accommodated into each nanoparticle, reduced photobleaching and environmental protection of the dye molecules thanks to the encapsulation in the silica matrix. In the second case, rare earths exhibit narrow emission bands (easy identification), large Stokes shifts (efficient discrimination of excitation and emission) and long luminescence lifetimes (possibility to perform time-delayed analysis) which can be efficiently used for the improvement of signal to noise ratio. The synthesis and characterization of good luminescent silica spheres either by organic dye-doping or by rare-earth-doping are investigated and reported. Moreover, their application in the DNA microarray technology in comparison to the use of standard molecular fluorophores or commercial quantum dots is discussed. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalization and bio

  9. 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.

  10. 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.

  11. DNA microarray integromics analysis platform.

    PubMed

    Waller, Tomasz; Gubała, Tomasz; Sarapata, Krzysztof; Piwowar, Monika; Jurkowski, Wiktor

    2015-01-01

    The study of interactions between molecules belonging to different biochemical families (such as lipids and nucleic acids) requires specialized data analysis methods. This article describes the DNA Microarray Integromics Analysis Platform, a unique web application that focuses on computational integration and analysis of "multi-omics" data. Our tool supports a range of complex analyses, including - among others - low- and high-level analyses of DNA microarray data, integrated analysis of transcriptomics and lipidomics data and the ability to infer miRNA-mRNA interactions. We demonstrate the characteristics and benefits of the DNA Microarray Integromics Analysis Platform using two different test cases. The first test case involves the analysis of the nutrimouse dataset, which contains measurements of the expression of genes involved in nutritional problems and the concentrations of hepatic fatty acids. The second test case involves the analysis of miRNA-mRNA interactions in polysaccharide-stimulated human dermal fibroblasts infected with porcine endogenous retroviruses. The DNA Microarray Integromics Analysis Platform is a web-based graphical user interface for "multi-omics" data management and analysis. Its intuitive nature and wide range of available workflows make it an effective tool for molecular biology research. The platform is hosted at https://lifescience.plgrid.pl/.

  12. DNA microarray-based mutation discovery and genotyping.

    PubMed

    Gresham, David

    2011-01-01

    DNA microarrays provide an efficient means of identifying single-nucleotide polymorphisms (SNPs) in DNA samples and characterizing their frequencies in individual and mixed samples. We have studied the parameters that determine the sensitivity of DNA probes to SNPs and found that the melting temperature (T (m)) of the probe is the primary determinant of probe sensitivity. An isothermal-melting temperature DNA microarray design, in which the T (m) of all probes is tightly distributed, can be implemented by varying the length of DNA probes within a single DNA microarray. I describe guidelines for designing isothermal-melting temperature DNA microarrays and protocols for labeling and hybridizing DNA samples to DNA microarrays for SNP discovery, genotyping, and quantitative determination of allele frequencies in mixed samples.

  13. DNA microarray technology in dermatology.

    PubMed

    Kunz, Manfred

    2008-03-01

    In recent years, DNA microarray technology has been used for the analysis of gene expression patterns in a variety of skin diseases, including malignant melanoma, psoriasis, lupus erythematosus, and systemic sclerosis. Many of the studies described herein confirmed earlier results on individual genes or functional groups of genes. However, a plethora of new candidate genes, gene patterns, and regulatory pathways have been identified. Major progresses were reached by the identification of a prognostic gene pattern in malignant melanoma, an immune signaling cluster in psoriasis, and a so-called interferon signature in systemic lupus erythematosus. In future, interference with genes or regulatory pathways with the use of different RNA interference technologies or targeted therapy may not only underscore the functional significance of microarray data but also may open interesting therapeutic perspectives. Large-scale gene expression analyses may also help to design more individualized treatment approaches of cutaneous diseases.

  14. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  15. Microarrays Made Simple: "DNA Chips" Paper Activity

    ERIC Educational Resources Information Center

    Barnard, Betsy

    2006-01-01

    DNA microarray technology is revolutionizing biological science. DNA microarrays (also called DNA chips) allow simultaneous screening of many genes for changes in expression between different cells. Now researchers can obtain information about genes in days or weeks that used to take months or years. The paper activity described in this article…

  16. 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.

  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 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.

  18. DNA Microarrays in Herbal Drug Research

    PubMed Central

    Chavan, Preeti; Joshi, Kalpana; Patwardhan, Bhushan

    2006-01-01

    Natural products are gaining increased applications in drug discovery and development. Being chemically diverse they are able to modulate several targets simultaneously in a complex system. Analysis of gene expression becomes necessary for better understanding of molecular mechanisms. Conventional strategies for expression profiling are optimized for single gene analysis. DNA microarrays serve as suitable high throughput tool for simultaneous analysis of multiple genes. Major practical applicability of DNA microarrays remains in DNA mutation and polymorphism analysis. This review highlights applications of DNA microarrays in pharmacodynamics, pharmacogenomics, toxicogenomics and quality control of herbal drugs and extracts. PMID:17173108

  19. Highly efficient factorial designs for cDNA microarray experiments: use of approximate theory together with a step-up step-down procedure.

    PubMed

    Zhang, Runchu; Mukerjee, Rahul

    2013-08-01

    A general method for obtaining highly efficient factorial designs of relatively small sizes is developed for cDNA microarray experiments. It allows the main effects and interactions to be of possibly unequal importance. First, the approximate theory is employed to get an optimal design measure which is then discretized. It is, however, observed that a naïve discretization may fail to yield an exact design of the stipulated size and, even when it yields such an exact design, there is often scope for improvement in efficiency. To address these issues, we propose a step-up/down procedure which is seen to work very well. The resulting designs turn out to be quite robust to possible dye-color effects and heteroscedasticity. We focus on the baseline and all-to-next parametrizations but our method works equally well also for hybrids of the two and other parametrizations.

  20. Analysis of DNA microarray expression data.

    PubMed

    Simon, Richard

    2009-06-01

    DNA microarrays are powerful tools for studying biological mechanisms and for developing prognostic and predictive classifiers for identifying the patients who require treatment and are best candidates for specific treatments. Because microarrays produce so much data from each specimen, they offer great opportunities for discovery and great dangers or producing misleading claims. Microarray based studies require clear objectives for selecting cases and appropriate analysis methods. Effective analysis of microarray data, where the number of measured variables is orders of magnitude greater than the number of cases, requires specialized statistical methods which have recently been developed. Recent literature reviews indicate that serious problems of analysis exist a substantial proportion of publications. This manuscript attempts to provide a non-technical summary of the key principles of statistical design and analysis for studies that utilize microarray expression profiling.

  1. Microarrays (DNA Chips) for the Classroom Laboratory

    ERIC Educational Resources Information Center

    Barnard, Betsy; Sussman, Michael; BonDurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-01-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The…

  2. Microarrays (DNA Chips) for the Classroom Laboratory

    ERIC Educational Resources Information Center

    Barnard, Betsy; Sussman, Michael; BonDurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-01-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The…

  3. Statistical approaches for the analysis of DNA methylation microarray data.

    PubMed

    Siegmund, Kimberly D

    2011-06-01

    Following the rapid development and adoption in DNA methylation microarray assays, we are now experiencing a growth in the number of statistical tools to analyze the resulting large-scale data sets. As is the case for other microarray applications, biases caused by technical issues are of concern. Some of these issues are old (e.g., two-color dye bias and probe- and array-specific effects), while others are new (e.g., fragment length bias and bisulfite conversion efficiency). Here, I highlight characteristics of DNA methylation that suggest standard statistical tools developed for other data types may not be directly suitable. I then describe the microarray technologies most commonly in use, along with the methods used for preprocessing and obtaining a summary measure. I finish with a section describing downstream analyses of the data, focusing on methods that model percentage DNA methylation as the outcome, and methods for integrating DNA methylation with gene expression or genotype data.

  4. Progress in the application of DNA microarrays.

    PubMed Central

    Lobenhofer, E K; Bushel, P R; Afshari, C A; Hamadeh, H K

    2001-01-01

    Microarray technology has been applied to a variety of different fields to address fundamental research questions. The use of microarrays, or DNA chips, to study the gene expression profiles of biologic samples began in 1995. Since that time, the fundamental concepts behind the chip, the technology required for making and using these chips, and the multitude of statistical tools for analyzing the data have been extensively reviewed. For this reason, the focus of this review will be not on the technology itself but on the application of microarrays as a research tool and the future challenges of the field. PMID:11673116

  5. 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

  6. 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…

  7. 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…

  8. Hybridization and Selective Release of DNA Microarrays

    SciTech Connect

    Beer, N R; Baker, B; Piggott, T; Maberry, S; Hara, C M; DeOtte, J; Benett, W; Mukerjee, E; Dzenitis, J; Wheeler, E K

    2011-11-29

    DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to

  9. 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.

  10. [DNA microarrays and their application in detecting and identifying intestinal pathogens].

    PubMed

    Jin, Da-Zhi; Wen, Si-Yuan; Wang, Sheng-Qi

    2006-06-01

    DNA microarrays offer many advantages of high throughout, automation, rapid detection, and so on. Therefore, this technology had been used in many fields such as molecular epidemiology of bacteria, microbial gene identification, disease mechanism, gene mutation, gene expression identification, DNA sequencing and medicine screening etc. The assays for identifying pathogens using DNA microarrays reported aboard recently are introduced. The application of DNA microarrays in detecting and identifying intestinal pathogens mainly includes three aspects: the identification of toxin and characteristic genes of pathogens, the identification of bacterial DNA or RNA directly, the simultaneous detection of a large number of intestinal pathogens with the target - gene of ribosomal RNA. Because of its high efficiency, DNA microarrays is superior to other biological method. Obviously DNA microarrays technology may be useful in identifying intestinal pathogens and have a wide prospect.

  11. Giant Magnetoresistive Sensors for DNA Microarray

    PubMed Central

    Xu, Liang; Yu, Heng; Han, Shu-Jen; Osterfeld, Sebastian; White, Robert L.; Pourmand, Nader; Wang, Shan X.

    2009-01-01

    Giant magnetoresistive (GMR) sensors are developed for a DNA microarray. Compared with the conventional fluorescent sensors, GMR sensors are cheaper, more sensitive, can generate fully electronic signals, and can be easily integrated with electronics and microfluidics. The GMR sensor used in this work has a bottom spin valve structure with an MR ratio of 12%. The single-strand target DNA detected has a length of 20 bases. Assays with DNA concentrations down to 10 pM were performed, with a dynamic range of 3 logs. A double modulation technique was used in signal detection to reduce the 1/f noise in the sensor while circumventing electromagnetic interference. The logarithmic relationship between the magnetic signal and the target DNA concentration can be described by the Temkin isotherm. Furthermore, GMR sensors integrated with microfluidics has great potential of improving the sensitivity to 1 pM or below, and the total assay time can be reduced to less than 1 hour. PMID:20824116

  12. Bacterial identification and subtyping using DNA microarray and DNA sequencing.

    PubMed

    Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

    2012-01-01

    The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

  13. Application of nanostructured biochips for efficient cell transfection microarrays

    NASA Astrophysics Data System (ADS)

    Akkamsetty, Yamini; Hook, Andrew L.; Thissen, Helmut; Hayes, Jason P.; Voelcker, Nicolas H.

    2007-01-01

    Microarrays, high-throughput devices for genomic analysis, can be further improved by developing materials that are able to manipulate the interfacial behaviour of biomolecules. This is achieved both spatially and temporally by smart materials possessing both switchable and patterned surface properties. A system had been developed to spatially manipulate both DNA and cell growth based upon the surface modification of highly doped silicon by plasma polymerisation and polyethylene grafting followed by masked laser ablation for formation of a pattered surface with both bioactive and non-fouling regions. This platform has been successfully applied to transfected cell microarray applications with the parallel expression of genes by utilising its ability to direct and limit both DNA and cell attachment to specific sites. One of the greatest advantages of this system is its application to reverse transfection, whereupon by utilising the switchable adsorption and desorption of DNA using a voltage bias, the efficiency of cell transfection can be enhanced. However, it was shown that application of a voltage also reduces the viability of neuroblastoma cells grown on a plasma polymer surface, but not human embryonic kidney cells. This suggests that the application of a voltage may not only result in the desorption of bound DNA but may also affect attached cells. The characterisation of a DNA microarray by contact printing has also been investigated.

  14. Overview of DNA microarrays: types, applications, and their future.

    PubMed

    Bumgarner, Roger

    2013-01-01

    This unit provides an overview of DNA microarrays. Microarrays are a technology in which thousands of nucleic acids are bound to a surface and are used to measure the relative concentration of nucleic acid sequences in a mixture via hybridization and subsequent detection of the hybridization events. This overview first discusses the history of microarrays and the antecedent technologies that led to their development. This is followed by discussion of the methods of manufacture of microarrays and the most common biological applications. The unit ends with a brief description of the limitations of microarrays and discusses how microarrays are being rapidly replaced by DNA sequencing technologies.

  15. Manufacturing DNA microarrays from unpurified PCR products

    PubMed Central

    Diehl, Frank; Beckmann, Boris; Kellner, Nadine; Hauser, Nicole C.; Diehl, Susanne; Hoheisel, Jörg D.

    2002-01-01

    For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(l-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products. PMID:12177307

  16. Integrated microfluidic biochips for DNA microarray analysis.

    PubMed

    Liu, Robin Hui; Dill, Kilian; Fuji, H Sho; McShea, Andy

    2006-03-01

    A fully integrated and self-contained microfluidic biochip device has been developed to automate the fluidic handling steps required to perform a gene expression study of the human leukemia cell line (K-562). The device consists of a DNA microarray semiconductor chip with 12,000 features and a microfluidic cartridge that consists of microfluidic pumps, mixers, valves, fluid channels and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. A single-color transcriptional analysis of K-562 cells with a series of calibration controls (spiked-in controls) was performed to characterize this new platform with regard to sensitivity, specificity and dynamic range. The device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than 3 orders of magnitude. Experiments also demonstrated that chip-to-chip variability was low, indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis.

  17. Use of Genomic DNA as A Reference in DNA Microarrays

    SciTech Connect

    Yang, Yunfeng

    2009-01-01

    DNA microarray has become a mainstream technology to explore gene expression profiles, identify novel genes involved in a biological process of interest and predict their function, and determine biomarkers that are relevant to a given phenotype or disease. Typical two-channel microarray studies use an experimental design called the complementary DNA (cDNA) reference method, in which samples from test and control conditions are compared directly on a microarray slide. A substantial limitation of this strategy is that it is nearly impossible to compare data between experiments because the reference sample composition is subjected to changes at the level of experimental design and thereby not consistent from one experiment to another. Using genomic DNA as common reference will effectively overcome this limitation. This chapter describes detailed methods to prepare genomic DNA of high quality, label with fluorescent dye, co-hybridize with cDNA samples, and the subsequent data analyses. In addition, notes are provided to help the readers to obtain optimal results using the procedure.

  18. Modeling background intensity in DNA microarrays

    NASA Astrophysics Data System (ADS)

    Kroll, K. M.; Barkema, G. T.; Carlon, E.

    2008-06-01

    DNA microarrays are devices that are able, in principle, to detect and quantify the presence of specific nucleic acid sequences in complex biological mixtures. The measurement consists in detecting fluorescence signals from several spots on the microarray surface onto which different probe sequences are grafted. One of the problems of the data analysis is that the signal contains a noisy background component due to nonspecific binding. We present a physical model for background estimation in Affymetrix Genechips. It combines two different approaches. The first is based on the sequence composition, specifically its sequence-dependent hybridization affinity. The second is based on the strong correlation of intensities from locations which are the physical neighbors of a specific spot on the chip. Both effects are incorporated in a background estimator which contains 24 free parameters, fixed by minimization on a training data set. In all data analyzed the sequence-specific parameters, obtained by minimization, are found to strongly correlate with empirically determined stacking free energies for RNA-DNA hybridization in solution. Moreover, there is an overall agreement with experimental background data and we show that the physics-based model that we propose performs on average better than purely statistical approaches for background calculations. The model thus provides an interesting alternative method for background subtraction schemes in Affymetrix Genechips.

  19. 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.

  20. DNA Microarray for Detection of Gastrointestinal Viruses

    PubMed Central

    Martínez, Miguel A.; Soto-del Río, María de los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y.; Greninger, Alexander L.; Contreras, Juan Francisco; López, Susana; Arias, Carlos F.

    2014-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 103 virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  1. DNA microarray for detection of gastrointestinal viruses.

    PubMed

    Martínez, Miguel A; Soto-Del Río, María de Los Dolores; Gutiérrez, Rosa María; Chiu, Charles Y; Greninger, Alexander L; Contreras, Juan Francisco; López, Susana; Arias, Carlos F; Isa, Pavel

    2015-01-01

    Gastroenteritis is a clinical illness of humans and other animals that is characterized by vomiting and diarrhea and caused by a variety of pathogens, including viruses. An increasing number of viral species have been associated with gastroenteritis or have been found in stool samples as new molecular tools have been developed. In this work, a DNA microarray capable in theory of parallel detection of more than 100 viral species was developed and tested. Initial validation was done with 10 different virus species, and an additional 5 species were validated using clinical samples. Detection limits of 1 × 10(3) virus particles of Human adenovirus C (HAdV), Human astrovirus (HAstV), and group A Rotavirus (RV-A) were established. Furthermore, when exogenous RNA was added, the limit for RV-A detection decreased by one log. In a small group of clinical samples from children with gastroenteritis (n = 76), the microarray detected at least one viral species in 92% of the samples. Single infection was identified in 63 samples (83%), and coinfection with more than one virus was identified in 7 samples (9%). The most abundant virus species were RV-A (58%), followed by Anellovirus (15.8%), HAstV (6.6%), HAdV (5.3%), Norwalk virus (6.6%), Human enterovirus (HEV) (9.2%), Human parechovirus (1.3%), Sapporo virus (1.3%), and Human bocavirus (1.3%). To further test the specificity and sensitivity of the microarray, the results were verified by reverse transcription-PCR (RT-PCR) detection of 5 gastrointestinal viruses. The RT-PCR assay detected a virus in 59 samples (78%). The microarray showed good performance for detection of RV-A, HAstV, and calicivirus, while the sensitivity for HAdV and HEV was low. Furthermore, some discrepancies in detection of mixed infections were observed and were addressed by reverse transcription-quantitative PCR (RT-qPCR) of the viruses involved. It was observed that differences in the amount of genetic material favored the detection of the most abundant

  2. Microarrays (DNA chips) for the classroom laboratory.

    PubMed

    Barnard, Betsy; Sussman, Michael; Bondurant, Sandra Splinter; Nienhuis, James; Krysan, Patrick

    2006-09-01

    We have developed and optimized the necessary laboratory materials to make DNA microarray technology accessible to all high school students at a fraction of both cost and data size. The primary component is a DNA chip/array that students "print" by hand and then analyze using research tools that have been adapted for classroom use. The primary adaptation is the use of a simulated cDNA target. The low density DNA array we discuss here was used to demonstrate differential expression of several Arabidopsis thaliana genes related to photosynthesis and photomorphogenesis. The methods we present here can be used with any biological organism whose sequence is known. Furthermore, these methods can be adapted to exhibit a variety of differential gene expression patterns under different experimental conditions. The materials and tools we discuss have been applied in classrooms at West High School in Madison, WI. We have also shared these materials with high school teachers attending professional development courses at the University of Wisconsin-Madison.

  3. Application of DNA microarrays in occupational health research.

    PubMed

    Koizumi, Shinji

    2004-01-01

    The profiling of gene expression patterns with DNA microarrays is recently being widely used not only in basic molecular biological studies but also in the practical fields. In clinical application, for example, this technique is expected to be quite useful in making a correct diagnosis. In the pharmacological area, the microarray analysis can be applied to drug discovery and individualized drug treatment. Although not so popular as these examples, DNA microarrays could also be a powerful tool in studies relevant to occupational health. This review will describe the outline of gene expression profiling with DNA microarrays and prospects in occupational health research.

  4. 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.

  5. Microarray analysis of DNA replication timing.

    PubMed

    Karnani, Neerja; Taylor, Christopher M; Dutta, Anindya

    2009-01-01

    Although all of the DNA in an eukaryotic cell replicates during the S-phase of cell cycle, there is a significant difference in the actual time in S-phase when a given chromosomal segment replicates. Methods are described here for generation of high-resolution temporal maps of DNA replication in synchronized human cells. This method does not require amplification of DNA before microarray hybridization and so avoids errors introduced during PCR. A major advantage of using this procedure is that it facilitates finer dissection of replication time in S-phase. Also, it helps delineate chromosomal regions that undergo biallelic or asynchronous replication, which otherwise are difficult to detect at a genome-wide scale by existing methods. The continuous TR50 (time of completion of 50% replication) maps of replication across chromosomal segments identify regions that undergo acute transitions in replication timing. These transition zones can play a significant role in identifying insulators that separate chromosomal domains with different chromatin modifications.

  6. 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.

  7. Metric learning for DNA microarray data analysis

    NASA Astrophysics Data System (ADS)

    Takeuchi, Ichiro; Nakagawa, Masao; Seto, Masao

    2009-12-01

    In many microarray studies, gene set selection is an important preliminary step for subsequent main task such as tumor classification, cancer subtype identification, etc. In this paper, we investigate the possibility of using metric learning as an alternative to gene set selection. We develop a simple metric learning algorithm aiming to use it for microarray data analysis. Exploiting a property of the algorithm, we introduce a novel approach for extending the metric learning to be adaptive. We apply the algorithm to previously studied microarray data on malignant lymphoma subtype identification.

  8. [DNA microarrays in parasitology and medical sciences].

    PubMed

    Jaros, Sławomir

    2006-01-01

    The article presents the current knowledge on the microarray technique and its applications in medical sciences and parasitology. The first part of the article is focused on the technical aspects (microarray preparation, different microarray platforms, probes preparation, hybridization and signal detection). The article also describes possible ways of proceeding during laboratory work on organism of which the genome sequence is not known or has been only partially sequenced. The second part of the review describes how microarray technique have been, or possibly will be, used for better understanding parasite life cycles and development, host-parasite relationship, comparative genomics of virulent organisms, develpoment vaccines against the most virulent parasites and host responses to infection.

  9. 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.

  10. Design of a combinatorial DNA microarray for protein-DNA interaction studies

    PubMed Central

    Mintseris, Julian; Eisen, Michael B

    2006-01-01

    Background Discovery of precise specificity of transcription factors is an important step on the way to understanding the complex mechanisms of gene regulation in eukaryotes. Recently, double-stranded protein-binding microarrays were developed as a potentially scalable approach to tackle transcription factor binding site identification. Results Here we present an algorithmic approach to experimental design of a microarray that allows for testing full specificity of a transcription factor binding to all possible DNA binding sites of a given length, with optimally efficient use of the array. This design is universal, works for any factor that binds a sequence motif and is not species-specific. Furthermore, simulation results show that data produced with the designed arrays is easier to analyze and would result in more precise identification of binding sites. Conclusion In this study, we present a design of a double stranded DNA microarray for protein-DNA interaction studies and show that our algorithm allows optimally efficient use of the arrays for this purpose. We believe such a design will prove useful for transcription factor binding site identification and other biological problems. PMID:17018151

  11. Development of a protein microarray using sequence-specific DNA binding domain on DNA chip surface

    SciTech Connect

    Choi, Yoo Seong; Pack, Seung Pil; Yoo, Young Je . E-mail: yjyoo@snu.ac.kr

    2005-04-22

    A protein microarray based on DNA microarray platform was developed to identify protein-protein interactions in vitro. The conventional DNA chip surface by 156-bp PCR product was prepared for a substrate of protein microarray. High-affinity sequence-specific DNA binding domain, GAL4 DNA binding domain, was introduced to the protein microarray as fusion partner of a target model protein, enhanced green fluorescent protein. The target protein was oriented immobilized directly on the DNA chip surface. Finally, monoclonal antibody of the target protein was used to identify the immobilized protein on the surface. This study shows that the conventional DNA chip can be used to make a protein microarray directly, and this novel protein microarray can be applicable as a tool for identifying protein-protein interactions.

  12. 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.

  13. Optimized LOWESS normalization parameter selection for DNA microarray data

    PubMed Central

    Berger, John A; Hautaniemi, Sampsa; Järvinen, Anna-Kaarina; Edgren, Henrik; Mitra, Sanjit K; Astola, Jaakko

    2004-01-01

    Background Microarray data normalization is an important step for obtaining data that are reliable and usable for subsequent analysis. One of the most commonly utilized normalization techniques is the locally weighted scatterplot smoothing (LOWESS) algorithm. However, a much overlooked concern with the LOWESS normalization strategy deals with choosing the appropriate parameters. Parameters are usually chosen arbitrarily, which may reduce the efficiency of the normalization and result in non-optimally normalized data. Thus, there is a need to explore LOWESS parameter selection in greater detail. Results and discussion In this work, we discuss how to choose parameters for the LOWESS method. Moreover, we present an optimization approach for obtaining the fraction of data points utilized in the local regression and analyze results for local print-tip normalization. The optimization procedure determines the bandwidth parameter for the local regression by minimizing a cost function that represents the mean-squared difference between the LOWESS estimates and the normalization reference level. We demonstrate the utility of the systematic parameter selection using two publicly available data sets. The first data set consists of three self versus self hybridizations, which allow for a quantitative study of the optimization method. The second data set contains a collection of DNA microarray data from a breast cancer study utilizing four breast cancer cell lines. Our results show that different parameter choices for the bandwidth window yield dramatically different calibration results in both studies. Conclusions Results derived from the self versus self experiment indicate that the proposed optimization approach is a plausible solution for estimating the LOWESS parameters, while results from the breast cancer experiment show that the optimization procedure is readily applicable to real-life microarray data normalization. In summary, the systematic approach to obtain critical

  14. Gene expression profiles in varicose veins using complementary DNA microarray.

    PubMed

    Lee, Seokjong; Lee, Wonchae; Choe, Yoonseok; Kim, Dowon; Na, Gunyeon; Im, Sanguk; Kim, Jinoh; Kim, Moonkyu; Kim, Jungchul; Cho, Joonyong

    2005-04-01

    There has been little information reported about the genetic event concerning the pathophysiology of varicose vein (VV). The purpose of this study was to examine the differentiation of gene expression in the wall of VV using complementary deoxyribonucleic acid (cDNA) microarrays. The study was performed with four pairs of VVs and control veins (CVs). cDNA specimens of VVs were prepared from the ribonucleic acid-isolated VVs of patients who underwent venous obliteration, using radiofrequency, as well as from CVs of those who underwent aortocoronary bypass grafting. Each set of VVs and CVs was hybridized with high-density microarray containing 3,063 human cDNAs. The finding of microarray hybridization were scanned, analyzed, and classified with the cluster program. Among 3,063 cDNA clones, 82 genes were up-regulated in VVs, and some of the up-regulated genes, which were detected by cDNA microarray, including transforming growth factor 3-induced gene (BIGH3), tubulin, lumican, actinin, collagen type I, versican, actin, and tropomyosin, belonged to extracelluar matrix molecules, cytoskeletal proteins, or myofibroblasts. Many up-regulated genes were found in Ws by applying cDNA microarray. These gene profiles suggested a pathway associated with fibrosis and that wound healing might be related to the pathophysiology of VVs.

  15. Molecular diagnosis and prognosis with DNA microarrays.

    PubMed

    Wiltgen, Marco; Tilz, Gernot P

    2011-05-01

    Microarray analysis makes it possible to determine thousands of gene expression values simultaneously. Changes in gene expression, as a response to diseases, can be detected allowing a better understanding and differentiation of diseases at a molecular level. By comparing different kinds of tissue, for example healthy tissue and cancer tissue, the microarray analysis indicates induced gene activity, repressed gene activity or when there is no change in the gene activity level. Fundamental patterns in gene expression are extracted by several clustering and machine learning algorithms. Certain kinds of cancer can be divided into subtypes, with different clinical outcomes, by their specific gene expression patterns. This enables a better diagnosis and tailoring of individual patient treatments.

  16. Hybridization chain reaction amplification for highly sensitive fluorescence detection of DNA with dextran coated microarrays.

    PubMed

    Chao, Jie; Li, Zhenhua; Li, Jing; Peng, Hongzhen; Su, Shao; Li, Qian; Zhu, Changfeng; Zuo, Xiaolei; Song, Shiping; Wang, Lianhui; Wang, Lihua

    2016-07-15

    Microarrays of biomolecules hold great promise in the fields of genomics, proteomics, and clinical assays on account of their remarkably parallel and high-throughput assay capability. However, the fluorescence detection used in most conventional DNA microarrays is still limited by sensitivity. In this study, we have demonstrated a novel universal and highly sensitive platform for fluorescent detection of sequence specific DNA at the femtomolar level by combining dextran-coated microarrays with hybridization chain reaction (HCR) signal amplification. Three-dimensional dextran matrix was covalently coated on glass surface as the scaffold to immobilize DNA recognition probes to increase the surface binding capacity and accessibility. DNA nanowire tentacles were formed on the matrix surface for efficient signal amplification by capturing multiple fluorescent molecules in a highly ordered way. By quantifying microscopic fluorescent signals, the synergetic effects of dextran and HCR greatly improved sensitivity of DNA microarrays, with a detection limit of 10fM (1×10(5) molecules). This detection assay could recognize one-base mismatch with fluorescence signals dropped down to ~20%. This cost-effective microarray platform also worked well with samples in serum and thus shows great potential for clinical diagnosis.

  17. DNA Microarray Detection of 18 Important Human Blood Protozoan Species.

    PubMed

    Chen, Mu-Xin; Ai, Lin; Chen, Jun-Hu; Feng, Xin-Yu; Chen, Shao-Hong; Cai, Yu-Chun; Lu, Yan; Zhou, Xiao-Nong; Chen, Jia-Xu; Hu, Wei

    2016-12-01

    Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species.

  18. DNA Microarray Detection of 18 Important Human Blood Protozoan Species

    PubMed Central

    Chen, Jun-Hu; Feng, Xin-Yu; Chen, Shao-Hong; Cai, Yu-Chun; Lu, Yan; Zhou, Xiao-Nong; Chen, Jia-Xu; Hu, Wei

    2016-01-01

    Background Accurate detection of blood protozoa from clinical samples is important for diagnosis, treatment and control of related diseases. In this preliminary study, a novel DNA microarray system was assessed for the detection of Plasmodium, Leishmania, Trypanosoma, Toxoplasma gondii and Babesia in humans, animals, and vectors, in comparison with microscopy and PCR data. Developing a rapid, simple, and convenient detection method for protozoan detection is an urgent need. Methodology/Principal Findings The microarray assay simultaneously identified 18 species of common blood protozoa based on the differences in respective target genes. A total of 20 specific primer pairs and 107 microarray probes were selected according to conserved regions which were designed to identify 18 species in 5 blood protozoan genera. The positive detection rate of the microarray assay was 91.78% (402/438). Sensitivity and specificity for blood protozoan detection ranged from 82.4% (95%CI: 65.9% ~ 98.8%) to 100.0% and 95.1% (95%CI: 93.2% ~ 97.0%) to 100.0%, respectively. Positive predictive value (PPV) and negative predictive value (NPV) ranged from 20.0% (95%CI: 2.5% ~ 37.5%) to 100.0% and 96.8% (95%CI: 95.0% ~ 98.6%) to 100.0%, respectively. Youden index varied from 0.82 to 0.98. The detection limit of the DNA microarrays ranged from 200 to 500 copies/reaction, similar to PCR findings. The concordance rate between microarray data and DNA sequencing results was 100%. Conclusions/Significance Overall, the newly developed microarray platform provides a convenient, highly accurate, and reliable clinical assay for the determination of blood protozoan species. PMID:27911895

  19. Performance comparison of SLFN training algorithms for DNA microarray classification.

    PubMed

    Huynh, Hieu Trung; Kim, Jung-Ja; Won, Yonggwan

    2011-01-01

    The classification of biological samples measured by DNA microarrays has been a major topic of interest in the last decade, and several approaches to this topic have been investigated. However, till now, classifying the high-dimensional data of microarrays still presents a challenge to researchers. In this chapter, we focus on evaluating the performance of the training algorithms of the single hidden layer feedforward neural networks (SLFNs) to classify DNA microarrays. The training algorithms consist of backpropagation (BP), extreme learning machine (ELM) and regularized least squares ELM (RLS-ELM), and an effective algorithm called neural-SVD has recently been proposed. We also compare the performance of the neural network approaches with popular classifiers such as support vector machine (SVM), principle component analysis (PCA) and fisher discriminant analysis (FDA).

  20. Development of DNA Microarrays for Metabolic Pathway and Bioprocess Monitoring

    SciTech Connect

    Gregory Stephanopoulos

    2004-07-31

    Transcriptional profiling experiments utilizing DNA microarrays to study the intracellular accumulation of PHB in Synechocystis has proved difficult in large part because strains that show significant differences in PHB which would justify global analysis of gene expression have not been isolated.

  1. 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

  2. Application of click chemistry to the production of DNA microarrays.

    PubMed

    Uszczyńska, Barbara; Ratajczak, Tomasz; Frydrych, Emilia; Maciejewski, Hieronim; Figlerowicz, Marek; Markiewicz, Wojciech T; Chmielewski, Marcin K

    2012-03-21

    The copper-catalyzed alkyne-azide cycloaddition (CuAAC) reaction was applied as the novel method of DNA immobilization on a modified solid support. The CuAAC click reaction enables the covalent binding of DNA modified with pentynyl groups at its 5'-end to azide-loaded slides. Click microarrays were produced using this approach and successfully employed in biological/model experiments.

  3. PNA microarrays for hybridisation of unlabelled DNA samples

    PubMed Central

    Brandt, Ole; Feldner, Julia; Stephan, Achim; Schröder, Markus; Schnölzer, Martina; Arlinghaus, Heinrich F.; Hoheisel, Jörg D.; Jacob, Anette

    2003-01-01

    Several strategies have been developed for the production of peptide nucleic acid (PNA) microarrays by parallel probe synthesis and selective coupling of full-length molecules. Such microarrays were used for direct detection of the hybridisation of unlabelled DNA by time-of-flight secondary ion mass spectrometry. PNAs were synthesised by an automated process on filter-bottom microtitre plates. The resulting molecules were released from the solid support and attached without any purification to microarray surfaces via the terminal amino group itself or via modifications, which had been chemically introduced during synthesis. Thus, only full-length PNA oligomers were attached whereas truncated molecules, produced during synthesis because of incomplete condensation reactions, did not bind. Different surface chemistries and fitting modifications of the PNA terminus were tested. For an examination of coupling selectivity, bound PNAs were cleaved off microarray surfaces and analysed by MALDI-TOF mass spectrometry. Additionally, hybridisation experiments were performed to compare the attachment chemistries, with fully acetylated PNAs spotted as controls. Upon hybridisation of unlabelled DNA to such microarrays, binding events could be detected by visualisation of phosphates, which are an integral part of nucleic acids but missing entirely in PNA probes. Overall best results in terms of selectivity and sensitivity were obtained with thiol-modified PNAs on maleimide surfaces. PMID:14500847

  4. Unravelling Microbial Communities with DNA-Microarrays: Challengesand Future Directions.

    SciTech Connect

    Wagner, Michael; Smidt, Hauke; Loy, Alexander; Zhou, Jizhong

    2007-03-08

    High-throughput technologies are urgently needed formonitoring the formidable biodiversity and functional capabilities ofmicroorganisms in the environment. Ten years ago, DNA microarrays,miniaturized platforms for highly parallel hybridization reactions, foundtheir way into environmental microbiology and raised great expectationsamong researchers in the field. In this article, we briefly summarize thestate-of-the-art of microarray approaches in microbial ecology researchand discuss in more detail crucial problems and promising solutions.Finally, we outline scenarios for an innovative combination ofmicroarrays with other molecular tools for structure-function analysis ofcomplex microbial communities.

  5. A Customized DNA Microarray for Microbial Source Tracking ...

    EPA Pesticide Factsheets

    It is estimated that more than 160, 000 miles of rivers and streams in the United States are impaired due to the presence of waterborne pathogens. These pathogens typically originate from human and other animal fecal pollution sources; therefore, a rapid microbial source tracking (MST) method is needed to facilitate water quality assessment and impaired water remediation. We report a novel qualitative DNA microarray technology consisting of 453 probes for the detection of general fecal and host-associated bacteria, viruses, antibiotic resistance, and other environmentally relevant genetic indicators. A novel data normalization and reduction approach is also presented to help alleviate false positives often associated with high-density microarray applications. To evaluate the performance of the approach, DNA and cDNA was isolated from swine, cattle, duck, goose and gull fecal reference samples, as well as soiled poultry liter and raw municipal sewage. Based on nonmetric multidimensional scaling analysis of results, findings suggest that the novel microarray approach may be useful for pathogen detection and identification of fecal contamination in recreational waters. The ability to simultaneously detect a large collection of environmentally important genetic indicators in a single test has the potential to provide water quality managers with a wide range of information in a short period of time. Future research is warranted to measure microarray performance i

  6. A Customized DNA Microarray for Microbial Source Tracking ...

    EPA Pesticide Factsheets

    It is estimated that more than 160, 000 miles of rivers and streams in the United States are impaired due to the presence of waterborne pathogens. These pathogens typically originate from human and other animal fecal pollution sources; therefore, a rapid microbial source tracking (MST) method is needed to facilitate water quality assessment and impaired water remediation. We report a novel qualitative DNA microarray technology consisting of 453 probes for the detection of general fecal and host-associated bacteria, viruses, antibiotic resistance, and other environmentally relevant genetic indicators. A novel data normalization and reduction approach is also presented to help alleviate false positives often associated with high-density microarray applications. To evaluate the performance of the approach, DNA and cDNA was isolated from swine, cattle, duck, goose and gull fecal reference samples, as well as soiled poultry liter and raw municipal sewage. Based on nonmetric multidimensional scaling analysis of results, findings suggest that the novel microarray approach may be useful for pathogen detection and identification of fecal contamination in recreational waters. The ability to simultaneously detect a large collection of environmentally important genetic indicators in a single test has the potential to provide water quality managers with a wide range of information in a short period of time. Future research is warranted to measure microarray performance i

  7. 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.

  8. Do DNA Microarrays Tell the Story of Gene Expression?

    PubMed Central

    Rosenfeld, Simon

    2010-01-01

    Poor reproducibility of microarray measurements is a major obstacle to their application as an instrument for clinical diagnostics. In this paper, several aspects of poor reproducibility are analyzed. All of them belong to the category of interpretive weaknesses of DNA microarray technology. First, the attention is drawn to the fact that absence of the information regarding post-transcriptional mRNA stability makes it impossible to evaluate the level of gene activity from the relative mRNA abundances, the quantities available from microarray measurements. Second, irreducible intracellular variability with persistent patterns of stochasticity and burstiness put natural limits to reproducibility. Third, strong interactions within intracellular biomolecular networks make it highly problematic to build a bridge between transcription rates of individual genes and structural fidelity of their genetic codes. For these reasons, the microarray measurements of relative mRNA abundances are more appropriate in laboratory settings as a tool for scientific research, hypotheses generating and producing the leads for subsequent validation through more sophisticated technologies. As to clinical settings, where firm conclusive diagnoses, not the leads for further experimentation, are required, microarrays still have a long way to go until they become a reliable instrument in patient-related decision making. PMID:20628535

  9. Using DNA Microarrays to Detect Multiple Pathogen Threats in Water.

    SciTech Connect

    Straub, Tim M.; Quinonez-Diaz, Maria D.; Valdez, Catherine O.; Call, Douglas R.; Chandler, Darrell P.

    2004-06-01

    Currently, there is no single method to collect, process, and analyze a water sample for all pathogenic microorganisms of interest. Some of the difficulties in developing a universal method include the physical differences between the major pathogen groups (viruses, bacteria, protozoa), efficiently concentrating large volume water samples to detect low target concentrations of certain pathogen groups, removing co-concentrated inhibitors from the sample, and standardizing a culture-independent endpoint detection method. Integrating the disparate technologies into a single, universal, simple method and detection system would represent a significant advance in public health and microbiological water quality analysis. Recent advances in sample collection, on-line sample processing and purification, and DNA microarray technologies may form the basis of a universal method to detect known and emerging waterborne pathogens. This review discusses some of the challenges in developing a universal pathogen detection method, current technology that may be employed to overcome these challenges, and the remaining needs for developing an integrated pathogen detection and monitoring system for source or finished water.

  10. Electrostatic readout of DNA microarrays with charged microspheres

    PubMed Central

    Clack, Nathan G; Salaita, Khalid; Groves, Jay T

    2014-01-01

    DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis1–3. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care4,5. Here, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-μm lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays. PMID:18587384

  11. Electrostatic readout of DNA microarrays with charged microspheres

    SciTech Connect

    Clack, Nathan G.; Salaita, Khalid; Groves, Jay T.

    2008-06-29

    DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care. In this paper, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-μm lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Lastly, because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays.

  12. 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.

  13. Viral Discovery and Sequence Recovery Using DNA Microarrays

    PubMed Central

    Wang, David; Urisman, Anatoly; Liu, Yu-Tsueng; Springer, Michael; Ksiazek, Thomas G; Erdman, Dean D; Mardis, Elaine R; Hickenbotham, Matthew; Magrini, Vincent; Eldred, James; Latreille, J. Phillipe; Wilson, Richard K; Ganem, Don

    2003-01-01

    Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease. PMID:14624234

  14. 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.

  15. Selective immobilization and detection of DNA on biopolymer supports for the design of microarrays.

    PubMed

    Kargl, R; Vorraber, V; Ribitsch, V; Köstler, S; Stana-Kleinschek, K; Mohan, T

    2015-06-15

    DNA immobilization for the manufacturing of microarrays requires sufficient probe density, low unspecific binding and high interaction efficiency with complementary strands that are detected from solutions. Many of these important parameters are affected by the surface chemistry and the blocking steps conducted during DNA spotting and hybridization. This work describes an alternative method to selectively immobilize probes and to detect DNA on biocompatible, hydrophilic cellulose coated supports with low unspecific binding, high selectivity and appropriate sensitivity. It takes advantage of a relatively selective adsorption of water soluble polysaccharides on a solid cellulose matrix. Single strands of DNA were conjugated to this soluble polysaccharide and subsequently micro-spotted on solid cellulose thin films that were coated on glass and polymer slides. This resulted in adsorptively bound DNA-probes that were used to detect complementary, labelled DNA strands with different lengths and sequences by hybridization. The interaction of the DNA-conjugates with cellulose surfaces and the selectivity of hybridization were investigated by a quartz crystal microbalance with dissipation monitoring (QCM-D) and fluorescence scanning. The method of non-covalent immobilization of DNA probes on an uncharged, non-reactive, hydrophilic support lowers the unspecific binding and the number of handling steps required to conduct the experiments for the detection of DNA on microarrays. Simultaneously selectivity, hybridization efficiency and detection limits are maintained. Copyright © 2015 Elsevier B.V. All rights reserved.

  16. Independent component analysis of Alzheimer's DNA microarray gene expression data

    PubMed Central

    Kong, Wei; Mou, Xiaoyang; Liu, Qingzhong; Chen, Zhongxue; Vanderburg, Charles R; Rogers, Jack T; Huang, Xudong

    2009-01-01

    Background Gene microarray technology is an effective tool to investigate the simultaneous activity of multiple cellular pathways from hundreds to thousands of genes. However, because data in the colossal amounts generated by DNA microarray technology are usually complex, noisy, high-dimensional, and often hindered by low statistical power, their exploitation is difficult. To overcome these problems, two kinds of unsupervised analysis methods for microarray data: principal component analysis (PCA) and independent component analysis (ICA) have been developed to accomplish the task. PCA projects the data into a new space spanned by the principal components that are mutually orthonormal to each other. The constraint of mutual orthogonality and second-order statistics technique within PCA algorithms, however, may not be applied to the biological systems studied. Extracting and characterizing the most informative features of the biological signals, however, require higher-order statistics. Results ICA is one of the unsupervised algorithms that can extract higher-order statistical structures from data and has been applied to DNA microarray gene expression data analysis. We performed FastICA method on DNA microarray gene expression data from Alzheimer's disease (AD) hippocampal tissue samples and consequential gene clustering. Experimental results showed that the ICA method can improve the clustering results of AD samples and identify significant genes. More than 50 significant genes with high expression levels in severe AD were extracted, representing immunity-related protein, metal-related protein, membrane protein, lipoprotein, neuropeptide, cytoskeleton protein, cellular binding protein, and ribosomal protein. Within the aforementioned categories, our method also found 37 significant genes with low expression levels. Moreover, it is worth noting that some oncogenes and phosphorylation-related proteins are expressed in low levels. In comparison to the PCA and support

  17. Scanometric analysis of DNA microarrays using DNA intercalator-conjugated gold nanoparticles.

    PubMed

    Cho, Hyunmin; Jung, Juyeon; Chung, Bong Hyun

    2012-08-07

    We introduce a scanometric detection method for the analysis of DNA microarrays using DNA intercalator-conjugated gold nanoparticles that can be analyzed with the naked eye or with an optical scanner after the enhancement of the AuNPs. Moreover, we successfully detected a hemagglutinin-subtyping DNA array using this method.

  18. DNA Microarray Technologies: A Novel Approach to Geonomic Research

    SciTech Connect

    Hinman, R.; Thrall, B.; Wong, K,

    2002-01-01

    A cDNA microarray allows biologists to examine the expression of thousands of genes simultaneously. Researchers may analyze the complete transcriptional program of an organism in response to specific physiological or developmental conditions. By design, a cDNA microarray is an experiment with many variables and few controls. One question that inevitably arises when working with a cDNA microarray is data reproducibility. How easy is it to confirm mRNA expression patterns? In this paper, a case study involving the treatment of a murine macrophage RAW 264.7 cell line with tumor necrosis factor alpha (TNF) was used to obtain a rough estimate of data reproducibility. Two trials were examined and a list of genes displaying either a > 2-fold or > 4-fold increase in gene expression was compiled. Variations in signal mean ratios between the two slides were observed. We can assume that erring in reproducibility may be compensated by greater inductive levels of similar genes. Steps taken to obtain results included serum starvation of cells before treatment, tests of mRNA for quality/consistency, and data normalization.

  19. 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

  20. DNA Microarray-Based PCR Ribotyping of Clostridium difficile

    PubMed Central

    Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2014-01-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. PMID:25411174

  1. DNA microarray-based PCR ribotyping of Clostridium difficile.

    PubMed

    Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2015-02-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray.

  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. Shrink-induced silica multiscale structures for enhanced fluorescence from DNA microarrays.

    PubMed

    Sharma, Himanshu; Wood, Jennifer B; Lin, Sophia; Corn, Robert M; Khine, Michelle

    2014-09-23

    We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30-45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays.

  4. DNA microarrays: translation of the genome from laboratory to clinic.

    PubMed

    Geschwind, Daniel H

    2003-05-01

    As the complete sequences of human and other mammalian genomes become available we are faced with the challenge of understanding how variation in sequence and gene expression contributes to neurological and psychiatric disorders. DNA microarrays, or DNA chips, provide the means to measure simultaneously where and when thousands of genes are expressed. Microarrays are changing the way that researchers approach work at the bench and have already yielded new insights into brain tumours, multiple sclerosis, acute neurological insults such as stroke and seizures, and schizophrenia. The study of disease-related changes in gene expression is the first step in the long process in translation of genome research to the clinic. Eventually, the changes observed in microarray studies will need to be independently confirmed and we wil need to understand how gene expression changes translate into functional effects at the cellular level in the nervous system. Progress in these studies will translate into array-based disease classification schemes and help optimise therapy for individual patients based on gene expression patterns or their genetic background.

  5. Recognition of multiple imbalanced cancer types based on DNA microarray data using ensemble classifiers.

    PubMed

    Yu, Hualong; Hong, Shufang; Yang, Xibei; Ni, Jun; Dan, Yuanyuan; Qin, Bin

    2013-01-01

    DNA microarray technology can measure the activities of tens of thousands of genes simultaneously, which provides an efficient way to diagnose cancer at the molecular level. Although this strategy has attracted significant research attention, most studies neglect an important problem, namely, that most DNA microarray datasets are skewed, which causes traditional learning algorithms to produce inaccurate results. Some studies have considered this problem, yet they merely focus on binary-class problem. In this paper, we dealt with multiclass imbalanced classification problem, as encountered in cancer DNA microarray, by using ensemble learning. We utilized one-against-all coding strategy to transform multiclass to multiple binary classes, each of them carrying out feature subspace, which is an evolving version of random subspace that generates multiple diverse training subsets. Next, we introduced one of two different correction technologies, namely, decision threshold adjustment or random undersampling, into each training subset to alleviate the damage of class imbalance. Specifically, support vector machine was used as base classifier, and a novel voting rule called counter voting was presented for making a final decision. Experimental results on eight skewed multiclass cancer microarray datasets indicate that unlike many traditional classification approaches, our methods are insensitive to class imbalance.

  6. Design of a combinatorial dna microarray for protein-dnainteraction studies

    SciTech Connect

    Mintseris, Julian; Eisen, Michael B.

    2006-07-07

    Background: Discovery of precise specificity oftranscription factors is an important step on the way to understandingthe complex mechanisms of gene regulation in eukaryotes. Recently,doublestranded protein-binding microarrays were developed as apotentially scalable approach to tackle transcription factor binding siteidentification. Results: Here we present an algorithmic approach toexperimental design of a microarray that allows for testing fullspecificity of a transcription factor binding to all possible DNA bindingsites of a given length, with optimally efficient use of the array. Thisdesign is universal, works for any factor that binds a sequence motif andis not species-specific. Furthermore, simulation results show that dataproduced with the designed arrays is easier to analyze and would resultin more precise identification of binding sites. Conclusion: In thisstudy, we present a design of a double stranded DNA microarray forprotein-DNA interaction studies and show that our algorithm allowsoptimally efficient use of the arrays for this purpose. We believe such adesign will prove useful for transcription factor binding siteidentification and other biological problems.

  7. Monitoring of dnaK gene expression in Porphyromonas gingivalis by oxygen stress using DNA microarray.

    PubMed

    Araki, Makoto; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Abiko, Yoshimitsu

    2004-06-01

    Porphyromonas gingivalis, a Gram-negative anaerobe associated with adult periodontitis, expresses numerous potential virulence factors. dnaK, a member of the heat shock protein family, functions as a molecular chaperone and plays a role in microbial pathogenicity. However, little is known regarding its gene expression caused by oxygen stress in P. gingivalis. In the present study, a custom-made DNA microarray was designed and used to monitor dnaK gene expression in P. gingivalis caused by oxygen stress. The results demonstrated that dnaK mRNA was up-regulated in a short time, and the DNA microarray results were confirmed by real-time polymerase chain reaction analysis. These findings suggest that oxygen stress stimulates gene expression of dnaK and may have a relationship to the aerotolerance activity of this organism as well as its expression of pathogenesis.

  8. DNA Microarray Wet Lab Simulation Brings Genomics into the High School Curriculum

    ERIC Educational Resources Information Center

    Campbell, A. Malcolm; Zanta, Carolyn A.; Heyer, Laurie J.; Kittinger, Ben; Gabric, Kathleen M.; Adler, Leslie

    2006-01-01

    We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH…

  9. DNA Microarray Wet Lab Simulation Brings Genomics into the High School Curriculum

    ERIC Educational Resources Information Center

    Campbell, A. Malcolm; Zanta, Carolyn A.; Heyer, Laurie J.; Kittinger, Ben; Gabric, Kathleen M.; Adler, Leslie

    2006-01-01

    We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH…

  10. [Software development in data analysis and mining for cDNA microarray].

    PubMed

    Wu, Bin; Wang, Jianguo; Wang, Miqu

    2007-12-01

    Data analysis and mining is a key issue to microarray technology and is usually implemented through software development. This paper summarizes the state-of-art software development in cDNA microarray data analysis and mining. The updated software developments are discussed in three stages: data inquisition from cDNA microarray tests, statistical treatment of cDNA data and data mining from gene network.

  11. 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.

  12. Feature extraction and signal processing for nylon DNA microarrays

    PubMed Central

    Lopez, F; Rougemont, J; Loriod, B; Bourgeois, A; Loï, L; Bertucci, F; Hingamp, P; Houlgatte, R; Granjeaud, S

    2004-01-01

    Background High-density DNA microarrays require automatic feature extraction methodologies and softwares. These can be a potential source of non-reproducibility of gene expression measurements. Variation in feature location or in signal integration methodology may be a significant contribution to the observed variance in gene expression levels. Results We explore sources of variability in feature extraction from DNA microarrays on Nylon membrane with radioactive detection. We introduce a mathematical model of the signal emission and derive methods for correcting biases such as overshining, saturation or variation in probe amount. We also provide a quality metric which can be used qualitatively to flag weak or untrusted signals or quantitatively to modulate the weight of each experiment or gene in higher level analyses (clustering or discriminant analysis). Conclusions Our novel feature extraction methodology, based on a mathematical model of the radioactive emission, reduces variability due to saturation, neighbourhood effects and variable probe amount. Furthermore, we provide a fully automatic feature extraction software, BZScan, which implements the algorithms described in this paper. PMID:15222896

  13. DNA Microarray Data Analysis: A Novel Biclustering Algorithm Approach

    NASA Astrophysics Data System (ADS)

    Tchagang, Alain B.; Tewfik, Ahmed H.

    2006-12-01

    Biclustering algorithms refer to a distinct class of clustering algorithms that perform simultaneous row-column clustering. Biclustering problems arise in DNA microarray data analysis, collaborative filtering, market research, information retrieval, text mining, electoral trends, exchange analysis, and so forth. When dealing with DNA microarray experimental data for example, the goal of biclustering algorithms is to find submatrices, that is, subgroups of genes and subgroups of conditions, where the genes exhibit highly correlated activities for every condition. In this study, we develop novel biclustering algorithms using basic linear algebra and arithmetic tools. The proposed biclustering algorithms can be used to search for all biclusters with constant values, biclusters with constant values on rows, biclusters with constant values on columns, and biclusters with coherent values from a set of data in a timely manner and without solving any optimization problem. We also show how one of the proposed biclustering algorithms can be adapted to identify biclusters with coherent evolution. The algorithms developed in this study discover all valid biclusters of each type, while almost all previous biclustering approaches will miss some.

  14. Bacterial DNA microarrays for clinical microbiology: the early logarithmic phase.

    PubMed

    Cassone, Marco; Giordano, Antonio; Pozzi, Gianni

    2007-01-01

    In this era of coexistence of high-throughput sequencing technologies and serious difficulties in the management of both common and novel infectious syndromes, new techniques which improve the study of micro-organisms is timely. In bacteriology, the most important subjects are bacterial pathogenicity, discovery of the genomic complexity of bacteria, and the epidemiology of antimicrobial resistance traits. From the clinical point of view, genetic testing is flanking phenotypic testing for the assessment of new, difficult to test antibiotic resistance traits, and for correlations with the microbial behaviour in vivo. The demand for faster, comprehensive and highly parallel microbial diagnostics is also cogent even at the basic laboratory level, where the ultimate objective is saving lives. In this setting, DNA microarrays offer a pivotal contribution by allowing performance of hybridization experiments in highly parallel formats, with an increasing reliability. Not only they are useful in deciphering host and microbial pathophysiology, they can also make the difference in the management of prognostic and therapeutic aspects of many diseases. Here, we provide an overview of the current use and the potential of DNA microarrays in clinical bacteriology, and several applications and technical solutions are discussed.

  15. [DNA microarrays (DNA chips) used in molecular medical research].

    PubMed

    Ørntoft, Torben F

    2003-02-17

    Microchip technology and mapping of the human genome has led to the invention of DNA-micro-array technology. In that technology thousands of genes are analysed in parallel on a small glass surface. The very large data material that is generated requires bioinformatic analysis in order to be transformed to understandable biological results. There are different forms of DNA-micro-arrays. Some are used for measuring gene expression, i.e. which genes are turned on and which genes are turned off. Others are used for sequencing, and finally some are used for measuring single nucleotide polymorphisms (SNPs). Some application possibilities of the arrays within the area of cell biology and classification of diseases are presented. Especially within the area of classification of cancer diseases the technology is promising, and a scientific breakthrough could occur in the area of classification of very aggressive cancers.

  16. Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

    PubMed Central

    Bulyk, Martha L.; Huang, Xiaohua; Choo, Yen; Church, George M.

    2001-01-01

    A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites. PMID:11404456

  17. Design issues in toxicogenomics using DNA microarray experiment

    SciTech Connect

    Lee, Kyoung-Mu; Kim, Ju-Han; Kang, Daehee . E-mail: dhkang@snu.ac.kr

    2005-09-01

    The methods of toxicogenomics might be classified into omics study (e.g., genomics, proteomics, and metabolomics) and population study focusing on risk assessment and gene-environment interaction. In omics study, microarray is the most popular approach. Genes falling into several categories (e.g., xenobiotics metabolism, cell cycle control, DNA repair etc.) can be selected up to 20,000 according to a priori hypothesis. The appropriate type of samples and species should be selected in advance. Multiple doses and varied exposure durations are suggested to identify those genes clearly linked to toxic response. Microarray experiments can be affected by numerous nuisance variables including experimental designs, sample extraction, type of scanners, etc. The number of slides might be determined from the magnitude and variance of expression change, false-positive rate, and desired power. Instead, pooling samples is an alternative. Online databases on chemicals with known exposure-disease outcomes and genetic information can aid the interpretation of the normalized results. Gene function can be inferred from microarray data analyzed by bioinformatics methods such as cluster analysis. The population study often adopts hospital-based or nested case-control design. Biases in subject selection and exposure assessment should be minimized, and confounding bias should also be controlled for in stratified or multiple regression analysis. Optimal sample sizes are dependent on the statistical test for gene-to-environment or gene-to-gene interaction. The design issues addressed in this mini-review are crucial in conducting toxicogenomics study. In addition, integrative approach of exposure assessment, epidemiology, and clinical trial is required.

  18. 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

  19. DNA microarrays for comparative genomic hybridization based on DOP-PCR amplification of BAC and PAC clones.

    PubMed

    Fiegler, Heike; Carr, Philippa; Douglas, Eleanor J; Burford, Deborah C; Hunt, Sarah; Scott, Carol E; Smith, James; Vetrie, David; Gorman, Patricia; Tomlinson, Ian P M; Carter, Nigel P

    2003-04-01

    We have designed DOP-PCR primers specifically for the amplification of large insert clones for use in the construction of DNA microarrays. A bioinformatic approach was used to construct primers that were efficient in the general amplification of human DNA but were poor at amplifying E. coli DNA, a common contaminant of DNA preparations from large insert clones. We chose the three most selective primers for use in printing DNA microarrays. DNA combined from the amplification of large insert clones by use of these three primers and spotted onto glass slides showed more than a sixfold increase in the human to E. coli hybridization ratio when compared to the standard DOP-PCR primer, 6MW. The microarrays reproducibly delineated previously characterized gains and deletions in a cancer cell line and identified a small gain not detected by use of conventional CGH. We also describe a method for the bulk testing of the hybridization characteristics of chromosome-specific clones spotted on microarrays by use of DNA amplified from flow-sorted chromosomes. Finally, we describe a set of clones selected from the publicly available Golden Path of the human genome at 1-Mb intervals and a view in the Ensembl genome browser from which data required for the use of these clones in array CGH and other experiments can be downloaded across the Internet.

  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. Fabrication of high quality cDNA microarray using a small amount of cDNA.

    PubMed

    Park, Chan Hee; Jeong, Ha Jin; Jung, Jae Jun; Lee, Gui Yeon; Kim, Sang-Chul; Kim, Tae Soo; Yang, Sang Hwa; Chung, Hyun Cheol; Rha, Sun Young

    2004-05-01

    DNA microarray technology has become an essential part of biological research. It enables the genome-scale analysis of gene expression in various types of model systems. Manufacturing high quality cDNA microarrays of microdeposition type depends on some key factors including a printing device, spotting pins, glass slides, spotting solution, and humidity during spotting. UsingEthe Microgrid II TAS model printing device, this study defined the optimal conditions for producing high density, high quality cDNA microarrays with the least amount of cDNA product. It was observed that aminosilane-modified slides were superior to other types of surface modified-slides. A humidity of 30+/-3% in a closed environment and the overnight drying of the spotted slides gave the best conditions for arraying. In addition, the cDNA dissolved in 30% DMSO gave the optimal conditions for spotting compared to the 1X ArrayIt, 3X SSC and 50% DMSO. Lastly, cDNA in the concentration range of 100-300 ng/ micro l was determined to be best for arraying and post-processing. Currently, the printing system in this study yields reproducible 9000 spots with a spot size 150 mm diameter, and a 200 nm spot spacing.

  2. Analysis of Mycobacterium leprae gene expression using DNA microarray.

    PubMed

    Akama, Takeshi; Tanigawa, Kazunari; Kawashima, Akira; Wu, Huhehasi; Ishii, Norihisa; Suzuki, Koichi

    2010-10-01

    Mycobacterium leprae, the causative agent of leprosy, does not grow under in vitro condition, making molecular analysis of this bacterium difficult. For this reason, bacteriological information regarding M. leprae gene function is limited compared with other mycobacterium species. In this study, we performed DNA microarray analysis to clarify the RNA expression profile of the Thai53 strain of M. leprae grown in footpads of hypertensive nude rats (SHR/NCrj-rnu). Of 1605 M. leprae genes, 315 showed signal intensity twofold higher than the median. These genes include Acyl-CoA metabolic enzymes and drug metabolic enzymes, which might be related to the virulence of M. leprae. In addition, consecutive RNA expression profile and in silico analyses enabled identification of possible operons within the M. leprae genome. The present results will shed light on M. leprae gene function and further our understanding of the pathogenesis of leprosy.

  3. 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

  4. Fully integrated miniature device for automated gene expression DNA microarray processing.

    PubMed

    Liu, Robin Hui; Nguyen, Tai; Schwarzkopf, Kevin; Fuji, H Sho; Petrova, Alla; Siuda, Tony; Peyvan, Kia; Bizak, Michael; Danley, David; McShea, Andy

    2006-03-15

    A DNA microarray with 12,000 features was integrated with a microfluidic cartridge to automate the fluidic handling steps required to carry out a gene expression study of the human leukemia cell line (K562). The fully integrated microfluidic device consists of microfluidic pumps/mixers, fluid channels, reagent chambers, and a DNA microarray silicon chip. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated into the cartridge to provide pumping of liquid solutions. The device was completely self-contained: no external pressure sources, fluid storage, mechanical pumps, mixers, or valves were necessary for fluid manipulation, thus eliminating possible sample contamination and simplifying device operation. Fluidic experiments were performed to study the on-chip washing efficiency and uniformity. A single-color transcriptional analysis of K562 cells with a series of calibration controls (spiked-in controls) to characterize this new platform with regard to sensitivity, specificity, and dynamic range was performed. The device detected sample RNAs with a concentration as low as 0.375 pM. Experiment also showed that the performance of the integrated microfluidic device is comparable with the conventional hybridization chambers with manual operations, indicating that the on-chip fluidic handling (washing and reaction) is highly efficient and can be automated with no loss of performance. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps in genomic analysis.

  5. Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays.

    PubMed

    von der Haar, Marcel; Heuer, Christopher; Pähler, Martin; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2016-11-30

    The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores' susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs.

  6. Optimization of Cyanine Dye Stability and Analysis of FRET Interaction on DNA Microarrays

    PubMed Central

    von der Haar, Marcel; Heuer, Christopher; Pähler, Martin; von der Haar, Kathrin; Lindner, Patrick; Scheper, Thomas; Stahl, Frank

    2016-01-01

    The application of DNA microarrays for high throughput analysis of genetic regulation is often limited by the fluorophores used as markers. The implementation of multi-scan techniques is limited by the fluorophores’ susceptibility to photobleaching when exposed to the scanner laser light. This paper presents combined mechanical and chemical strategies which enhance the photostability of cyanine 3 and cyanine 5 as part of solid state DNA microarrays. These strategies are based on scanning the microarrays while the hybridized DNA is still in an aqueous solution with the presence of a reductive/oxidative system (ROXS). Furthermore, the experimental setup allows for the analysis and eventual normalization of Förster-resonance-energy-transfer (FRET) interaction of cyanine-3/cyanine-5 dye combinations on the microarray. These findings constitute a step towards standardization of microarray experiments and analysis and may help to increase the comparability of microarray experiment results between labs. PMID:27916881

  7. Reduction of autofluorescence on DNA microarrays and slide surfaces by treatment with sodium borohydride.

    PubMed

    Raghavachari, Nalini; Bao, Yijia P; Li, Guangshan; Xie, Xinying; Müller, Uwe R

    2003-01-15

    Microarray technology is currently being used extensively in functional genomics research and modern drug discovery and development. Henceforward, tremendous application potential for this technology exists in the fields of clinical diagnostics and prognostics, pathology, and toxicology for high-throughput analysis of "disease" gene expression. However, the major hurdle now in this technology is not the performance of the arrays but rather the efficient reproducibility of the hybridization signal intensity in a fluorescence-based analysis. The sensitivity of fluorescence detection on an array is to a large extent limited by the amount of background signal arising due to nonspecifically bound probes and fluorescence that is intrinsically associated with the chip substrate and/or the attached target DNA, the so-called autofluorescence. Here, we describe a simple and efficient method to reduce autofluorescence from undetermined sources on coated glass slides with and without DNA arrays. This sodium borohydride-mediated reduction process resulted in significantly lower and more even background fluorescence. This in turn extended the dynamic range of detection and reduced the average coefficient of variation of fluorescent signal ratios on DNA microarrays in addition to improving the detection of genes that are expressed at a low level.

  8. E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.

    PubMed

    Urisman, Anatoly; Fischer, Kael F; Chiu, Charles Y; Kistler, Amy L; Beck, Shoshannah; Wang, David; DeRisi, Joseph L

    2005-01-01

    DNA microarrays may be used to identify microbial species present in environmental and clinical samples. However, automated tools for reliable species identification based on observed microarray hybridization patterns are lacking. We present an algorithm, E-Predict, for microarray-based species identification. E-Predict compares observed hybridization patterns with theoretical energy profiles representing different species. We demonstrate the application of the algorithm to viral detection in a set of clinical samples and discuss its relevance to other metagenomic applications.

  9. Shrink-Induced Silica Multiscale Structures for Enhanced Fluorescence from DNA Microarrays

    PubMed Central

    2015-01-01

    We describe a manufacturable and scalable method for fabrication of multiscale wrinkled silica (SiO2) structures on shrink-wrap film to enhance fluorescence signals in DNA fluorescence microarrays. We are able to enhance the fluorescence signal of hybridized DNA by more than 120 fold relative to a planar glass slide. Notably, our substrate has improved detection sensitivity (280 pM) relative to planar glass slide (11 nM). Furthermore, this is accompanied by a 30–45 times improvement in the signal-to-noise ratio (SNR). Unlike metal enhanced fluorescence (MEF) based enhancements, this is a far-field and uniform effect based on surface concentration and photophysical effects from the nano- to microscale SiO2 structures. Notably, the photophysical effects contribute an almost 2.5 fold enhancement over the concentration effects alone. Therefore, this simple and robust method offers an efficient technique to enhance the detection capabilities of fluorescence based DNA microarrays. PMID:25191785

  10. Uropathogenic Escherichia coli virulence genes: invaluable approaches for designing DNA microarray probes.

    PubMed

    Jahandeh, Nadia; Ranjbar, Reza; Behzadi, Payam; Behzadi, Elham

    2015-01-01

    The pathotypes of uropathogenic Escherichia coli (UPEC) cause different types of urinary tract infections (UTIs). The presence of a wide range of virulence genes in UPEC enables us to design appropriate DNA microarray probes. These probes, which are used in DNA microarray technology, provide us with an accurate and rapid diagnosis and definitive treatment in association with UTIs caused by UPEC pathotypes. The main goal of this article is to introduce the UPEC virulence genes as invaluable approaches for designing DNA microarray probes. Main search engines such as Google Scholar and databases like NCBI were searched to find and study several original pieces of literature, review articles, and DNA gene sequences. In parallel with in silico studies, the experiences of the authors were helpful for selecting appropriate sources and writing this review article. There is a significant variety of virulence genes among UPEC strains. The DNA sequences of virulence genes are fabulous patterns for designing microarray probes. The location of virulence genes and their sequence lengths influence the quality of probes. The use of selected virulence genes for designing microarray probes gives us a wide range of choices from which the best probe candidates can be chosen. DNA microarray technology provides us with an accurate, rapid, cost-effective, sensitive, and specific molecular diagnostic method which is facilitated by designing microarray probes. Via these tools, we are able to have an accurate diagnosis and a definitive treatment regarding UTIs caused by UPEC pathotypes.

  11. 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

  12. 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.

  13. An efficient ensemble learning method for gene microarray classification.

    PubMed

    Osareh, Alireza; Shadgar, Bita

    2013-01-01

    The gene microarray analysis and classification have demonstrated an effective way for the effective diagnosis of diseases and cancers. However, it has been also revealed that the basic classification techniques have intrinsic drawbacks in achieving accurate gene classification and cancer diagnosis. On the other hand, classifier ensembles have received increasing attention in various applications. Here, we address the gene classification issue using RotBoost ensemble methodology. This method is a combination of Rotation Forest and AdaBoost techniques which in turn preserve both desirable features of an ensemble architecture, that is, accuracy and diversity. To select a concise subset of informative genes, 5 different feature selection algorithms are considered. To assess the efficiency of the RotBoost, other nonensemble/ensemble techniques including Decision Trees, Support Vector Machines, Rotation Forest, AdaBoost, and Bagging are also deployed. Experimental results have revealed that the combination of the fast correlation-based feature selection method with ICA-based RotBoost ensemble is highly effective for gene classification. In fact, the proposed method can create ensemble classifiers which outperform not only the classifiers produced by the conventional machine learning but also the classifiers generated by two widely used conventional ensemble learning methods, that is, Bagging and AdaBoost.

  14. A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

    PubMed Central

    Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

    2012-01-01

    We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

  15. 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.

  16. Identification of conserved core xylem gene sets: conifer cDNA microarray development, transcript profiling and computational analyses.

    PubMed

    Pavy, Nathalie; Boyle, Brian; Nelson, Colleen; Paule, Charles; Giguère, Isabelle; Caron, Sébastien; Parsons, Lee S; Dallaire, Nancy; Bedon, Frank; Bérubé, Hugo; Cooke, Janice; Mackay, John

    2008-01-01

    One approach for investigating the molecular basis of wood formation is to integrate microarray profiling data sets and sequence analyses, comparing tree species with model plants such as Arabidopsis. Conifers may be included in comparative studies thanks to large-scale expressed sequence tag (EST) analyses, which enable the development of cDNA microarrays with very significant genome coverage. A microarray of 10,400 low-redundancy sequences was designed starting from white spruce (Picea glauca (Moench.) Voss) cDNAs. Computational procedures that were developed to ensure broad transcriptome coverage and efficient PCR amplification were used to select cDNA clones, which were re-sequenced in the microarray manufacture process. White spruce transcript profiling experiments that compared secondary xylem to phloem and needles identified 360 xylem-preferential gene sequences. The functional annotations of all differentially expressed sequences were highly consistent with the results of similar analyses carried out in angiosperm trees and herbaceous plants. Computational analyses comparing the spruce microarray sequences and core xylem gene sets from Arabidopsis identified 31 transcripts that were highly conserved in angiosperms and gymnosperms, in terms of both sequence and xylem expression. Several other spruce sequences have not previously been linked to xylem differentiation (including genes encoding TUBBY-like domain proteins (TLPs) and a gibberellin insensitive (gai) gene sequence) or were shown to encode proteins of unknown function encompassing diverse conserved domains of unknown function.

  17. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

    EPA Science Inventory

    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology
    Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  18. SPERM RNA AMPLIFICATION FOR GENE EXPRESSION PROFILING BY DNA MICROARRAY TECHNOLOGY

    EPA Science Inventory

    Sperm RNA Amplification for Gene Expression Profiling by DNA Microarray Technology
    Hongzu Ren, Kary E. Thompson, Judith E. Schmid and David J. Dix, Reproductive Toxicology Division, NHEERL, Office of Research and Development, US Environmental Protection Agency, Research Triang...

  19. Detecting variants with Metabolic Design, a new software tool to design probes for explorative functional DNA microarray development

    PubMed Central

    2010-01-01

    Background Microorganisms display vast diversity, and each one has its own set of genes, cell components and metabolic reactions. To assess their huge unexploited metabolic potential in different ecosystems, we need high throughput tools, such as functional microarrays, that allow the simultaneous analysis of thousands of genes. However, most classical functional microarrays use specific probes that monitor only known sequences, and so fail to cover the full microbial gene diversity present in complex environments. We have thus developed an algorithm, implemented in the user-friendly program Metabolic Design, to design efficient explorative probes. Results First we have validated our approach by studying eight enzymes involved in the degradation of polycyclic aromatic hydrocarbons from the model strain Sphingomonas paucimobilis sp. EPA505 using a designed microarray of 8,048 probes. As expected, microarray assays identified the targeted set of genes induced during biodegradation kinetics experiments with various pollutants. We have then confirmed the identity of these new genes by sequencing, and corroborated the quantitative discrimination of our microarray by quantitative real-time PCR. Finally, we have assessed metabolic capacities of microbial communities in soil contaminated with aromatic hydrocarbons. Results show that our probe design (sensitivity and explorative quality) can be used to study a complex environment efficiently. Conclusions We successfully use our microarray to detect gene expression encoding enzymes involved in polycyclic aromatic hydrocarbon degradation for the model strain. In addition, DNA microarray experiments performed on soil polluted by organic pollutants without prior sequence assumptions demonstrate high specificity and sensitivity for gene detection. Metabolic Design is thus a powerful, efficient tool that can be used to design explorative probes and monitor metabolic pathways in complex environments, and it may also be used to

  20. Compressed sensing methods for DNA microarrays, RNA interference, and metagenomics.

    PubMed

    Rao, Aditya; P, Deepthi; Renumadhavi, C H; Chandra, M Girish; Srinivasan, Rajgopal

    2015-02-01

    Compressed sensing (CS) is a sparse signal sampling methodology for efficiently acquiring and reconstructing a signal from relatively few measurements. Recent work shows that CS is well-suited to be applied to problems in genomics, including probe design in microarrays, RNA interference (RNAi), and taxonomic assignment in metagenomics. The principle of using different CS recovery methods in these applications has thus been established, but a comprehensive study of using a wide range of CS methods has not been done. For each of these applications, we apply three hitherto unused CS methods, namely, l1-magic, CoSaMP, and l1-homotopy, in conjunction with CS measurement matrices such as randomly generated CS m matrix, Hamming matrix, and projective geometry-based matrix. We find that, in RNAi, the l1-magic (the standard package for l1 minimization) and l1-homotopy methods show significant reduction in reconstruction error compared to the baseline. In metagenomics, we find that l1-homotopy as well as CoSaMP estimate concentration with significantly reduced time when compared to the GPSR and WGSQuikr methods.

  1. 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.

  2. DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii

    DTIC Science & Technology

    2004-11-15

    DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii Chien-Chung Chao Rickettsiae Diseases...TITLE AND SUBTITLE DNA Microarray Analysis of Human Monocytes Early Response Genes upon Infection with Rickettsia rickettsii 5a. CONTRACT NUMBER 5b...ANSI Std Z39-18 Rickettsiae • Gram negative coccobacillary bacteria • Obligate intracellular organisms • Arthropod-borne • Cause febrile diseases (mild

  3. Comparative analysis of amplified and nonamplified RNA for hybridization in cDNA microarray.

    PubMed

    Gomes, Luciana I; Silva, Ricardo L A; Stolf, Beatriz S; Cristo, Elier B; Hirata, Roberto; Soares, Fernando A; Reis, Luiz F L; Neves, E Jordão; Carvalho, Alex F

    2003-10-15

    Limiting amounts of RNA is a major issue in cDNA microarray, especially when one is dealing with fresh tissue samples. Here we describe a protocol based on template switch and T7 amplification that led to efficient and linear amplification of 1300x. Using a glass-array containing 368 genes printed in three or six replicas covering a wide range of expression levels and ratios, we determined quality and reproducibility of the data obtained from one nonamplified and two independently amplified RNAs (aRNA) derived from normal and tumor samples using replicas with dye exchange (dye-swap measurements). Overall, signal-to-noise ratio improved when we used aRNA (1.45-fold for channel 1 and 2.02-fold for channel 2), increasing by 6% the number of spots with meaningful data. Measurements arising from independent aRNA samples showed strong correlation among themselves (r(2)=0.962) and with those from the nonamplified sample (r(2)=0.975), indicating the reproducibility and fidelity of the amplification procedure. Measurement differences, i.e, spots with poor correlation between amplified and nonamplified measurements, did not show association with gene sequence, expression intensity, or expression ratio and can, therefore, be compensated with replication. In conclusion, aRNA can be used routinely in cDNA microarray analysis, leading to improved quality of data with high fidelity and reproducibility.

  4. Capturing genomic signatures of DNA sequence variation using a standard anonymous microarray platform

    PubMed Central

    Cannon, C. H.; Kua, C. S.; Lobenhofer, E. K.; Hurban, P.

    2006-01-01

    Comparative genomics, using the model organism approach, has provided powerful insights into the structure and evolution of whole genomes. Unfortunately, only a small fraction of Earth's biodiversity will have its genome sequenced in the foreseeable future. Most wild organisms have radically different life histories and evolutionary genomics than current model systems. A novel technique is needed to expand comparative genomics to a wider range of organisms. Here, we describe a novel approach using an anonymous DNA microarray platform that gathers genomic samples of sequence variation from any organism. Oligonucleotide probe sequences placed on a custom 44 K array were 25 bp long and designed using a simple set of criteria to maximize their complexity and dispersion in sequence probability space. Using whole genomic samples from three known genomes (mouse, rat and human) and one unknown (Gonystylus bancanus), we demonstrate and validate its power, reliability, transitivity and sensitivity. Using two separate statistical analyses, a large numbers of genomic ‘indicator’ probes were discovered. The construction of a genomic signature database based upon this technique would allow virtual comparisons and simple queries could generate optimal subsets of markers to be used in large-scale assays, using simple downstream techniques. Biologists from a wide range of fields, studying almost any organism, could efficiently perform genomic comparisons, at potentially any phylogenetic level after performing a small number of standardized DNA microarray hybridizations. Possibilities for refining and expanding the approach are discussed. PMID:17000641

  5. Development of a DNA microarray for species identification of quarantine aphids.

    PubMed

    Lee, Won Sun; Choi, Hwalran; Kang, Jinseok; Kim, Ji-Hoon; Lee, Si Hyeock; Lee, Seunghwan; Hwang, Seung Yong

    2013-12-01

    Aphid pests are being brought into Korea as a result of increased crop trading. Aphids exist on growth areas of plants, and thus plant growth is seriously affected by aphid pests. However, aphids are very small and have several sexual morphs and life stages, so it is difficult to identify species on the basis of morphological features. This problem was approached using DNA microarray technology. DNA targets of the cytochrome c oxidase subunit I gene were generated with a fluorescent dye-labelled primer and were hybridised onto a DNA microarray consisting of specific probes. After analysing the signal intensity of the specific probes, the unique patterns from the DNA microarray, consisting of 47 species-specific probes, were obtained to identify 23 aphid species. To confirm the accuracy of the developed DNA microarray, ten individual blind samples were used in blind trials, and the identifications were completely consistent with the sequencing data of all individual blind samples. A microarray has been developed to distinguish aphid species. DNA microarray technology provides a rapid, easy, cost-effective and accurate method for identifying aphid species for pest control management. © 2013 Society of Chemical Industry.

  6. Magnetic Scanometric DNA Microarray Detection of Methyl Tertiary Butyl Ether Degrading Bacteria for Environmental Monitoring

    PubMed Central

    Chan, Mei-Lin; Jaramillo, Gerardo; Hristova, Krassimira R.; Horsley, David A.

    2010-01-01

    A magnetoresistive biosensing platform based on a single magnetic tunnel junction (MTJ) scanning probe and DNA microarrays labeled with magnetic particles has been developed to provide an inexpensive, sensitive and reliable detection of DNA. The biosensing platform was demonstrated on a DNA microarray assay for quantifying bacteria capable of degrading methyl tertiary-butyl ether (MTBE), where concentrations as low as 10 pM were detectable. Synthetic probe bacterial DNA was immobilized on a microarray glass slide surface, hybridized with the 48 base pair long biotinylated target DNA and subsequently incubated with streptavidin-coated 2.8 μm diameter magnetic particles. The biosensing platform then makes use of a micron-sized MTJ sensor that was raster scanned across a 3 mm by 5 mm glass slide area to capture the stray magnetic field from the tagged DNA and extract two dimensional magnetic field images of the microarray. The magnetic field output is then averaged over each 100 μm diameter DNA array spot to extract the magnetic spot intensity, analogous to the fluorescence spot intensity used in conventional optical scanners. The magnetic scanning result is compared with results from a commercial laser scanner and particle coverage optical counting to demonstrate the dynamic range and linear sensitivity of the biosensing platform as a potentially inexpensive, sensitive and portable alternative for DNA microarray detection for field applications. PMID:20889328

  7. DNA Microarray Characterization of Pathogens Associated with Sexually Transmitted Diseases

    PubMed Central

    Cao, Boyang; Wang, Suwei; Tian, Zhenyang; Hu, Pinliang; Feng, Lu; Wang, Lei

    2015-01-01

    This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs. PMID:26208181

  8. DNA microarray technology for target identification and validation.

    PubMed

    Jayapal, Manikandan; Melendez, Alirio J

    2006-01-01

    1. Microarrays, a recent development, provide a revolutionary platform to analyse thousands of genes at once. They have enormous potential in the study of biological processes in health and disease and, perhaps, microarrays have become crucial tools in diagnostic applications and drug discovery. 2. Microarray based studies have provided the essential impetus for biomedical experiments, such as identification of disease-causing genes in malignancies and regulatory genes in the cell cycle mechanism. Microarrays can identify genes for new and unique potential drug targets, predict drug responsiveness for individual patients and, finally, initiate gene therapy and prevention strategies. 3. The present article reviews the principles and technological concerns, as well as the steps involved in obtaining and analysing of data. Furthermore, applications of microarray based experiments in drug target identifications and validation strategies are discussed. 4. To exemplify how this tool can be useful, in the present review we provide an overview of some of the past and potential future aspects of microarray technology and present a broad overview of this rapidly growing field.

  9. DNA Microarray Characterization of Pathogens Associated with Sexually Transmitted Diseases.

    PubMed

    Cao, Boyang; Wang, Suwei; Tian, Zhenyang; Hu, Pinliang; Feng, Lu; Wang, Lei

    2015-01-01

    This study established a multiplex PCR-based microarray to detect simultaneously a diverse panel of 17 sexually transmitted diseases (STDs)-associated pathogens including Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma, Herpes simplex virus (HSV) types 1 and 2, and Human papillomavirus (HPV) types 6, 11, 16, 18, 31, 33, 35, 39, 54 and 58. The target genes are 16S rRNA gene for N. gonorrhoeae, M. genitalium, M. hominism, and Ureaplasma, the major outer membrane protein gene (ompA) for C. trachomatis, the glycoprotein B gene (gB) for HSV; and the L1 gene for HPV. A total of 34 probes were selected for the microarray including 31 specific probes, one as positive control, one as negative control, and one as positional control probe for printing reference. The microarray is specific as the commensal and pathogenic microbes (and closely related organisms) in the genitourinary tract did not cross-react with the microarray probes. The microarray is 10 times more sensitive than that of the multiplex PCR. Among the 158 suspected HPV specimens examined, the microarray showed that 49 samples contained HPV, 21 samples contained Ureaplasma, 15 contained M. hominis, four contained C. trachomatis, and one contained N. gonorrhoeae. This work reports the development of the first high through-put detection system that identifies common pathogens associated with STDs from clinical samples, and paves the way for establishing a time-saving, accurate and high-throughput diagnostic tool for STDs.

  10. Characterization and simulation of cDNA microarray spots using a novel mathematical model

    PubMed Central

    Kim, Hye Young; Lee, Seo Eun; Kim, Min Jung; Han, Jin Il; Kim, Bo Kyung; Lee, Yong Sung; Lee, Young Seek; Kim, Jin Hyuk

    2007-01-01

    Background The quality of cDNA microarray data is crucial for expanding its application to other research areas, such as the study of gene regulatory networks. Despite the fact that a number of algorithms have been suggested to increase the accuracy of microarray gene expression data, it is necessary to obtain reliable microarray images by improving wet-lab experiments. As the first step of a cDNA microarray experiment, spotting cDNA probes is critical to determining the quality of spot images. Results We developed a governing equation of cDNA deposition during evaporation of a drop in the microarray spotting process. The governing equation included four parameters: the surface site density on the support, the extrapolated equilibrium constant for the binding of cDNA molecules with surface sites on glass slides, the macromolecular interaction factor, and the volume constant of a drop of cDNA solution. We simulated cDNA deposition from the single model equation by varying the value of the parameters. The morphology of the resulting cDNA deposit can be classified into three types: a doughnut shape, a peak shape, and a volcano shape. The spot morphology can be changed into a flat shape by varying the experimental conditions while considering the parameters of the governing equation of cDNA deposition. The four parameters were estimated by fitting the governing equation to the real microarray images. With the results of the simulation and the parameter estimation, the phenomenon of the formation of cDNA deposits in each type was investigated. Conclusion This study explains how various spot shapes can exist and suggests which parameters are to be adjusted for obtaining a good spot. This system is able to explore the cDNA microarray spotting process in a predictable, manageable and descriptive manner. We hope it can provide a way to predict the incidents that can occur during a real cDNA microarray experiment, and produce useful data for several research applications

  11. Improvement in the amine glass platform by bubbling method for a DNA microarray.

    PubMed

    Jee, Seung Hyun; Kim, Jong Won; Lee, Ji Hyeong; Yoon, Young Soo

    2015-01-01

    A glass platform with high sensitivity for sexually transmitted diseases microarray is described here. An amino-silane-based self-assembled monolayer was coated on the surface of a glass platform using a novel bubbling method. The optimized surface of the glass platform had highly uniform surface modifications using this method, as well as improved hybridization properties with capture probes in the DNA microarray. On the basis of these results, the improved glass platform serves as a highly reliable and optimal material for the DNA microarray. Moreover, in this study, we demonstrated that our glass platform, manufactured by utilizing the bubbling method, had higher uniformity, shorter processing time, lower background signal, and higher spot signal than the platforms manufactured by the general dipping method. The DNA microarray manufactured with a glass platform prepared using bubbling method can be used as a clinical diagnostic tool.

  12. Genomewide expression analysis in amino acid-producing bacteria using DNA microarrays.

    PubMed

    Polen, Tino; Wendisch, Volker F

    2004-01-01

    DNA microarray technology has become an important research tool for biotechnology and microbiology. It is now possible to characterize genetic diversity and gene expression in a genomewide manner. DNA microarrays have been applied extensively to study the biology of many bacteria including Escherichia coli, but only recently have they been developed for the Gram-positive Corynebacterium glutamicum. Both bacteria are widely used for biotechnological amino acid production. In this article, in addition to the design and generation of microarrays as well as their use in hybridization experiments and subsequent data analysis, we describe recent applications of DNA microarray technology regarding amino acid production in C. glutamicum and E. coli. We also discuss the impact of functional genomics studies on fundamental as well as applied aspects of amino acid production with C. glutamicum and E. coli.

  13. Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm

    PubMed Central

    Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

    2015-01-01

    DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively. PMID:26284175

  14. Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm.

    PubMed

    Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

    2015-01-01

    DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively.

  15. Detection of Alicyclobacillus species in fruit juice using a random genomic DNA microarray chip.

    PubMed

    Jang, Jun Hyeong; Kim, Sun-Joong; Yoon, Bo Hyun; Ryu, Jee-Hoon; Gu, Man Bock; Chang, Hyo-Ihl

    2011-06-01

    This study describes a method using a DNA microarray chip to rapidly and simultaneously detect Alicyclobacillus species in orange juice based on the hybridization of genomic DNA with random probes. Three food spoilage bacteria were used in this study: Alicyclobacillus acidocaldarius, Alicyclobacillus acidoterrestris, and Alicyclobacillus cycloheptanicus. The three Alicyclobacillus species were adjusted to 2 × 10(3) CFU/ml and inoculated into pasteurized 100% pure orange juice. Cy5-dCTP labeling was used for reference signals, and Cy3-dCTP was labeled for target genomic DNA. The molar ratio of 1:1 of Cy3-dCTP and Cy5-dCTP was used. DNA microarray chips were fabricated using randomly fragmented DNA of Alicyclobacillus spp. and were hybridized with genomic DNA extracted from Bacillus spp. Genomic DNA extracted from Alicyclobacillus spp. showed a significantly higher hybridization rate compared with DNA of Bacillus spp., thereby distinguishing Alicyclobacillus spp. from Bacillus spp. The results showed that the microarray DNA chip containing randomly fragmented genomic DNA was specific and clearly identified specific food spoilage bacteria. This microarray system is a good tool for rapid and specific detection of thermophilic spoilage bacteria, mainly Alicyclobacillus spp., and is useful and applicable to the fruit juice industry.

  16. Synergistic effects of epoxy- and amine-silanes on microarray DNA immobilization and hybridization.

    PubMed

    Chiu, Sung-Kay; Hsu, Mandy; Ku, Wei-Chi; Tu, Ching-Yu; Tseng, Yu-Tien; Lau, Wai-Kwan; Yan, Rong-Yih; Ma, Jing-Tyan; Tzeng, Chi-Meng

    2003-09-15

    Most microarray slides are manufactured or coated with a layer of poly(L-lysine) or with silanes with different chemical functional groups, for the attachment of nucleic acids on to their surfaces. The efficiency with which nucleic acids bind to these surfaces is not high, because they can be washed away, especially in the case of spotting oligonucleotides. In view of this, we have developed a method to increase the binding capacity and efficiency of hybridization of DNA on to derivatized glass surfaces. This makes use of the synergistic effect of two binding interactions between the nucleic acids and the coating chemicals on the surface of the glass slides. The enhanced binding allows the nucleic acids to be bound tightly and to survive stringency washes. When immobilized, DNA exhibits a higher propensity for hybridization on the surface than on slides with only one binding chemical. By varying the silane concentrations, we have shown that maximal DNA oligonucleotide binding on glass surfaces occurs when the percentage composition of both of the surface-coating chemicals falls to 0.2%, which is different from that on binding PCR products. This new mixture-combination approach for nucleic-acid binding allows signals from immobilization and hybridization to have higher signal-to-noise ratios than for other silane-coated methods.

  17. Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses

    PubMed Central

    2011-01-01

    Background The toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples. Results We took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with

  18. Automation of cDNA microarray hybridization and washing yields improved data quality.

    PubMed

    Yauk, Carole; Berndt, Lynn; Williams, Andrew; Douglas, George R

    2005-07-29

    Microarray technology allows the analysis of whole-genome transcription within a single hybridization, and has become a standard research tool. It is extremely important to minimize variation in order to obtain high quality microarray data that can be compared among experiments and laboratories. The majority of facilities implement manual hybridization approaches for microarray studies. We developed an automated method for cDNA microarray hybridization that uses equivalent pre-hybridization, hybridization and washing conditions to the suggested manual protocol. The automated method significantly decreased variability across microarray slides compared to manual hybridization. Although normalized signal intensities for buffer-only spots across the chips were identical, significantly reduced variation and inter-quartile ranges were obtained using the automated workstation. This decreased variation led to improved correlation among technical replicates across slides in both the Cy3 and Cy5 channels.

  19. High-efficiency microarray of 3-D carbon MEMS electrodes for pathogen detection systems

    NASA Astrophysics Data System (ADS)

    Kassegne, Sam; Wondimu, Berhanu; Majzoub, Mohammad; Shin, Jiae

    2008-11-01

    Molecular diagnostic applications for pathogen detections require the ability to separate pathogens such as bacteria, viruses, etc., from a biological sample of blood or saliva. Over the past several years, conventional two-dimensional active microarrays have been used with success for the manipulation of biomolecules including DNA. However, they have a major drawback of inability to process relatively 'largevolume' samples useful in infectious disease diagnostics applications. This paper presents an active microarray of three-dimensional carbon electrodes that exploits electrokinetic forces for transport, accumulation, and hybridization of charged bio-molecules with an added advantage of large volume capability. Tall 3-dimensional carbon microelectrode posts are fabricated using C-MEMS (Carbon MEMS) technology that is emerging as a very exciting research area since carbon has fascinating physical, chemical, mechanical and electrical properties in addition to its low cost. The chip fabricated using CMEMS technology is packaged and its efficiency of separation and accumulation of charged particle established by manipulating negatively charged polycarboxylate 2 μm beads in 50 mM histidine buffer.

  20. Biostatistical Considerations of the Use of Genomic DNA Reference in Microarrays

    SciTech Connect

    Yang, Yunfeng; Zhu, Mengxia; Wu, Liyou; Zhou, Jizhong

    2008-01-01

    Using genomic DNA as common reference in microarray experiments has recently been tested by different laboratories (2, 3, 5, 7, 9, 20, 24-26). While some reported that experimental results of microarrays using genomic DNA reference conforms nicely to those obtained by cDNA: cDNA co-hybridization method, others acquired poor results. We hypothesize that these conflicting reports could be resolved by biostatistical analyses. To test it, microarray experiments were performed in a -proteobacterium Shewanella oneidensis. Pair-wise comparison of three experimental conditions was obtained either by direct cDNA: cDNA co-hybridization, or by indirect calculation through a Shewanella genomic DNA reference. Several major biostatistical techniques were exploited to reduce the amount of inconsistency between both methods and the results were assessed. We discovered that imposing the constraint of minimal number of replicates, logarithmic transformation and random error analyses could significantly improve the data quality. Therefore, it is useful to adopt these biostatistical techniques for microarray data analysis using genomic DNA as reference.

  1. FPGA based system for automatic cDNA microarray image processing.

    PubMed

    Belean, Bogdan; Borda, Monica; Le Gal, Bertrand; Terebes, Romulus

    2012-07-01

    Automation is an open subject in DNA microarray image processing, aiming reliable gene expression estimation. The paper presents a novel shock filter based approach for automatic microarray grid alignment. The proposed method brings up significantly reduced computational complexity compared to state of the art approaches, while similar results in terms of accuracy are achieved. Based on this approach, we also propose an FPGA based system for microarray image analysis that eliminates the shortcomings of existing software platforms: user intervention, increased computational time and cost. Our system includes application-specific architectures which involve algorithm parallelization, aiming fast and automated cDNA microarray image processing. The proposed automated image processing chain is implemented both on a general purpose processor and using the developed hardware architectures as co-processors in a FPGA based system. The comparative results included in the last section show that an important gain in terms of computational time is obtained using hardware based implementations.

  2. [DNA microarrays--perspective of application for drug effectivity and safety evaluation].

    PubMed

    Roman, Iza

    2008-01-01

    Microarray technology provides a unique tool for the determination of gene expression at the level of messenger RNA (mRNA). Microarray has been successfully applied to the high throughput simultaneous expression of many thousands of genes in a single experiment. One important application of DNA microarray technology, within the context of drugs effectiveness and safety evaluation studies, is its use as a screening tool for the identification of biochemical pathways, potential targets for novel molecular therapeutics, for the identification of molecular mechanisms of toxicity and to understand and predict individual drug sensitivity and resistance. The purpose of this review is presentation of the utility of DNA microarray technology in all phases of the drug discovery process.

  3. 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.

  4. Fundamental Patterns Underlying Neurotoxicity Revealed by DNA Microarray Expression Profiling

    DTIC Science & Technology

    2004-09-01

    microarray analysis of the dopaminergic cell line, SN4741 , revealed induction of stress indices following MPP* treatment (Chun et al., 2001). To...response to a wide range of cellular stresses including oxidative insult of the nigral dopaminergic cell line SN4741 with hydrogen peroxide or MPP* (Salinas

  5. 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…

  6. 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…

  7. Novel multistranded, alternative, plasmid and helical transitional DNA and RNA microarrays: implications for therapeutics.

    PubMed

    Gagna, Claude E; Lambert, W Clark

    2009-05-01

    Novel multistranded and alternative DNA, RNA and plasmid microarrays (transitional structural nucleic acid microarrays) have been developed that allows for the immobilization of intact, nondenatured, double-stranded DNA, double-stranded RNA, and alternative and multistranded nucleic acids. It also allows for the study of transitional changes that occur in the structure of DNA and RNA. Alternative types of DNA, RNA and multistranded nucleic acids are immobilized by a variety of different surface chemistries (i.e., noncovalent or covalent) onto a novel substrate surface. This technology represents the next generation of microarrays, which will aid in the characterization of nucleic acid structure and function, and accelerate the discovery of drugs that bind to nucleic acids. In addition, we demonstrate four novel techniques that are the first practical applications of the microarray, that is, transitional structural chemogenomics, transitional structural chemoproteomics, transitional structural pharmacogenomics and transitional structural pharmacoproteomics. These novel nucleic acid microarrays, together with pharmacogenomics, can be used to improve the study of DNA and RNA structure, gene expression, drug development and treatment of various diseases.

  8. The emergence and diffusion of DNA microarray technology

    PubMed Central

    Lenoir, Tim; Giannella, Eric

    2006-01-01

    The network model of innovation widely adopted among researchers in the economics of science and technology posits relatively porous boundaries between firms and academic research programs and a bi-directional flow of inventions, personnel, and tacit knowledge between sites of university and industry innovation. Moreover, the model suggests that these bi-directional flows should be considered as mutual stimulation of research and invention in both industry and academe, operating as a positive feedback loop. One side of this bi-directional flow – namely; the flow of inventions into industry through the licensing of university-based technologies – has been well studied; but the reverse phenomenon of the stimulation of university research through the absorption of new directions emanating from industry has yet to be investigated in much detail. We discuss the role of federal funding of academic research in the microarray field, and the multiple pathways through which federally supported development of commercial microarray technologies have transformed core academic research fields. Our study confirms the picture put forward by several scholars that the open character of networked economies is what makes them truly innovative. In an open system innovations emerge from the network. The emergence and diffusion of microarray technologies we have traced here provides an excellent example of an open system of innovation in action. Whether they originated in a startup company environment that operated like a think-tank, such as Affymax, the research labs of a large firm, such as Agilent, or within a research university, the inventors we have followed drew heavily on knowledge resources from all parts of the network in bringing microarray platforms to light. Federal funding for high-tech startups and new industrial development was important at several phases in the early history of microarrays, and federal funding of academic researchers using microarrays was fundamental

  9. Manufacturing DNA microarrays of high spot homogeneity and reduced background signal

    PubMed Central

    Diehl, Frank; Grahlmann, Susanne; Beier, Markus; Hoheisel, Jörg D.

    2001-01-01

    Analyses on DNA microarrays depend considerably on spot quality and a low background signal of the glass support. By using betaine as an additive to a spotting solution made of saline sodium citrate, both the binding efficiency of spotted PCR products and the homogeneity of the DNA spots is improved significantly on aminated surfaces such as glass slides coated with the widely used poly-l-lysine or aminosilane. In addition, non-specific background signal is markedly diminished. Concomitantly, during the arraying procedure, the betaine reduces evaporation from the microtitre dish wells, which hold the PCR products. Subsequent blocking of the chip surface with succinic anhydride was improved considerably in the presence of the non-polar, non-aqueous solvent 1,2-dichloroethane and the acylating catalyst N-methylimidazole. This procedure prevents the overall background signal that occurs with the frequently applied aqueous solvent 1-methyl-2-pyrrolidone in borate buffer because of DNA that re-dissolves from spots during the blocking process, only to bind again across the entire glass surface. PMID:11266573

  10. DNA methylation analysis using CpG microarrays is impaired in benzopyrene exposed cells

    SciTech Connect

    Sadikovic, Bekim; Andrews, Joseph; Rodenhiser, David I.

    2007-12-15

    Epigenetic alterations have emerged as a key mechanism involved in tumorigenesis. These disruptions are partly due to environmental factors that change normal DNA methylation patterns necessary for transcriptional regulation and chromatin compaction. Microarray technologies are allowing environmentally susceptible epigenetic patterns to be mapped and the precise targets of environmentally induced alterations to be identified. Previously, we observed BaP-induced epigenetic events and cell cycle disruptions in breast cancer cell lines that included time- and concentration-dependent loss of proliferation as well as sequence-specific hypo- and hypermethylation events. In this present report, we further characterized epigenetic changes in BaP-exposed MCF-7 cells. We analyzed DNA methylation on a CpG island microarray platform with over 5400 unique genomic regions. Depleted and enriched microarray targets, representative of putative DNA methylation changes, were identified across the genome; however, subsequent sodium bisulfite analyses revealed no changes in DNA methylation at a number of these loci. Instead, we found that the identification of DNA methylation changes using this restriction enzyme-based microarray approach corresponded with the regions of DNA bound by the BaP derived DNA adducts. This DNA adduct formation occurs at both methylated and unmethylated CpG dinucleotides and affects PCR amplification during sample preparation. Our data suggest that caution should be exercised when interpreting data from comparative microarray experiments that rely on enzymatic reactions. These results are relevant to genome screening approaches involving environmental exposures in which DNA adduct formation at specific nucleotide sites may bias target acquisition and compromise the correct identification of epigenetically responsive genes.

  11. A glance at DNA microarray technology and applications.

    PubMed

    Saei, Amir Ata; Omidi, Yadollah

    2011-01-01

    Because of huge impacts of "OMICS" technologies in life sciences, many researchers aim to implement such high throughput approach to address cellular and/or molecular functions in response to any influential intervention in genomics, proteomics, or metabolomics levels. However, in many cases, use of such technologies often encounters some cybernetic difficulties in terms of knowledge extraction from a bunch of data using related softwares. In fact, there is little guidance upon data mining for novices. The main goal of this article is to provide a brief review on different steps of microarray data handling and mining for novices and at last to introduce different PC and/or web-based softwares that can be used in preprocessing and/or data mining of microarray data. To pursue such aim, recently published papers and microarray softwares were reviewed. It was found that defining the true place of the genes in cell networks is the main phase in our understanding of programming and functioning of living cells. This can be obtained with global/selected gene expression profiling. Studying the regulation patterns of genes in groups, using clustering and classification methods helps us understand different pathways in the cell, their functions, regulations and the way one component in the system affects the other one. These networks can act as starting points for data mining and hypothesis generation, helping us reverse engineer.

  12. A Glance at DNA Microarray Technology and Applications

    PubMed Central

    Saei, Amir Ata; Omidi, Yadollah

    2011-01-01

    Introduction Because of huge impacts of “OMICS” technologies in life sciences, many researchers aim to implement such high throughput approach to address cellular and/or molecular functions in response to any influential intervention in genomics, proteomics, or metabolomics levels. However, in many cases, use of such technologies often encounters some cybernetic difficulties in terms of knowledge extraction from a bunch of data using related softwares. In fact, there is little guidance upon data mining for novices. The main goal of this article is to provide a brief review on different steps of microarray data handling and mining for novices and at last to introduce different PC and/or web-based softwares that can be used in preprocessing and/or data mining of microarray data. Methods To pursue such aim, recently published papers and microarray softwares were reviewed. Results It was found that defining the true place of the genes in cell networks is the main phase in our understanding of programming and functioning of living cells. This can be obtained with global/selected gene expression profiling. Conclusion Studying the regulation patterns of genes in groups, using clustering and classification methods helps us understand different pathways in the cell, their functions, regulations and the way one component in the system affects the other one. These networks can act as starting points for data mining and hypothesis generation, helping us reverse engineer. PMID:23678411

  13. Profiling of differentially expressed genes in human gingival epithelial cells and fibroblasts by DNA microarray.

    PubMed

    Abiko, Yoshimitsu; Hiratsuka, Koichi; Kiyama-Kishikawa, Michiko; Tsushima, Katsumasa; Ohta, Mitsuhiro; Sasahara, Hiroshige

    2004-03-01

    Gingival epithelial cells and fibroblasts play important roles and have a harmonious relationship under normal and disease conditions, but the precise differences between theses cells remain unknown. To study the differences in gene expression between human gingival epithelial cells (HGE) and human gingival fibroblasts (HGF), mRNA was recovered from primary cultured cells and analyzed using cDNA microarray technology. The cDNA retro-transcribed from equal quantities of mRNA was labeled with the fluorescent dyes Cy5 and Cy3. The mixed probes were then hybridized with 7276 genes on the DNA microarray, after which fluorescence signals were scanned and further analyzed using GeneSpring software. Of the 7276 genes screened, 469 showed expression levels that were more than 2-fold greater in HGE than in HGF, while 293 showed expression levels that were more than 2-fold greater in HGF than in HGE. To confirm the reliability of the microarray results, keratin K5 and desmocolin, and vimentin and gp130, which showed higher mRNA levels in HGE and HGF, respectively, were selected and their mRNA levels were further analyzed by RT-PCR. The results of RT-PCR correlated well with those of microarray analysis. The present findings using a DNA microarray to detect differences in the gene expression profiles of HGE and HGF may be beneficial for genetic diagnosis of periodontal tissue metabolism and periodontal diseases.

  14. Easy and fast detection and genotyping of high-risk human papillomavirus by dedicated DNA microarrays.

    PubMed

    Albrecht, Valérie; Chevallier, Anne; Magnone, Virginie; Barbry, Pascal; Vandenbos, Fanny; Bongain, André; Lefebvre, Jean-Claude; Giordanengo, Valérie

    2006-11-01

    Persistent cervical high-risk human papillomavirus (HPV) infection is correlated with an increased risk of developing a high-grade cervical intraepithelial lesion. A two-step method was developed for detection and genotyping of high-risk HPV. DNA was firstly amplified by asymmetrical PCR in the presence of Cy3-labelled primers and dUTP. Labelled DNA was then genotyped using DNA microarray hybridization. The current study evaluated the technical efficacy of laboratory-designed HPV DNA microarrays for high-risk HPV genotyping on 57 malignant and non-malignant cervical smears. The approach was evaluated for a broad range of cytological samples: high-grade squamous intraepithelial lesions (HSIL), low-grade squamous intraepithelial lesions (LSIL) and atypical squamous cells of high-grade (ASC-H). High-risk HPV was also detected in six atypical squamous cells of undetermined significance (ASC-US) samples; among them only one cervical specimen was found uninfected, associated with no histological lesion. The HPV oligonucleotide DNA microarray genotyping detected 36 infections with a single high-risk HPV type and 5 multiple infections with several high-risk types. Taken together, these results demonstrate the sensitivity and specificity of the HPV DNA microarray approach. This approach could improve clinical management of patients with cervical cytological abnormalities.

  15. Application of L-cystein derivative to DNA microarray.

    PubMed

    Nakauchi, Gen; Inaki, Yoshiaki; Kitaoka, Shiho; Yokoyama, Chieko; Tanabe, Tadashi

    2002-01-01

    S-carboxymethyl-L-cystein derivatives of nucleic acid bases were prepared as DNA chip probe. These compounds in vitro have been found to form stable complex with oligo-DNA and RNA. This paper deals with preparing new DNA chip using L-cystein derivative synthetic nucleotides as probe and immobilized it to quartz plate by photosensitive PVA. Then the chip exposed with FITC labeled target DNA was observed by confocal fluorescence microscope.

  16. Rapid and sensitive detection of UGT1A1 polymorphisms associated with irinotecan toxicity by a novel DNA microarray.

    PubMed

    Tsunedomi, Ryouichi; Hazama, Shoichi; Okayama, Naoko; Oka, Masaaki; Nagano, Hiroaki

    2017-07-01

    Recent developments in the field of human genomics have greatly enhanced the potential for precision and personalized medicine. We have developed a novel DNA microarray, using a 3-mm square chip coated with diamond-like carbon to enhance the signal-to-background ratio, for use as an in vitro diagnostic tool in precision medicine. To verify the genotyping effectiveness of this newly developed DNA microarray we examined UDP-glucuronosyltransferase 1A1 (UGT1A1) polymorphisms in DNA extracted from patients with metastatic colorectal cancer. It is established that the polymorphisms of UGT1A1*28 and UGT1A1*6 are significantly associated with severe toxicity induced by the anti-cancer drug irinotecan. For each sample, the results obtained with the novel microarray platform were compared with those obtained using other, more established, methods, including direct sequencing and the Invader assay. The polymorphisms tested included a single nucleotide substitution (UGT1A1*6) and a TA-repeat polymorphism (UGT1A1*28), both of which were detected simultaneously and accurately using our method. Moreover, our method required 1.5-fold less time to assay and 20-fold less sample than those required by the Invader assay. In summary, our newly developed DNA microarray is more practical than established methods, and is at least as accurate; this will increase the efficiency of polymorphism detection prior to diagnosis and the commencement of treatment, and can feasibly be applied in precision medicine. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  17. 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

  18. Sequential interim analyses of survival data in DNA microarray experiments

    PubMed Central

    2011-01-01

    Background Discovery of biomarkers that are correlated with therapy response and thus with survival is an important goal of medical research on severe diseases, e.g. cancer. Frequently, microarray studies are performed to identify genes of which the expression levels in pretherapeutic tissue samples are correlated to survival times of patients. Typically, such a study can take several years until the full planned sample size is available. Therefore, interim analyses are desirable, offering the possibility of stopping the study earlier, or of performing additional laboratory experiments to validate the role of the detected genes. While many methods correcting the multiple testing bias introduced by interim analyses have been proposed for studies of one single feature, there are still open questions about interim analyses of multiple features, particularly of high-dimensional microarray data, where the number of features clearly exceeds the number of samples. Therefore, we examine false discovery rates and power rates in microarray experiments performed during interim analyses of survival studies. In addition, the early stopping based on interim results of such studies is evaluated. As stop criterion we employ the achieved average power rate, i.e. the proportion of detected true positives, for which a new estimator is derived and compared to existing estimators. Results In a simulation study, pre-specified levels of the false discovery rate are maintained in each interim analysis, where reduced levels as used in classical group sequential designs of one single feature are not necessary. Average power rates increase with each interim analysis, and many studies can be stopped prior to their planned end when a certain pre-specified power rate is achieved. The new estimator for the power rate slightly deviates from the true power rate but is comparable to other estimators. Conclusions Interim analyses of microarray experiments can provide evidence for early stopping of

  19. ArrayQuest: a web resource for the analysis of DNA microarray data

    PubMed Central

    Argraves, Gary L; Jani, Saurin; Barth, Jeremy L; Argraves, W Scott

    2005-01-01

    Background Numerous microarray analysis programs have been created through the efforts of Open Source software development projects. Providing browser-based interfaces that allow these programs to be executed over the Internet enhances the applicability and utility of these analytic software tools. Results Here we present ArrayQuest, a web-based DNA microarray analysis process controller. Key features of ArrayQuest are that (1) it is capable of executing numerous analysis programs such as those written in R, BioPerl and C++; (2) new analysis programs can be added to ArrayQuest Methods Library at the request of users or developers; (3) input DNA microarray data can be selected from public databases (i.e., the Medical University of South Carolina (MUSC) DNA Microarray Database or Gene Expression Omnibus (GEO)) or it can be uploaded to the ArrayQuest center-point web server into a password-protected area; and (4) analysis jobs are distributed across computers configured in a backend cluster. To demonstrate the utility of ArrayQuest we have populated the methods library with methods for analysis of Affymetrix DNA microarray data. Conclusion ArrayQuest enables browser-based implementation of DNA microarray data analysis programs that can be executed on a Linux-based platform. Importantly, ArrayQuest is a platform that will facilitate the distribution and implementation of new analysis algorithms and is therefore of use to both developers of analysis applications as well as users. ArrayQuest is freely available for use at . PMID:16321157

  20. ArrayQuest: a web resource for the analysis of DNA microarray data.

    PubMed

    Argraves, Gary L; Jani, Saurin; Barth, Jeremy L; Argraves, W Scott

    2005-12-01

    Numerous microarray analysis programs have been created through the efforts of Open Source software development projects. Providing browser-based interfaces that allow these programs to be executed over the Internet enhances the applicability and utility of these analytic software tools. Here we present ArrayQuest, a web-based DNA microarray analysis process controller. Key features of ArrayQuest are that (1) it is capable of executing numerous analysis programs such as those written in R, BioPerl and C++; (2) new analysis programs can be added to ArrayQuest Methods Library at the request of users or developers; (3) input DNA microarray data can be selected from public databases (i.e., the Medical University of South Carolina (MUSC) DNA Microarray Database or Gene Expression Omnibus (GEO)) or it can be uploaded to the ArrayQuest center-point web server into a password-protected area; and (4) analysis jobs are distributed across computers configured in a backend cluster. To demonstrate the utility of ArrayQuest we have populated the methods library with methods for analysis of Affymetrix DNA microarray data. ArrayQuest enables browser-based implementation of DNA microarray data analysis programs that can be executed on a Linux-based platform. Importantly, ArrayQuest is a platform that will facilitate the distribution and implementation of new analysis algorithms and is therefore of use to both developers of analysis applications as well as users. ArrayQuest is freely available for use at http://proteogenomics.musc.edu/arrayquest.html.

  1. 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.

  2. Fabrication of a microarray using a combination of the large circular sense and antisense DNA.

    PubMed

    Doh, Kyung-Oh; Lee, Yun-Han; Han, Kil-Hwan; Uhm, Seok-Yong; Kim, Jong-Pil; Bae, Yun-Ui; Park, Jeong-Hoh; Moon, Ik-Jae; Park, Jong-Gu

    2010-01-01

    In the present study, single-stranded large circular (LC)-sense molecules were utilized as probes for DNA microarrays and showed stronger binding signals than those of PCR-amplified cDNA probes. A microarray experiment using 284 LC-sense DNA probes found 6 upregulated and 7 downregulated genes in A549 cells as compared to WI38VA13 cells. Repeated experiments showed largely consistent results, and microarray data strongly correlated with data acquired from quantitative real-time RT-PCR. A large array comprising 5,079 LC-sense DNA was prepared, and analysis of the mean differential expression from dye-swap experiments revealed 332 upregulated and 509 downregulated genes in A549 cells compared to WI38VA13 cells. Subsequent functional analysis using an LC-antisense library of overexpressed genes identified 28 genes involved in A549 cell growth. These experiments demonstrated the proper features of LC-sense molecules as probe DNA for microarray and the potential utility of the combination of LC-sense and -antisense libraries for an effective functional validation of genes.

  3. 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

  4. Microarray long oligo probe designing for Escherichia coli: an in-silico DNA marker extraction.

    PubMed

    Behzadi, Payam; Najafi, Ali; Behzadi, Elham; Ranjbar, Reza

    2016-01-01

    Urinary tract infections are predominant diseases which may be caused by different pathogenic microorganisms, particularly Escherichia coli (E.coli). DNA microarray technology is an accurate, rapid, sensitive, and specific diagnostic tool which may lead to definite diagnosis and treatment of several infectious diseases. DNA microarray is a multi-process method in which probe designing plays an important. Therefore, the authors of the present study have tried to design a range of effective and proper long oligo microarray probes for detection and identification of different strains of pathogenic E.coli and in particular, uropathogenic E.coli (UPEC). E.coli O26 H11 11368 uid41021 was selected as the standard strain for probe designing. This strain encompasses the largest nucleotide sequence and the most number of genes among other pathogenic strains of E.coli. For performing this in silico survey, NCBI database, GReview Server, PanSeq Server, Oligoanalyzer tool, and AlleleID 7.7 were used to design accurate, appropriate, effective, and flexible long oligo microarray probes. Moreover, the genome of E.coli and its closely related microorganisms were compared. In this study, 15 long oligo microarray probes were designed for detecting and identifying different strains of E.coli such as UPEC. These probes possessed the best physico-chemical characteristics. The functional and structural properties of the designed probes were recognized by practical tools and softwares. The use of reliable advanced technologies and methodologies for probe designing guarentees the high quality of microarray probes and makes DNA microarray technology more flexible and an effective diagnostic technique.

  5. Temperature scans/cycles for the detection of low abundant DNA point mutations on microarrays.

    PubMed

    Pingel, Julia; Buhot, Arnaud; Calemczuk, Roberto; Livache, Thierry

    2012-01-15

    The possibility to detect low abundant DNA point mutations is essential for early cancer diagnosis and/or prognosis. Furthermore, in order to be less invasive, the somatic mutations are not only sought in tumor extract samples but also from body fluids or stools rendering their content even more diluted compared to the wild type sequences. In this short communication, we propose two protocols based on temperature scans or cycles for the enrichment of the mutation strands hybridized on microarrays. We predict numerically and confirm experimentally a 10-fold increase in the fraction of mutated DNA hybridized on the microarray compared to the sample content. Coupled to more standard solution phase enrichment techniques, it would be possible to lower by one order of magnitude the current detection limit with the advantage of multiple mutation detections offered by the microarray technology. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. A 7872 cDNA microarray and its use in bovine functional genomics.

    PubMed

    Everts, Robin E; Band, Mark R; Liu, Z Lewis; Kumar, Charu G; Liu, Lei; Loor, Juan J; Oliveira, Rosane; Lewin, Harris A

    2005-05-15

    The strategy used to create and annotate a 7872 cDNA microarray from cattle placenta and spleen cDNA sequences is described. This microarray contains approximately 6300 unique genes, as determined by BLASTN and TBLASTX similarity search against the human and mouse UniGene and draft human genome sequence databases (build 34). Sequences on the array were annotated with gene ontology (GO) terms, thereby facilitating data analysis and interpretation. A total of 3244 genes were annotated with GO terms. The array is rich in sequences encoding transcription factors, signal transducers and cell cycle regulators. Current research being conducted with this array is described, and an overview of planned improvements in our microarray platform for cattle functional genomics is presented.

  7. A DNA Microarray-Based Assay to Detect Dual Infection with Two Dengue Virus Serotypes

    PubMed Central

    Díaz-Badillo, Alvaro; de Lourdes Muñoz, María; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G.; Martínez-Muñoz, Jorge P.; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-01-01

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples. PMID:24776933

  8. A DNA microarray-based assay to detect dual infection with two dengue virus serotypes.

    PubMed

    Díaz-Badillo, Alvaro; Muñoz, María de Lourdes; Perez-Ramirez, Gerardo; Altuzar, Victor; Burgueño, Juan; Mendoza-Alvarez, Julio G; Martínez-Muñoz, Jorge P; Cisneros, Alejandro; Navarrete-Espinosa, Joel; Sanchez-Sinencio, Feliciano

    2014-04-25

    Here; we have described and tested a microarray based-method for the screening of dengue virus (DENV) serotypes. This DNA microarray assay is specific and sensitive and can detect dual infections with two dengue virus serotypes and single-serotype infections. Other methodologies may underestimate samples containing more than one serotype. This technology can be used to discriminate between the four DENV serotypes. Single-stranded DNA targets were covalently attached to glass slides and hybridised with specific labelled probes. DENV isolates and dengue samples were used to evaluate microarray performance. Our results demonstrate that the probes hybridized specifically to DENV serotypes; with no detection of unspecific signals. This finding provides evidence that specific probes can effectively identify single and double infections in DENV samples.

  9. 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.

  10. Optimal cDNA microarray design using expressed sequence tags for organisms with limited genomic information

    PubMed Central

    Chen, Yian A; Mckillen, David J; Wu, Shuyuan; Jenny, Matthew J; Chapman, Robert; Gross, Paul S; Warr, Gregory W; Almeida, Jonas S

    2004-01-01

    Background Expression microarrays are increasingly used to characterize environmental responses and host-parasite interactions for many different organisms. Probe selection for cDNA microarrays using expressed sequence tags (ESTs) is challenging due to high sequence redundancy and potential cross-hybridization between paralogous genes. In organisms with limited genomic information, like marine organisms, this challenge is even greater due to annotation uncertainty. No general tool is available for cDNA microarray probe selection for these organisms. Therefore, the goal of the design procedure described here is to select a subset of ESTs that will minimize sequence redundancy and characterize potential cross-hybridization while providing functionally representative probes. Results Sequence similarity between ESTs, quantified by the E-value of pair-wise alignment, was used as a surrogate for expected hybridization between corresponding sequences. Using this value as a measure of dissimilarity, sequence redundancy reduction was performed by hierarchical cluster analyses. The choice of how many microarray probes to retain was made based on an index developed for this research: a sequence diversity index (SDI) within a sequence diversity plot (SDP). This index tracked the decreasing within-cluster sequence diversity as the number of clusters increased. For a given stage in the agglomeration procedure, the EST having the highest similarity to all the other sequences within each cluster, the centroid EST, was selected as a microarray probe. A small dataset of ESTs from Atlantic white shrimp (Litopenaeus setiferus) was used to test this algorithm so that the detailed results could be examined. The functional representative level of the selected probes was quantified using Gene Ontology (GO) annotations. Conclusions For organisms with limited genomic information, combining hierarchical clustering methods to analyze ESTs can yield an optimal cDNA microarray design. If

  11. Multiplex PCR, amplicon size and hybridization efficiency on the NanoChip electronic microarray.

    PubMed

    Børsting, Claus; Sanchez, Juan J; Morling, Niels

    2004-04-01

    We tested the SNP typing protocol developed for the NanoChip electronic microarray by analyzing the four Y chromosome loci SRY1532, SRY8299, TAT, and 92R7. Amplicons of different lengths containing the same locus were purified and addressed to the NanoChip array and fluorescently labelled reporter probes were hybridized to the amplicons. We demonstrated that as little as 10-30 fmol of 50 bp DNA amplicons was sufficient to obtain strong and reproducible results. The hybridization to 50 bp amplicons was up to 10 times more efficient than the hybridization to 200 bp amplicons containing the same SNP. Hybridization to individual amplicons in multiplexes was less efficient suggesting that intramolecular and intermolecular interactions may block access to the target sequence on the NanoChip array. We observed a high risk of contamination with amplicons shorter than 60 bp and therefore, we recommend the use of 60-200 bp amplicons for SNP typing analysis on the NanoChip platform. In a comparative study, we typed the 5 Y chromosome loci M173, 92R7, P25, SRY1532, and M9 in 400 males using the NanoChip SNP typing protocol and the SNaPshot kit. Concording results were obtained for all samples demonstrating the accuracy of the NanoChip SNP typing protocol.

  12. 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

  13. Electronic hybridization detection in microarray format and DNA genotyping

    PubMed Central

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-01-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device. PMID:24569823

  14. Electronic hybridization detection in microarray format and DNA genotyping

    NASA Astrophysics Data System (ADS)

    Blin, Antoine; Cissé, Ismaïl; Bockelmann, Ulrich

    2014-02-01

    We describe an approach to substituting a fluorescence microarray with a surface made of an arrangement of electrolyte-gated field effect transistors. This was achieved using a dedicated blocking of non-specific interactions and comparing threshold voltage shifts of transistors exhibiting probe molecules of different base sequence. We apply the approach to detection of the 35delG mutation, which is related to non-syndromic deafness and is one of the most frequent mutations in humans. The process involves barcode sequences that are generated by Tas-PCR, a newly developed replication reaction using polymerase blocking. The barcodes are recognized by hybridization to surface attached probes and are directly detected by the semiconductor device.

  15. 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...

  16. USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    EPA Science Inventory

    USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION
    IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    John C. Rockett1, J. Christopher Luft1, J. Brian Garges1, M. Stacey Ricci2, Pasquale Patrizio2, Norman B. Hecht2 and David J. Dix1
    Reproductive Toxicology Divisio...

  17. Simultaneous detection of multiple fish pathogens using a naked-eye readable DNA microarray.

    PubMed

    Chang, Chin-I; Hung, Pei-Hsin; Wu, Chia-Che; Cheng, Ta Chih; Tsai, Jyh-Ming; Lin, King-Jung; Lin, Chung-Yen

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 10(3) CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens.

  18. Direct identification of chlamydiae from clinical samples using a DNA microarray assay: a validation study.

    PubMed

    Borel, Nicole; Kempf, Evelyne; Hotzel, Helmut; Schubert, Evelyn; Torgerson, Paul; Slickers, Peter; Ehricht, Ralf; Tasara, Taurai; Pospischil, Andreas; Sachse, Konrad

    2008-02-01

    While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.

  19. Simultaneous Detection of Multiple Fish Pathogens Using a Naked-Eye Readable DNA Microarray

    PubMed Central

    Chang, Chin-I; Hung, Pei-Hsin; Wu, Chia-Che; Cheng, Ta Chih; Tsai, Jyh-Ming; Lin, King-Jung; Lin, Chung-Yen

    2012-01-01

    We coupled 16S rDNA PCR and DNA hybridization technology to construct a microarray for simultaneous detection and discrimination of eight fish pathogens (Aeromonas hydrophila, Edwardsiella tarda, Flavobacterium columnare, Lactococcus garvieae, Photobacterium damselae, Pseudomonas anguilliseptica, Streptococcus iniae and Vibrio anguillarum) commonly encountered in aquaculture. The array comprised short oligonucleotide probes (30 mer) complementary to the polymorphic regions of 16S rRNA genes for the target pathogens. Targets annealed to the microarray probes were reacted with streptavidin-conjugated alkaline phosphatase and nitro blue tetrazolium/5-bromo-4-chloro-3′-indolylphosphate, p-toluidine salt (NBT/BCIP), resulting in blue spots that are easily visualized by the naked eye. Testing was performed against a total of 168 bacterial strains, i.e., 26 representative collection strains, 81 isolates of target fish pathogens, and 61 ecologically or phylogenetically related strains. The results showed that each probe consistently identified its corresponding target strain with 100% specificity. The detection limit of the microarray was estimated to be in the range of 1 pg for genomic DNA and 103 CFU/mL for pure pathogen cultures. These high specificity and sensitivity results demonstrate the feasibility of using DNA microarrays in the diagnostic detection of fish pathogens. PMID:22736973

  20. USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    EPA Science Inventory

    USING DNA MICROARRAYS TO CHARACTERIZE GENE EXPRESSION
    IN TESTES OF FERTILE AND INFERTILE HUMANS AND MICE

    John C. Rockett1, J. Christopher Luft1, J. Brian Garges1, M. Stacey Ricci2, Pasquale Patrizio2, Norman B. Hecht2 and David J. Dix1
    Reproductive Toxicology Divisio...

  1. Comparison of DNA ploidy status and DNA ploidy-related parameters in malignant melanoma tissue microarrays and full sections.

    PubMed

    Korabiowska, Monika; Cordon-Cardo, Carlos; Buschmann, Nadine; Stachura, Jerzy; Fischer, Gösta; Brinck, Ulrich

    2004-07-01

    A new high-throughput tissue-arraying technique, now frequently used in tumor pathology, requires standardization of methods of DNA analysis, previously applied in full histological sections. The main objectives of this study were to evaluate DNA ploidy status and DNA ploidy-related parameters using the CAS200 image analyzer in malignant melanoma tissue microarrays and to compare them with full histological sections. Comparison of DNA ploidy-related parameters, including percentage of diploid cells, percentage of aneuploid cells between 2c and 4c, percentage of tetraploid cells, percentage of aneuploid cells between 4c and 8c, percentage of octaploid cells, percentage of 16-ploid cells, and 5c exceeding rate, did not reveal any significant differences between malignant melanoma tissue microarrays and full sections. The DNA ploidy status according to Auer differed in 1 out of 59 cases investigated. Our study demonstrated that it is possible to evaluate DNA ploidy status and DNA ploidy-related parameters in tissue microarrays, which is of practical relevance to tumor pathology.

  2. Robust embryo identification using first polar body single nucleotide polymorphism microarray-based DNA fingerprinting.

    PubMed

    Treff, Nathan R; Su, Jing; Kasabwala, Natasha; Tao, Xin; Miller, Kathleen A; Scott, Richard T

    2010-05-01

    This study sought to validate a novel, minimally invasive system for embryo tracking by single nucleotide polymorphism microarray-based DNA fingerprinting of the first polar body. First polar body-based assignments of which embryos implanted and were delivered after multiple ET were 100% consistent with previously validated embryo DNA fingerprinting-based assignments. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  3. A newly developed DNA microarray is useful to assess induction of cytochromes p450 in the cynomolgus monkey.

    PubMed

    Ise, Ryota; Uehara, Shotaro; Akiyama, Hideo; Kondo, Satoshi; Iwasaki, Kazuhide; Nagata, Ryoichi; Nobumasa, Hitoshi; Yamazaki, Hiroshi; Uno, Yasuhiro

    2011-06-01

    Cytochromes P450 (P450s or CYPs) are a gene family of highly homologous genes and include the CYP1-4 family, which is relevant to drug metabolism. In the cynomolgus monkey (which is frequently used in drug metabolism studies), numerous CYPs (mfCYPs) have been identified in the CYP1-4 family. DNA microarrays are useful for high-throughput screening assays; however, there is a potential problem with cross-hybridization of highly homologous genes in the gene family. This problem might be solved with the use of low-density DNA microarrays, with which specific validation can be performed for the genes on the microarray. We have developed a DNA microarray for the 20 mfCYPs and have evaluated and validated its specificity and usefulness. First, in both DNA microarray and quantitative polymerase chain reaction (qPCR) analyses, hepatic expression of each mfCYP correlated well, and similar tissue expression patterns were observed for five representative mfCYPs, confirming the specificity of the DNA microarray. Second, the usefulness of this DNA microarray was validated by induction analysis of mfCYPs in primary hepatocytes, which successfully detected known responders, but also novel responders (mfCYP2C43, mfCYP2C75, and mfCYP3A5 for rifampicin), as confirmed by qPCR analysis. This DNA microarray can thus be utilized for high-throughput assays during drug development.

  4. The microarray explorer tool for data mining of cDNA microarrays: application for the mammary gland.

    PubMed

    Lemkin, P F; Thornwall, G C; Walton, K D; Hennighausen, L

    2000-11-15

    The Microarray Explorer (MAExplorer) is a versatile Java-based data mining bioinformatic tool for analyzing quantitative cDNA expression profiles across multiple microarray platforms and DNA labeling systems. It may be run as either a stand-alone application or as a Web browser applet over the Internet. With this program it is possible to (i) analyze the expression of individual genes, (ii) analyze the expression of gene families and clusters, (iii) compare expression patterns and (iv) directly access other genomic databases for clones of interest. Data may be downloaded as required from a Web server or in the case of the stand-alone version, reside on the user's computer. Analyses are performed in real-time and may be viewed and directly manipulated in images, reports, scatter plots, histograms, expression profile plots and cluster analyses plots. A key feature is the clone data filter for constraining a working set of clones to those passing a variety of user-specified logical and statistical tests. Reports may be generated with hypertext Web access to UniGene, GenBank and other Internet databases for sets of clones found to be of interest. Users may save their explorations on the Web server or local computer and later recall or share them with other scientists in this groupware Web environment. The emphasis on direct manipulation of clones and sets of clones in graphics and tables provides a high level of interaction with the data, making it easier for investigators to test ideas when looking for patterns. We have used the MAExplorer to profile gene expression patterns of 1500 duplicated genes isolated from mouse mammary tissue. We have identified genes that are preferentially expressed during pregnancy and during lactation. One gene we identified, carbonic anhydrase III, is highly expressed in mammary tissue from virgin and pregnant mice and in gene knock-out mice with underdeveloped mammary epithelium. Other genes, which include those encoding milk proteins

  5. 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.

  6. A multichannel order-statistic technique for cDNA microarray image processing.

    PubMed

    Lukac, Rastislav; Plataniotis, Konstantinos N; Smolka, Bogdan; Venetsanopoulos, Anastasios N

    2004-12-01

    This paper introduces an automated image processing procedure capable of processing complementary deoxyribonucleic acid (cDNA) microarray images. Microarray data is contaminated by noise and suffers from broken edges and visual artifacts. Without the utilization of a filter, subsequent tasks such as spot identification and gene expression determination cannot be completed. By employing, in a unique cascade processing cycle, nonlinear filtering solutions based on robust order statistics, the procedure: 1) removes both background and high-frequency corrupting noise and 2) correctly identifies edges and spots in cDNA microarray data. The proposed solution operates directly on the microarray data, does not rely on explicit data normalization or spot separation preprocessing, and operates in a robust manner without using heuristically determined design parameters. Other routine microarray processing operations such as shape manipulations and grid adjustments can be used in conjunction with the developed solution in the processing pipeline. Experimentation reported in this paper indicates that the proposed solution yields excellent performance by removing noise and enhancing spot location determination.

  7. Toward 'smart' DNA microarrays: algorithms for improving data quality and statistical inference

    NASA Astrophysics Data System (ADS)

    Bakewell, David J. G.; Wit, Ernst

    2007-12-01

    DNA microarrays are a laboratory tool for understanding biological processes at the molecular scale and future applications of this technology include healthcare, agriculture, and environment. Despite their usefulness, however, the information microarrays make available to the end-user is not used optimally, and the data is often noisy and of variable quality. This paper describes the use of hierarchical Maximum Likelihood Estimation (MLE) for generating algorithms that improve the quality of microarray data and enhance statistical inference about gene behavior. The paper describes examples of recent work that improves microarray performance, demonstrated using data from both Monte Carlo simulations and published experiments. One example looks at the variable quality of cDNA spots on a typical microarray surface. It is shown how algorithms, derived using MLE, are used to "weight" these spots according to their morphological quality, and subsequently lead to improved detection of gene activity. Another example, briefly discussed, addresses the "noisy data about too many genes" issue confronting many analysts who are also interested in the collective action of a group of genes, often organized as a pathway or complex. Preliminary work is described where MLE is used to "share" variance information across a pre-assigned group of genes of interest, leading to improved detection of gene activity.

  8. Use of a DNA Microarray for Simultaneous Detection of Antibiotic Resistance Genes among Staphylococcal Clinical Isolates▿

    PubMed Central

    Zhu, Ling-Xiang; Zhang, Zhi-Wei; Wang, Can; Yang, Hua-Wei; Jiang, Di; Zhang, Qiong; Mitchelson, Keith; Cheng, Jing

    2007-01-01

    We developed a multiplex asymmetric PCR (MAPCR)-based DNA microarray assay for characterization of the clinically relevant antibiotic resistance genes leading to penicillin, methicillin, aminoglycoside, macrolide, lincosamide, and streptogramin B (MLSB) resistance in staphylococci. The DNA-based assay involves detection of specific conserved regions of the mecA, blaZ (methicillin and penicillin resistance), aac(6′)-Ie-aph(2‴) (aminoglycoside resistance), ermA and ermC genes (MLSB resistance), and the msrA gene (macrolide and streptogramin B resistance). The microarray uses a variable sequence region of the 16S rRNA gene to broadly differentiate between Staphylococcus aureus and other coagulase-negative staphylococci (CoNS). The performance of the microarray was validated with a total of 178 clinically important S. aureus and 237 CoNS isolates, with correlations of 100% for S. aureus to CoNS discrimination and more than 90% for antibiotic resistance between the genotypic analysis determined by the microarray and the phenotype determined by standard methods of species identification and susceptibility testing. The major discrepant results were 17 mecA-positive CoNS and 60 aac(6′)-Ie-aph(2‴)-positive CoNS isolates measured by microarray that were susceptible to the corresponding antibiotics based on disk diffusion assay. Overall, this microarray-based assay offers a simultaneous, fast (≤5 h), and accurate identification of antibiotic resistance genes from a single colony, as well as species classification. Our extensive validation of the microarray suggests that it may be a useful tool to complement phenotypic susceptibility testing in clinical laboratories and to survey the spread of antibiotic resistance determinants in epidemiological studies. PMID:17728472

  9. Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays.

    PubMed

    Panicker, Gitika; Call, Douglas R; Krug, Melissa J; Bej, Asim K

    2004-12-01

    This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50 degrees C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 10(2) to 10(3) CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

  10. A gDNA microarray for genotyping salvia species.

    PubMed

    Olarte, Alexandra; Mantri, Nitin; Nugent, Gregory; Wohlmuth, Hans; Li, Chun Guang; Xue, Charlie; Pang, Edwin

    2013-07-01

    Salvia is an important genus from the Lamiaceae with approximately 1,000 species. This genus is distributed globally and cultivated for ornamental, culinary, and medicinal uses. We report the construction of the first fingerprinting array for Salvia species enriched with polymorphic and divergent DNA sequences and demonstrate the potential of this array for fingerprinting several economically important members of this genus. In order to generate the Salvia subtracted diversity array (SDA) a suppression subtractive hybridization (SSH) was performed between a pool of Salvia species and a pool of angiosperms and non-angiosperms to selectively isolate Salvia-specific sequences. A total of 285-subtracted genomic DNA (gDNA) fragments were amplified and arrayed. DNA fingerprints were obtained for fifteen Salvia genotypes including three that were not part of the original subtraction pool. Hierarchical cluster analysis indicated that the Salvia-specific SDA was capable of differentiating S. officinalis and S. miltiorrhiza from their closely related species and was also able to reveal genetic relationships consistent with geographical origins. In addition, this approach was capable of isolating highly polymorphic sequences from chloroplast and nuclear DNA without preliminary sequence information. Therefore, SDA is a powerful technique for fingerprinting non-model plants and for identifying new polymorphic loci that may be developed as potential molecular markers.

  11. DNA microarray analysis of genes differentially expressed in adipocyte differentiation.

    PubMed

    Yin, Chunyan; Xiao, Yanfeng; Zhang, Wei; Xu, Erdi; Liu, Weihua; Yi, Xiaoqing; Chang, Ming

    2014-06-01

    In the present study, the human liposarcoma cell line SW872 was used to identify global changes in gene expression profiles occurring during adipogenesis. We further explored some of the genes expressed during the late phase of adipocyte differentiation. These genes may play a major role in promoting excessive proliferation and accumulation of lipid droplets, which contribute to the development of obesity. By using microarray-based technology, we examined differential gene expression in early differentiated adipocytes and late differentiated adipocytes. Validated genes exhibited a greater than or equal to 10-fold increase in the late phase of adipocyte differentiation by polymerase chain reaction (RT-PCR). Compared with undifferentiated preadipocytes, we found that 763 genes were increased in early differentiated adipocytes, and 667 genes were increased in later differentiated adipocytes. Furthermore, 21 genes were found being expressed 10-fold higher in the late phase of adipocyte differentiation. The results were in accordance with the RTPCR test, which validated 11 genes, namely, CIDEC, PID1, LYRM1, ADD1, PPAR?2, ANGPTL4, ADIPOQ, ACOX1, FIP1L1, MAP3K2 and PEX14. Most of these genes were found being expressed in the later phase of adipocyte differentiation involved in obesity-related diseases. The findings may help to better understand the mechanism of obesity and related diseases.

  12. Xylella fastidiosa gene expression analysis by DNA microarrays

    PubMed Central

    2009-01-01

    Xylella fastidiosa genome sequencing has generated valuable data by identifying genes acting either on metabolic pathways or in associated pathogenicity and virulence. Based on available information on these genes, new strategies for studying their expression patterns, such as microarray technology, were employed. A total of 2,600 primer pairs were synthesized and then used to generate fragments using the PCR technique. The arrays were hybridized against cDNAs labeled during reverse transcription reactions and which were obtained from bacteria grown under two different conditions (liquid XDM2 and liquid BCYE). All data were statistically analyzed to verify which genes were differentially expressed. In addition to exploring conditions for X. fastidiosa genome-wide transcriptome analysis, the present work observed the differential expression of several classes of genes (energy, protein, amino acid and nucleotide metabolism, transport, degradation of substances, toxins and hypothetical proteins, among others). The understanding of expressed genes in these two different media will be useful in comprehending the metabolic characteristics of X. fastidiosa, and in evaluating how important certain genes are for the functioning and survival of these bacteria in plants. PMID:21637690

  13. Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays

    PubMed Central

    Guimond, Chantal; Trudel, Nathalie; Brochu, Christian; Marquis, Nathalie; Fadili, Amal El; Peytavi, Régis; Briand, Guylaine; Richard, Dave; Messier, Nadine; Papadopoulou, Barbara; Corbeil, Jacques; Bergeron, Michel G.; Légaré, Danielle; Ouellette, Marc

    2003-01-01

    In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes γ-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance. PMID:14530437

  14. Modulation of gene expression in Leishmania drug resistant mutants as determined by targeted DNA microarrays.

    PubMed

    Guimond, Chantal; Trudel, Nathalie; Brochu, Christian; Marquis, Nathalie; El Fadili, Amal; Peytavi, Régis; Briand, Guylaine; Richard, Dave; Messier, Nadine; Papadopoulou, Barbara; Corbeil, Jacques; Bergeron, Michel G; Légaré, Danielle; Ouellette, Marc

    2003-10-15

    In the protozoan parasite Leishmania, drug resistance can be a complex phenomenon. Several metabolic pathways and membrane transporters are implicated in the resistance phenotype. To monitor the expression of these genes, we generated custom DNA microarrays with PCR fragments corresponding to 44 genes involved with drug resistance. Transcript profiling of arsenite and antimony resistant mutants with these arrays pinpointed a number of genes overexpressed in mutants, including the ABC transporter PGPA, the glutathione biosynthesis genes gamma-glutamylcysteine synthetase (GSH1) and the glutathione synthetase (GSH2). Competitive hybridisations with total RNA derived from sensitive and methotrexate resistant cells revealed the overexpression of genes coding for dihydrofolate reductase (DHFR-TS), pteridine reductase (PTR1) and S-adenosylmethionine synthase (MAT2) and a down regulation of one gene of the folate transporter (FT) family. By labelling the DNA of sensitive and resistant parasites we could also detect several gene amplification events using DNA microarrays including the amplification of the S-adenosyl homocysteine hydrolase gene (SAHH). Alteration in gene expression detected by microarrays was validated by northern blot analysis, while Southern blots indicated that most genes overexpressed were also amplified, although other mechanisms were also present. The microarrays were useful in the study of resistant parasites to pinpoint several genes linked to drug resistance.

  15. Preparation of fluorescent-dye-labeled cDNA from RNA for microarray hybridization.

    PubMed

    Ares, Manuel

    2014-01-01

    This protocol describes how to prepare fluorescently labeled cDNA for hybridization to microarrays. It consists of two steps: first, a mixture of anchored oligo(dT) and random hexamers is used to prime amine-modified cDNA synthesis by reverse transcriptase using a modified deoxynucleotide with a reactive amine group (aminoallyl-dUTP) and an RNA sample as a template. Second, the cDNA is purified and exchanged into bicarbonate buffer so that the amine groups in the cDNA react with the dye N-hydroxysuccinimide (NHS) esters, covalently joining the dye to the cDNA. The dye-coupled cDNA is purified again, and the amount of dye incorporated per microgram of cDNA is determined.

  16. DNA Microarrays for Aptamer Identification and Structural Characterization

    DTIC Science & Technology

    2012-09-01

    Zhou, T., Gulari, E., “ Microfluidic Reactor Array Device for Massively Parallel in Situ Synthesis of Oligonucleotides,” Sensors and Actuators B...Soh, H. T., “Quantitative Selection of DNA Aptamers Through Microfluidic Selection and High- throughput Sequencing,” Proc Natl Acad Sci., 107, 2010

  17. A three-dimensional waveguide substrate for DNA-microarrays based on macroporous silicon

    NASA Astrophysics Data System (ADS)

    Dertinger, Stephan K.; Klühr, Marco; Sauermann, Alexander; Thein, Kerstin

    2005-06-01

    In this paper we present a three-dimensional waveguide structure with unique optical and fluidic properties and demonstrate its application as a substrate for DNA microarrays. The structure is fabricated by thermal oxidation of a macroporous silicon membrane with a periodic pattern of discrete microchannels running perpendicular through the substrate. Partial oxidation generates compartments with channel walls that are completely converted into SiO2 but leaves a rectangular grid of silicon walls separating the SiO2 compartments. We demonstrate that the SiO2 walls act as optical waveguides and the opaque silicon walls divide the substrate into optically isolated compartments. In DNA microarray experiments, we show that the silicon walls of the compartments prevent cross talk between adjacent DNA spots. The structure is compatible with all conventional read-out techniques such as fluorescence, chemiluminescence, and precipitation staining.

  18. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization1

    PubMed Central

    Beheshti, Ben; Braude, Ilan; Marrano, Paula; Thorner, Paul; Zielenska, Maria; Squire, Jeremy A

    2003-01-01

    Abstract Conventional comparative genomic hybridization (CGH) profiling of neuroblastomas has identified many genomic aberrations, although the limited resolution has precluded a precise localization of sequences of interest within amplicons. To map high copy number genomic gains in clinically matched stage IV neuroblastomas, CGH analysis using a 19,200-feature cDNA microarray was used. A dedicated (freely available) algorithm was developed for rapid in silico determination of chromosomal localizations of microarray cDNA targets, and for generation of an ideogram-type profile of copy number changes. Using these methodologies, novel gene amplifications undetectable by chromosome CGH were identified, and larger MYCN amplicon sizes (in one tumor up to 6 Mb) than those previously reported in neuroblastoma were identified. The genes HPCAL1, LPIN1/KIAA0188, NAG, and NSE1/LOC151354 were found to be coamplified with MYCN. To determine whether stage IV primary tumors could be further subclassified based on their genomic copy number profiles, hierarchical clustering was performed. Cluster analysis of microarray CGH data identified three groups: 1) no amplifications evident, 2) a small MYCN amplicon as the only detectable imbalance, and 3) a large MYCN amplicon with additional gene amplifications. Application of CGH to cDNA microarray targets will help to determine both the variation of amplicon size and help better define amplification-dependent and independent pathways of progression in neuroblastoma. PMID:12659670

  19. DNA Microarray-Based Typing of Streptococcus agalactiae Isolates

    PubMed Central

    Nitschke, Heike; Slickers, Peter; Müller, Elke; Ehricht, Ralf

    2014-01-01

    Streptococcus agalactiae frequently colonizes the urogenital tract, and it is a major cause of bacterial septicemia, meningitis, and pneumonia in newborns. For typing purposes, a microarray targeting group B streptococcus (GBS) virulence-associated markers and resistance genes was designed and validated with reference strains, as well as clinical and veterinary isolates. Selected isolates were also subjected to multilocus sequence typing. It was observed that putative typing markers, such as alleles of the alpha-like protein or capsule types, vary independently of each other, and they also vary independently from the affiliation to their multilocus sequence typing (MLST)-defined sequence types. Thus, it is not possible to assign isolates to sequence types based on the identification of a single distinct marker, such as a capsule type or alp allele. This suggests the occurrence of frequent genomic recombination. For array-based typing, a set of 11 markers (bac, alp, pil1 locus, pepS8, fbsB, capsule locus, hylB, abiG-I/-II plus Q8DZ34, pil2 locus, nss plus srr plus rogB2, and rgfC/A/D/B) was defined that provides a framework for splitting the tested 448 S. agalactiae isolates into 76 strains that clustered mainly according to MLST-defined clonal complexes. There was evidence for region- and host-specific differences in the population structure of S. agalactiae, as well as an overrepresentation of strains related to sequence type 17 among the invasive isolates. The arrays and typing scheme described here proved to be a convenient tool for genotyping large numbers of clinical/veterinary isolates and thus might help obtain insight into the epidemiology of S. agalactiae. PMID:25165085

  20. ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

    PubMed Central

    Moulton, Kevin R.; Taylor, Amber W.; Rowlen, Kathy L.; Dawson, Erica D.

    2011-01-01

    DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective. The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is ˜1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection. Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one

  1. Direct immobilization of DNA oligomers onto the amine-functionalized glass surface for DNA microarray fabrication through the activation-free reaction of oxanine

    PubMed Central

    Pack, Seung Pil; Kamisetty, Nagendra Kumar; Nonogawa, Mitsuru; Devarayapalli, Kamakshaiah Charyulu; Ohtani, Kairi; Yamada, Kazunari; Yoshida, Yasuko; Kodaki, Tsutomu; Makino, Keisuke

    2007-01-01

    Oxanine having an O-acylisourea structure was explored to see if its reactivity with amino group is useful in DNA microarray fabrication. By the chemical synthesis, a nucleotide unit of oxanine (Oxa-N) was incorporated into the 5′-end of probe DNA with or without the -(CH2)n- spacers (n = 3 and 12) and found to immobilize the probe DNA covalently onto the NH2-functionalized glass slide by one-pot reaction, producing the high efficiency of the target hybridization. The methylene spacer, particularly the longer one, generated higher efficiency of the target recognition although there was little effect on the amount of the immobilized DNA oligomers. The post-spotting treatment was also carried out under the mild conditions (at 25 or 42°C) and the efficiencies of the immobilization and the target recognition were evaluated similarly, and analogous trends were obtained. It has also been determined under the mild conditions that the humidity and time of the post-spotting treatment, pH of the spotting solution and the synergistic effects with UV-irradiation largely contribute to the desired immobilization and resulting target recognition. Immobilization of DNA oligomer by use of Oxa-N on the NH2-functionalized surface without any activation step would be employed as one of the advanced methods for generating DNA-conjugated solid surface. PMID:17715142

  2. 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.

  3. 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

  4. Gene expression analysis of perennial ryegrass (Lolium perenne) using cDNA microarrays

    NASA Astrophysics Data System (ADS)

    Ong, Eng-Kok; Sawbridge, Tim; Webster, Tracie; Emmerling, Michael; Nguyen, Nga; Nunan, Katrina; O'Neill, Matthew; O'Toole, Fiona; Rhodes, Carolyn; Simmonds, Jason; Tian, Pei; Wearne, Katherine; Winkworth, Amanda; Spangenberg, German

    2003-07-01

    Perennial ryegrass (Lolium perenne) is a major forage grass of temperate pastures. A genomics program has been undertaken generating over 52,000 expressed sequence tags (ESTs). Cluster analysis of the ESTs identified approximately 14,600 ryegrass unigenes. In this report, we described the application of ryegrass unigene cDNAs to produce ryegrass 15K microarray. Fifteen microarray hybridisations were performed with labeled total RNA isolated from a variety of plant organs and developmental stages. In a proof of concept, gene expression profiling of ryegrass ESTs using the 15K unigene microarrays has been established using several known genes and two cluster analysis approaches (parallel coordinate planes plot and hierarchical clustering). The expression profile of the known genes (e.g. rubisco and invertase) corresponds well with published data. The microarray expression profile of a ryegrass putative root specific kinase gene was also verified with Northern blotting. This combination of DNA microarray hybridisations and cluster analysis can be applied as a tool for the identification of novel sequences of unknown function.

  5. The role of DNA microarrays in the evaluation of fetal death.

    PubMed

    Reddy, Uma M; Page, Grier P; Saade, George R

    2012-04-01

    Fetal death occurs in 15% of clinically recognized pregnancies. Cytogenetic abnormalities are present in 50% of spontaneous abortions (fetal deaths < 20 weeks) whereas the rate is 6% to 13% for stillbirths (fetal deaths ≥ 20 weeks). Microarray has been demonstrated to increase the diagnosis of genetic abnormalities by providing coverage of the entire genome at a higher density, detecting as small as 50 to 100 kb deletions or duplications, known as copy number changes. Microarray is particularly suited for evaluation of fetal death because DNA can still be analyzed in macerated fetuses and nonviable tissue, two situations where culturing and karyotyping is known to have low yield. Microarray has already proven successful in providing additional genetic information beyond karyotype in spontaneous abortion. The few studies on the use of microarray in stillbirth evaluation have been promising, demonstrating an increase in the diagnosis of clinically relevant genetic abnormalities when compared with karyotype. As the cost and technology improve, microarray may ultimately become the first line screen for genetic abnormalities in stillbirth. The accurate diagnosis of a genetic abnormality as the cause for fetal death may provide closure for families, prevent unnecessary treatments, and enable clinicians to more accurately counsel and manage subsequent pregnancies.

  6. Quantitative comparison of the HSV-1 and HSV-2 transcriptomes using DNA microarray analysis

    SciTech Connect

    Aguilar, J.S. . E-mail: jsaguila@uci.edu; Devi-Rao, G.V.; Rice, M.K.; Sunabe, J.; Ghazal, P.; Wagner, E.K.

    2006-04-25

    The genomes of human herpes virus type-1 and type-2 share a high degree of sequence identity; yet, they exhibit important differences in pathology in their natural human host as well as in animal host and cell cultures. Here, we report the comparative analysis of the time and relative abundance profiles of the transcription of each virus type (their transcriptomes) using parallel infections and microarray analysis using HSV-1 probes which hybridize with high efficiency to orthologous HSV-2 transcripts. We have confirmed that orthologous transcripts belong to the same kinetic class; however, the temporal pattern of accumulation of 4 transcripts (U{sub L}4, U{sub L}29, U{sub L}30, and U{sub L}31) differs in infections between the two virus types. Interestingly, the protein products of these transcripts are all involved in nuclear organization and viral DNA localization. We discuss the relevance of these findings and whether they may have potential roles in the pathological differences of HSV-1 and HSV-2.

  7. 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

  8. 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.

  9. SNP-microarrays can accurately identify the presence of an individual in complex forensic DNA mixtures.

    PubMed

    Voskoboinik, Lev; Ayers, Sheri B; LeFebvre, Aaron K; Darvasi, Ariel

    2015-05-01

    Common forensic and mass disaster scenarios present DNA evidence that comprises a mixture of several contributors. Identifying the presence of an individual in such mixtures has proven difficult. In the current study, we evaluate the practical usefulness of currently available "off-the-shelf" SNP microarrays for such purposes. We found that a set of 3000 SNPs specifically selected for this purpose can accurately identify the presence of an individual in complex DNA mixtures of various compositions. For example, individuals contributing as little as 5% to a complex DNA mixture can be robustly identified even if the starting DNA amount was as little as 5.0ng and had undergone whole-genome amplification (WGA) prior to SNP analysis. The work presented in this study represents proof-of-principle that our previously proposed approach, can work with real "forensic-type" samples. Furthermore, in the absence of a low-density focused forensic SNP microarray, the use of standard, currently available high-density SNP microarrays can be similarly used and even increase statistical power due to the larger amount of available information.

  10. Differential gene expression in recombinant Pichia pastoris analysed by heterologous DNA microarray hybridisation

    PubMed Central

    Sauer, Michael; Branduardi, Paola; Gasser, Brigitte; Valli, Minoska; Maurer, Michael; Porro, Danilo; Mattanovich, Diethard

    2004-01-01

    Background Pichia pastoris is a well established yeast host for heterologous protein expression, however, the physiological and genetic information about this yeast remains scanty. The lack of a published genome sequence renders DNA arrays unavailable, thereby hampering more global investigations of P. pastoris from the beginning. Here, we examine the suitability of Saccharomyces cerevisiae DNA microarrays for heterologous hybridisation with P. pastoris cDNA. Results We could show that it is possible to obtain new and valuable information about transcriptomic regulation in P. pastoris by probing S. cerevisiae DNA microarrays. The number of positive signals was about 66 % as compared to homologous S. cerevisiae hybridisation, and both the signal intensities and gene regulations correlated with high significance between data obtained from P. pastoris and S. cerevisiae samples. The differential gene expression patterns upon shift from glycerol to methanol as carbon source were investigated in more detail. Downregulation of TCA cycle genes and a decrease of genes related to ribonucleotide and ribosome synthesis were among the major effects identified. Conclusions We could successfully demonstrate that heterologous microarray hybridisations allow deep insights into the transcriptomic regulation processes of P. pastoris. The observed downregulation of TCA cycle and ribosomal synthesis genes correlates to a significantly lower specific growth rate during the methanol feed phase. PMID:15610561

  11. Use of a DNA microarray for detection and identification of bacterial pathogens associated with fishery products.

    PubMed

    Cao, Boyang; Li, Rongrong; Xiong, Songjin; Yao, Fangfang; Liu, Xiangqian; Wang, Min; Feng, Lu; Wang, Lei

    2011-12-01

    We established a microarray for the simultaneous detection and identification of diverse putative pathogens often associated with fishery products by targeting specific genes of Listeria monocytogenes, Salmonella, Shigella, Staphylococcus aureus, Streptococcus pyogenes, Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica and the 16S-23S rRNA gene internal transcribed spacer (ITS) region of Proteus mirabilis and Proteus vulgaris. The microarray contained 26 specific probes and was tested against a total of 123 target bacterial strains that included 55 representative strains, 68 clinical isolates, and 45 strains of other bacterial species that belonged to 8 genera and 34 species, and it was shown to be specific and reproducible. A detection sensitivity of 10 ng DNA or 10 CFU/ml for pure cultures of each target organism demonstrated that the assay was highly sensitive and reproducible. Mock and real fishery product samples were tested by the microarray, and the accuracy was 100%. The DNA microarray method described in this communication is specific, sensitive, and reliable and has several advantages over traditional methods of bacterial culture and antiserum agglutination assays.

  12. Electronic microarray analysis of 16S rDNA amplicons for bacterial detection.

    PubMed

    Barlaan, Edward A; Sugimori, Miho; Furukawa, Seiji; Takeuchi, Kazuhisa

    2005-01-12

    Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.

  13. A novel neural network approach to cDNA microarray image segmentation.

    PubMed

    Wang, Zidong; Zineddin, Bachar; Liang, Jinling; Zeng, Nianyin; Li, Yurong; Du, Min; Cao, Jie; Liu, Xiaohui

    2013-07-01

    Microarray technology has become a great source of information for biologists to understand the workings of DNA which is one of the most complex codes in nature. Microarray images typically contain several thousands of small spots, each of which represents a different gene in the experiment. One of the key steps in extracting information from a microarray image is the segmentation whose aim is to identify which pixels within an image represent which gene. This task is greatly complicated by noise within the image and a wide degree of variation in the values of the pixels belonging to a typical spot. In the past there have been many methods proposed for the segmentation of microarray image. In this paper, a new method utilizing a series of artificial neural networks, which are based on multi-layer perceptron (MLP) and Kohonen networks, is proposed. The proposed method is applied to a set of real-world cDNA images. Quantitative comparisons between the proposed method and commercial software GenePix(®) are carried out in terms of the peak signal-to-noise ratio (PSNR). This method is shown to not only deliver results comparable and even superior to existing techniques but also have a faster run time.

  14. A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

    PubMed Central

    2012-01-01

    Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These

  15. An automated microfluidic system for single-stranded DNA preparation and magnetic bead-based microarray analysis

    PubMed Central

    Wang, Shuaiqin; Sun, Yujia; Liu, Yan; Xiang, Guangxin; Wang, Lei; Cheng, Jing; Liu, Peng

    2015-01-01

    We present an integrated microfluidic device capable of performing single-stranded DNA (ssDNA) preparation and magnetic bead-based microarray analysis with a white-light detection for detecting mutations that account for hereditary hearing loss. The entire operation process, which includes loading of streptavidin-coated magnetic beads (MBs) and biotin-labeled polymerase chain reaction products, active dispersion of the MBs with DNA for binding, alkaline denaturation of DNA, dynamic hybridization of the bead-labeled ssDNA to a tag array, and white-light detection, can all be automatically accomplished in a single chamber of the microchip, which was operated on a self-contained instrument with all the necessary components for thermal control, fluidic control, and detection. Two novel mixing valves with embedded polydimethylsiloxane membranes, which can alternately generate a 3-μl pulse flow at a peak rate of around 160 mm/s, were integrated into the chip for thoroughly dispersing magnetic beads in 2 min. The binding efficiency of biotinylated oligonucleotides to beads was measured to be 80.6% of that obtained in a tube with the conventional method. To critically test the performance of this automated microsystem, we employed a commercial microarray-based detection kit for detecting nine mutation loci that account for hereditary hearing loss. The limit of detection of the microsystem was determined as 2.5 ng of input K562 standard genomic DNA using this kit. In addition, four blood samples obtained from persons with mutations were all correctly typed by our system in less than 45 min per run. The fully automated, “amplicon-in-answer-out” operation, together with the white-light detection, makes our system an excellent platform for low-cost, rapid genotyping in clinical diagnosis. PMID:25825617

  16. An MCMC Algorithm for Target Estimation in Real-Time DNA Microarrays

    NASA Astrophysics Data System (ADS)

    Vikalo, Haris; Gokdemir, Mahsuni

    2010-12-01

    DNA microarrays detect the presence and quantify the amounts of nucleic acid molecules of interest. They rely on a chemical attraction between the target molecules and their Watson-Crick complements, which serve as biological sensing elements (probes). The attraction between these biomolecules leads to binding, in which probes capture target analytes. Recently developed real-time DNA microarrays are capable of observing kinetics of the binding process. They collect noisy measurements of the amount of captured molecules at discrete points in time. Molecular binding is a random process which, in this paper, is modeled by a stochastic differential equation. The target analyte quantification is posed as a parameter estimation problem, and solved using a Markov Chain Monte Carlo technique. In simulation studies where we test the robustness with respect to the measurement noise, the proposed technique significantly outperforms previously proposed methods. Moreover, the proposed approach is tested and verified on experimental data.

  17. VARAN: a web server for variability analysis of DNA microarray experiments.

    PubMed

    Golfier, G; Dang, M Tran; Dauphinot, L; Graison, E; Rossier, J; Potier, M-C

    2004-07-10

    Here, we describe a tool for VARiability Analysis of DNA microarrays experiments (VARAN), a freely available Web server that performs a signal intensity based analysis of the log2 expression ratio variability deduced from DNA microarray data (one or two channels). Two modules are proposed: VARAN generator to compute a sliding windows analysis of the experimental variability (mean and SD) and VARAN analyzer to compare experimental data with an asymptotic variability model previously built with the generator module from control experiments. Both modules provide normalized intensity signals with five possible methods, log ratio values and a list of genes showing significant variations between conditions. http://www.bionet.espci.fr/varan/ http://www.bionet.espci.fr/varan/help.html

  18. DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae.

    PubMed

    Yasokawa, Daisuke; Murata, Satomi; Iwahashi, Yumiko; Kitagawa, Emiko; Kishi, Katsuyuki; Okumura, Yukihiro; Iwahashi, Hitoshi

    2010-05-01

    Zinc pyrithione has been used in anti-dandruff shampoos and in anti-fouling paint on ships. However, little is known of its mode of action. We characterized the effects of sub-lethal concentrations of zinc pyrithione (Zpt) on Saccharomyces cerevisiae using DNA microarrays. The majority of the strongly upregulated genes are related to iron transport, and many of the strongly downregulated genes are related to the biosynthesis of cytochrome (heme). These data suggest that Zpt induces severe iron starvation. To confirm the DNA microarray data, we supplemented cultures containing Zpt with iron, and the growth of the yeast was restored significantly. From these results, we propose that the principal toxicity of zinc pyrithione arises from iron starvation.

  19. Assessing Differential Expression Measurements by Highly Parallel Pyrosequencing and DNA Microarrays: A Comparative Study

    PubMed Central

    Ariño, Joaquín; Casamayor, Antonio; Pérez, Julián Perez; Pedrola, Laia; Álvarez-Tejado, Miguel; Marbà, Martina; Santoyo, Javier

    2013-01-01

    Abstract To explore the feasibility of pyrosequencing for quantitative differential gene expression analysis we have performed a comparative study of the results of the sequencing experiments to those obtained by a conventional DNA microarray platform. A conclusion from our analysis is that, over a threshold of 35 normalized reads per gene, the measurements of gene expression display a good correlation with the references. The observed concordance between pyrosequencing and DNA microarray platforms beyond the threshold was of 0.8, measured as a Pearson's correlation coefficient. In differential gene expression the initial aim is the quantification the differences among transcripts when comparing experimental conditions. Thus, even in a scenario of low coverage the concordance in the measurements is quite acceptable. On the other hand, the comparatively longer read size obtained by pyrosequencing allows detecting unconventional splicing forms. PMID:21919703

  20. DNA microarray analysis of the liver of mice treated with cobalt chloride.

    PubMed

    Matsumoto, Kanako; Fujishiro, Hitomi; Satoh, Masahiko; Himeno, Seiichiro

    2010-12-01

    To investigate the in vivo effects of cobalt chloride on gene expression at early time points, DNA microarray analysis was performed on the liver of mice injected subcutaneously with cobalt chloride. The liver tissue samples were taken 0.5, 1, and 3 hr after injection. Of the 14 genes up-regulated at 0.5 hr after injection, 7 are related to immunological responses, and 4 of the 7 were found to be involved in the activation of interferon.

  1. Screening insertion libraries for mutations in many genes simultaneously using DNA microarrays

    PubMed Central

    Mahalingam, Ramamurthy; Fedoroff, Nina

    2001-01-01

    We describe a method to screen pools of DNA from multiple transposon lines for insertions in many genes simultaneously. We use thermal asymmetric interlaced–PCR, a hemispecific PCR amplification protocol that combines nested, insertion-specific primers with degenerate primers, to amplify DNA flanking the transposons. In reconstruction experiments with previously characterized Arabidopsis lines carrying insertions of the maize Dissociation (Ds) transposon, we show that fluorescently labeled, transposon-flanking fragments overlapping ORFs hybridize to cognate expressed sequence tags (ESTs) on a DNA microarray. We further show that insertions can be detected in DNA pools from as many as 100 plants representing different transposon lines and that all of the tested, transposon-disrupted genes whose flanking fragments can be amplified individually also can be detected when amplified from the pool. The ability of a transposon-flanking fragment to hybridize declines rapidly with decreasing homology to the spotted DNA fragment, so that only ESTs with >90% homology to the transposon-disrupted gene exhibit significant cross-hybridization. Because thermal asymmetric interlaced–PCR fragments tend to be short, use of the present method favors recovery of insertions in and near genes. We apply the technique to screening pools of new Ds lines using cDNA microarrays containing ESTs for ≈1,000 stress-induced and -repressed Arabidopsis genes. PMID:11416215

  2. Programmable and automated bead-based microfluidics for versatile DNA microarrays under isothermal conditions.

    PubMed

    Penchovsky, Robert

    2013-06-21

    Advances in modern genomic research depend heavily on applications of various devices for automated high- or ultra-throughput arrays. Micro- and nanofluidics offer possibilities for miniaturization and integration of many different arrays onto a single device. Therefore, such devices are becoming a platform of choice for developing analytical instruments for modern biotechnology. This paper presents an implementation of a bead-based microfluidic platform for fully automated and programmable DNA microarrays. The devices are designed to work under isothermal conditions as DNA immobilization and hybridization transfer are performed under steady temperature using reversible pH alterations of reaction solutions. This offers the possibility for integration of more selection modules onto a single chip compared to maintaining a temperature gradient. This novel technology allows integration of many modules on a single reusable chip reducing the application cost. The method takes advantage of demonstrated high-speed DNA hybridization kinetics and denaturation on beads under flow conditions, high-fidelity of DNA hybridization, and small sample volumes are needed. The microfluidic devices are applied for a single nucleotide polymorphism analysis and DNA sequencing by synthesis without the need for fluorescent removal step. Apart from that, the microfluidic platform presented is applicable to many areas of modern biotechnology, including biosensor devices, DNA hybridization microarrays, molecular computation, on-chip nucleic acid selection, high-throughput screening of chemical libraries for drug discovery.

  3. 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.

  4. MASQOT: a method for cDNA microarray spot quality control

    PubMed Central

    Bylesjö, Max; Eriksson, Daniel; Sjödin, Andreas; Sjöström, Michael; Jansson, Stefan; Antti, Henrik; Trygg, Johan

    2005-01-01

    Background cDNA microarray technology has emerged as a major player in the parallel detection of biomolecules, but still suffers from fundamental technical problems. Identifying and removing unreliable data is crucial to prevent the risk of receiving illusive analysis results. Visual assessment of spot quality is still a common procedure, despite the time-consuming work of manually inspecting spots in the range of hundreds of thousands or more. Results A novel methodology for cDNA microarray spot quality control is outlined. Multivariate discriminant analysis was used to assess spot quality based on existing and novel descriptors. The presented methodology displays high reproducibility and was found superior in identifying unreliable data compared to other evaluated methodologies. Conclusion The proposed methodology for cDNA microarray spot quality control generates non-discrete values of spot quality which can be utilized as weights in subsequent analysis procedures as well as to discard spots of undesired quality using the suggested threshold values. The MASQOT approach provides a consistent assessment of spot quality and can be considered an alternative to the labor-intensive manual quality assessment process. PMID:16223442

  5. Development of a Daphnia magna DNA microarray for evaluating the toxicity of environmental chemicals.

    PubMed

    Watanabe, Hajime; Takahashi, Eri; Nakamura, Yuko; Oda, Shigeto; Tatarazako, Norihisa; Iguchi, Taisen

    2007-04-01

    Toxic chemical contaminants have a variety of detrimental effects on various species, and the impact of pollutants on ecosystems has become an urgent issue. However, the majority of studies regarding the effects of chemical contaminants have focused on vertebrates. Among aquatic organisms, Daphnia magna has been used extensively to evaluate organism- and population-level responses of invertebrates to pollutants in acute toxicity or reproductive toxicity tests. Although these types of tests can provide information concerning hazardous concentrations of chemicals, they provide no information about their mode of action. Recent advances in molecular genetic techniques have provided tools to better understand the responses of aquatic organisms to pollutants. In the present study, we adapted some of the techniques of molecular genetics to develop new tools, which form the basis for an ecotoxicogenomic assessment of D. magna. Based on a Daphnia expressed sequence tag database, we developed an oligonucleotide-based DNA microarray with high reproducibility. The DNA microarray was used to evaluate gene expression profiles of neonatal daphnids exposed to several different chemicals: Copper sulfate, hydrogen peroxide, pentachlorophenol, or beta-naphthoflavone. Exposure to these chemicals resulted in characteristic patterns of gene expression that were chemical-specific, indicating that the Daphnia DNA microarray can be used for classification of toxic chemicals and for development of a mechanistic understanding of chemical toxicity on a common freshwater organism.

  6. Development and application of a DNA microarray-based yeast two-hybrid system

    PubMed Central

    Suter, Bernhard; Fontaine, Jean-Fred; Yildirimman, Reha; Raskó, Tamás; Schaefer, Martin H.; Rasche, Axel; Porras, Pablo; Vázquez-Álvarez, Blanca M.; Russ, Jenny; Rau, Kirstin; Foulle, Raphaele; Zenkner, Martina; Saar, Kathrin; Herwig, Ralf; Andrade-Navarro, Miguel A.; Wanker, Erich E.

    2013-01-01

    The yeast two-hybrid (Y2H) system is the most widely applied methodology for systematic protein–protein interaction (PPI) screening and the generation of comprehensive interaction networks. We developed a novel Y2H interaction screening procedure using DNA microarrays for high-throughput quantitative PPI detection. Applying a global pooling and selection scheme to a large collection of human open reading frames, proof-of-principle Y2H interaction screens were performed for the human neurodegenerative disease proteins huntingtin and ataxin-1. Using systematic controls for unspecific Y2H results and quantitative benchmarking, we identified and scored a large number of known and novel partner proteins for both huntingtin and ataxin-1. Moreover, we show that this parallelized screening procedure and the global inspection of Y2H interaction data are uniquely suited to define specific PPI patterns and their alteration by disease-causing mutations in huntingtin and ataxin-1. This approach takes advantage of the specificity and flexibility of DNA microarrays and of the existence of solid-related statistical methods for the analysis of DNA microarray data, and allows a quantitative approach toward interaction screens in human and in model organisms. PMID:23275563

  7. DNA Microarray Wet Lab Simulation Brings Genomics into the High School Curriculum

    PubMed Central

    Zanta, Carolyn A.; Heyer, Laurie J.; Kittinger, Ben; Gabric, Kathleen M.; Adler, Leslie

    2006-01-01

    We have developed a wet lab DNA microarray simulation as part of a complete DNA microarray module for high school students. The wet lab simulation has been field tested with high school students in Illinois and Maryland as well as in workshops with high school teachers from across the nation. Instead of using DNA, our simulation is based on pH indicators, which offer many ideal teaching characteristics. The simulation requires no specialized equipment, is very inexpensive, is very reliable, and takes very little preparation time. Student and teacher assessment data indicate the simulation is popular with both groups, and students show significant learning gains. We include many resources with this publication, including all prelab introductory materials (e.g., a paper microarray activity), the student handouts, teachers notes, and pre- and postassessment tools. We did not test the simulation on other student populations, but based on teacher feedback, the simulation also may fit well in community college and in introductory and nonmajors' college biology curricula. PMID:17146040

  8. Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry

    PubMed Central

    Zhu, Chenggang; Zhu, Xiangdong; Landry, James P.; Cui, Zhaomeng; Li, Quanfu; Dang, Yongjun; Mi, Lan; Zheng, Fengyun; Fei, Yiyan

    2016-01-01

    Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%. PMID:26999137

  9. Developing an Efficient and General Strategy for Immobilization of Small Molecules onto Microarrays Using Isocyanate Chemistry.

    PubMed

    Zhu, Chenggang; Zhu, Xiangdong; Landry, James P; Cui, Zhaomeng; Li, Quanfu; Dang, Yongjun; Mi, Lan; Zheng, Fengyun; Fei, Yiyan

    2016-03-16

    Small-molecule microarray (SMM) is an effective platform for identifying lead compounds from large collections of small molecules in drug discovery, and efficient immobilization of molecular compounds is a pre-requisite for the success of such a platform. On an isocyanate functionalized surface, we studied the dependence of immobilization efficiency on chemical residues on molecular compounds, terminal residues on isocyanate functionalized surface, lengths of spacer molecules, and post-printing treatment conditions, and we identified a set of optimized conditions that enable us to immobilize small molecules with significantly improved efficiencies, particularly for those molecules with carboxylic acid residues that are known to have low isocyanate reactivity. We fabricated microarrays of 3375 bioactive compounds on isocyanate functionalized glass slides under these optimized conditions and confirmed that immobilization percentage is over 73%.

  10. An evaluation of two-channel ChIP-on-chip and DNA methylation microarray normalization strategies.

    PubMed

    Adriaens, Michiel E; Jaillard, Magali; Eijssen, Lars M T; Mayer, Claus-Dieter; Evelo, Chris T A

    2012-01-25

    The combination of chromatin immunoprecipitation with two-channel microarray technology enables genome-wide mapping of binding sites of DNA-interacting proteins (ChIP-on-chip) or sites with methylated CpG di-nucleotides (DNA methylation microarray). These powerful tools are the gateway to understanding gene transcription regulation. Since the goals of such studies, the sample preparation procedures, the microarray content and study design are all different from transcriptomics microarrays, the data pre-processing strategies traditionally applied to transcriptomics microarrays may not be appropriate. Particularly, the main challenge of the normalization of "regulation microarrays" is (i) to make the data of individual microarrays quantitatively comparable and (ii) to keep the signals of the enriched probes, representing DNA sequences from the precipitate, as distinguishable as possible from the signals of the un-enriched probes, representing DNA sequences largely absent from the precipitate. We compare several widely used normalization approaches (VSN, LOWESS, quantile, T-quantile, Tukey's biweight scaling, Peng's method) applied to a selection of regulation microarray datasets, ranging from DNA methylation to transcription factor binding and histone modification studies. Through comparison of the data distributions of control probes and gene promoter probes before and after normalization, and assessment of the power to identify known enriched genomic regions after normalization, we demonstrate that there are clear differences in performance between normalization procedures. T-quantile normalization applied separately on the channels and Tukey's biweight scaling outperform other methods in terms of the conservation of enriched and un-enriched signal separation, as well as in identification of genomic regions known to be enriched. T-quantile normalization is preferable as it additionally improves comparability between microarrays. In contrast, popular normalization

  11. Nonlinear matching measure for the analysis of on-off type DNA microarray images

    NASA Astrophysics Data System (ADS)

    Kim, Jong D.; Park, Misun; Kim, Jongwon

    2003-07-01

    In this paper, we propose a new nonlinear matching measure for automatic analysis of the on-off type DNA microarray images in which the hybridized spots are detected by the template matching method. The targeting spots of HPV DNA chips are designed for genotyping the human papilloma virus(HPV). The proposed measure is obtained by binarythresholding over the whole template region and taking the number of white pixels inside the spotted area. This measure is evaluated in terms of the accuracy of the estimated marker location to show better performance than the normalized covariance.

  12. Development of a DNA Microarray for Molecular Identification of All 46 Salmonella O Serogroups

    PubMed Central

    Guo, Dan; Liu, Bin; Liu, Fenxia; Cao, Boyang; Chen, Min; Hao, Xiyan; Feng, Lu

    2013-01-01

    Salmonella is a major cause of food-borne disease in many countries. Serotype determination of Salmonella is important for disease assessment, infection control, and epidemiological surveillance. In this study, a microarray system that targets the O antigen-specific genes was developed for simultaneously detecting and identifying all 46 Salmonella O serogroups. Of these, 40 serogroups can be confidently identified, and the remaining 6, in three pairs (serogroups O67 and B, E1 and E4, and A and D1), need to be further distinguished from each other using PCR methods or conventional serotyping methods. The microarray was shown to be highly specific when evaluated against 293 Salmonella strains, 186 Shigella strains, representative Escherichia coli strains, and 10 strains of other bacterial species. The assay correctly identified 288 (98%) of the Salmonella strains. The detection sensitivity was determined to be 50 ng genomic DNA per sample. By testing simulated samples in a tomato background, 2 to 8 CFU per gram inoculated could be detected after enrichment. This newly developed microarray assay is the first molecular protocol that can be used for the comprehensive detection and identification of all 46 Salmonella O serogroups. Compared to the traditional serogrouping method, the microarray provides a reliable, high-throughput, and sensitive approach that can be used for rapid identification of multiple Salmonella O serogroups simultaneously. PMID:23524674

  13. A hill-climbing approach for automatic gridding of cDNA microarray images.

    PubMed

    Rueda, Luis; Vidyadharan, Vidya

    2006-01-01

    Image and statistical analysis are two important stages of cDNA microarrays. Of these, gridding is necessary to accurately identify the location of each spot while extracting spot intensities from the microarray images and automating this procedure permits high-throughput analysis. Due to the deficiencies of the equipment used to print the arrays, rotations, misalignments, high contamination with noise and artifacts, and the enormous amount of data generated, solving the gridding problem by means of an automatic system is not trivial. Existing techniques to solve the automatic grid segmentation problem cover only limited aspects of this challenging problem and require the user to specify the size of the spots, the number of rows and columns in the grid, and boundary conditions. In this paper, a hill-climbing automatic gridding and spot quantification technique is proposed which takes a microarray image (or a subgrid) as input and makes no assumptions about the size of the spots, rows, and columns in the grid. The proposed method is based on a hill-climbing approach that utilizes different objective functions. The method has been found to effectively detect the grids on microarray images drawn from databases from GEO and the Stanford genomic laboratories.

  14. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes.

    PubMed

    Yang, G P; Ross, D T; Kuang, W W; Brown, P O; Weigel, R J

    1999-03-15

    Comparing patterns of gene expression in cell lines and tissues has important applications in a variety of biological systems. In this study we have examined whether the emerging technology of cDNA microarrays will allow a high throughput analysis of expression of cDNA clones generated by suppression subtractive hybridization (SSH). A set of cDNA clones including 332 SSH inserts amplified by PCR was arrayed using robotic printing. The cDNA arrays were hybridized with fluorescent labeled probes prepared from RNA from ER-positive (MCF7 and T47D) and ER-negative (MDA-MB-231 and HBL-100) breast cancer cell lines. Ten clones were identified that were over-expressed by at least a factor of five in the ER-positive cell lines. Northern blot analysis confirmed over-expression of these 10 cDNAs. Sequence analysis identified four of these clones as cytokeratin 19, GATA-3, CD24 and glutathione-S-transferase mu-3. Of the remaining six cDNA clones, four clones matched EST sequences from two different genes and two clones were novel sequences. Flow cytometry and immunofluorescence confirmed that CD24 protein was over-expressed in the ER-positive cell lines. We conclude that SSH and microarray technology can be successfully applied to identify differentially expressed genes. This approach allowed the identification of differentially expressed genes without the need to obtain previously cloned cDNAs.

  15. Complete gene expression profiling of Saccharopolyspora erythraea using GeneChip DNA microarrays

    PubMed Central

    Peano, Clelia; Bicciato, Silvio; Corti, Giorgio; Ferrari, Francesco; Rizzi, Ermanno; Bonnal, Raoul JP; Bordoni, Roberta; Albertini, Alberto; Bernardi, Luigi Rossi; Donadio, Stefano; De Bellis, Gianluca

    2007-01-01

    Background The Saccharopolyspora erythraea genome sequence, recently published, presents considerable divergence from those of streptomycetes in gene organization and function, confirming the remarkable potential of S. erythraea for producing many other secondary metabolites in addition to erythromycin. In order to investigate, at whole transcriptome level, how S. erythraea genes are modulated, a DNA microarray was specifically designed and constructed on the S. erythraea strain NRRL 2338 genome sequence, and the expression profiles of 6494 ORFs were monitored during growth in complex liquid medium. Results The transcriptional analysis identified a set of 404 genes, whose transcriptional signals vary during growth and characterize three distinct phases: a rapid growth until 32 h (Phase A); a growth slowdown until 52 h (Phase B); and another rapid growth phase from 56 h to 72 h (Phase C) before the cells enter the stationary phase. A non-parametric statistical method, that identifies chromosomal regions with transcriptional imbalances, determined regional organization of transcription along the chromosome, highlighting differences between core and non-core regions, and strand specific patterns of expression. Microarray data were used to characterize the temporal behaviour of major functional classes and of all the gene clusters for secondary metabolism. The results confirmed that the ery cluster is up-regulated during Phase A and identified six additional clusters (for terpenes and non-ribosomal peptides) that are clearly regulated in later phases. Conclusion The use of a S. erythraea DNA microarray improved specificity and sensitivity of gene expression analysis, allowing a global and at the same time detailed picture of how S. erythraea genes are modulated. This work underlines the importance of using DNA microarrays, coupled with an exhaustive statistical and bioinformatic analysis of the results, to understand the transcriptional organization of the chromosomes

  16. Development of a DNA microarray for detection and identification of Legionella pneumophila and ten other pathogens in drinking water.

    PubMed

    Zhou, Guangpeng; Wen, Shaoping; Liu, Yanwei; Li, Rongrong; Zhong, Xiaoying; Feng, Lu; Wang, Lei; Cao, Boyang

    2011-01-31

    The safety and accessibility of drinking water are major concerns throughout the world. Consumption of water contaminated with infectious agents, toxic chemicals or radiological hazards represents a significant health risk and is strongly associated with mortality. Therefore, we have developed an oligonucleotide-based microarray using the sequences of 16S-23S rDNA internal transcribed spacer regions (ITS) and the gyrase subunit B gene (gyrB) found in the most prevalent and devastating waterborne pathogenic agents. This new diagnostic contains 26 specific probes and can simultaneously detect Aeromonas hydrophila, Klebsiella pneumoniae, Legionella pneumophila, Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Staphylococcus aureus, Vibrio choleraeo, Vibrio parahaemolyticus, Yersinia enterocolitica and Leptospira interrogans. Testing was carried out against a total of 218 bacterial strains, including 53 representative strains, 103 clinical isolates and 62 strains of other bacterial species belonging to 10 genera and 48 species. The results were specific and reproducible, with a detection sensitivity of 0.1 ng DNA or 10(4)CFU/ml achieved for pure cultures of each target organism. The diluted cultures and real drinking water samples were tested by the microarray with 100% accuracy. This novel diagnostic method is superior in time- and labor-efficiency to conventional bacterial culture and antiserum agglutination, and can be readily applied to epidemiological surveillance and other food safety applications.

  17. Development of a prostate cDNA microarray and statistical gene expression analysis package.

    PubMed

    Carlisle, A J; Prabhu, V V; Elkahloun, A; Hudson, J; Trent, J M; Linehan, W M; Williams, E D; Emmert-Buck, M R; Liotta, L A; Munson, P J; Krizman, D B

    2000-05-01

    A cDNA microarray comprising 5184 different cDNAs spotted onto nylon membrane filters was developed for prostate gene expression studies. The clones used for arraying were identified by cluster analysis of > 35 000 prostate cDNA library-derived expressed sequence tags (ESTs) present in the dbEST database maintained by the National Center for Biotechnology Information. Total RNA from two cell lines, prostate line 8.4 and melanoma line UACC903, was used to make radiolabeled probe for filter hybridizations. The absolute intensity of each individual cDNA spot was determined by phosphorimager scanning and evaluated by a bioinformatics package developed specifically for analysis of cDNA microarray experimentation. Results indicated 89% of the genes showed intensity levels above background in prostate cells compared with only 28% in melanoma cells. Replicate probe preparations yielded results with correlation values ranging from r = 0.90 to 0.93 and coefficient of variation ranging from 16 to 28%. Findings indicate that among others, the keratin 5 and vimentin genes were differentially expressed between these two divergent cell lines. Follow-up northern blot analysis verified these two expression changes, thereby demonstrating the reliability of this system. We report the development of a cDNA microarray system that is sensitive and reliable, demonstrates a low degree of variability, and is capable of determining verifiable gene expression differences between two distinct human cell lines. This system will prove useful for differential gene expression analysis in prostate-derived cells and tissue.

  18. Microarray technology for the study of DNA damage by low-energy electrons

    NASA Astrophysics Data System (ADS)

    Solomun, T.; Hultschig, C.; Illenberger, E.

    2005-08-01

    The damage induced to a model DNA (dT{25}) immobilized on a gold surface by the interaction of low-energy (1 eV) electrons was studied by means of microarray technology. High quality single-stranded DNA arrays were hybridized with a dye-marked complementary strand after irradiation with electrons and the normalized fluorescence data were used to quantify the DNA damage. The data clearly show the sensitivity of the method. A significant loss of genetic information was already observed at dose as low as few hundred of electrons per immobilized oligonucleotide. The results imply that single stranded DNA and RNA are appreciably more sensitive to radiation and the attack of secondary electrons during replication, transcription or translation stages than the current radiation damage models envisage.

  19. Systematic Expression Profiling of the Mouse Transcriptome Using RIKEN cDNA Microarrays

    PubMed Central

    Bono, Hidemasa; Yagi, Ken; Kasukawa, Takeya; Nikaido, Itoshi; Tominaga, Naoko; Miki, Rika; Mizuno, Yosuke; Tomaru, Yasuhiro; Goto, Hitoshi; Nitanda, Hiroyuki; Shimizu, Daisuke; Makino, Hirochika; Morita, Tomoyuki; Fujiyama, Junshin; Sakai, Takehito; Shimoji, Takashi; Hume, David A.; Hayashizaki, Yoshihide; Okazaki, Yasushi

    2003-01-01

    The number of known mRNA transcripts in the mouse has been greatly expanded by the RIKEN Mouse Gene Encyclopedia project. Validation of their reproducible expression in a tissue is an important contribution to the study of functional genomics. In this report, we determine the expression profile of 57,931 clones on 20 mouse tissues using cDNA microarrays. Of these 57,931 clones, 22,928 clones correspond to the FANTOM2 clone set. The set represents 20,234 transcriptional units (TUs) out of 33,409 TUs in the FANTOM2 set. We identified 7206 separate clones that satisfied stringent criteria for tissue-specific expression. Gene Ontology terms were assigned for these 7206 clones, and the proportion of `molecular function' ontology for each tissue-specific clone was examined. These data will provide insights into the function of each tissue. Tissue-specific gene expression profiles obtained using our cDNA microarrays were also compared with the data extracted from the GNF Expression Atlas based on Affymetrix microarrays. One major outcome of the RIKEN transcriptome analysis is the identification of numerous nonprotein-coding mRNAs. The expression profile was also used to obtain evidence of expression for putative noncoding RNAs. In addition, 1926 clones (70%) of 2768 clones that were categorized as “unknown EST,” and 1969 (58%) clones of 3388 clones that were categorized as “unclassifiable” were also shown to be reproducibly expressed. PMID:12819129

  20. Comparative genomics profiling of clinical isolates of Aeromonas salmonicida using DNA microarrays

    PubMed Central

    Nash, John HE; Findlay, Wendy A; Luebbert, Christian C; Mykytczuk, Oksana L; Foote, Simon J; Taboada, Eduardo N; Carrillo, Catherine D; Boyd, Jessica M; Colquhoun, Duncan J; Reith, Michael E; Brown, Laura L

    2006-01-01

    Background Aeromonas salmonicida has been isolated from numerous fish species and shows wide variation in virulence and pathogenicity. As part of a larger research program to identify virulence genes and candidates for vaccine development, a DNA microarray was constructed using a subset of 2024 genes from the draft genome sequence of A. salmonicida subsp. salmonicida strain A449. The microarray included genes encoding known virulence-associated factors in A. salmonicida and homologs of virulence genes of other pathogens. We used microarray-based comparative genomic hybridizations (M-CGH) to compare selected A. salmonicida sub-species and other Aeromonas species from different hosts and geographic locations. Results Results showed variable carriage of virulence-associated genes and generally increased variation in gene content across sub-species and species boundaries. The greatest variation was observed among genes associated with plasmids and transposons. There was little correlation between geographic region and degree of variation for all isolates tested. Conclusion We have used the M-CGH technique to identify subsets of conserved genes from amongst this set of A. salmonicida virulence genes for further investigation as potential vaccine candidates. Unlike other bacterial characterization methods that use a small number of gene or DNA-based functions, M-CGH examines thousands of genes and/or whole genomes and thus is a more comprehensive analytical tool for veterinary or even human health research. PMID:16522207

  1. 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

  2. Typing and Subtyping Influenza Virus Using DNA Microarrays and Multiplex Reverse Transcriptase PCR

    PubMed Central

    Li, Jiping; Chen, Shu; Evans, David H.

    2001-01-01

    A model DNA microarray has been prepared and shown to facilitate typing and subtyping of human influenza A and B viruses. Reverse transcriptase PCR was used to prepare cDNAs encoding ∼500-bp influenza virus gene fragments, which were then cloned, sequenced, reamplified, and spotted to form a glass-bound microarray. These target DNAs included multiple fragments of the hemagglutinin, neuraminidase, and matrix protein genes. Cy3- or Cy5-labeled fluorescent probes were then hybridized to these target DNAs, and the arrays were scanned to determine the probe binding site(s). The hybridization pattern agreed perfectly with the known grid location of each target, and the signal-to-background ratio varied from 5 to 30. No cross-hybridization could be detected beyond that expected from the limited degree of sequence overlap between different probes and targets. At least 100 to 150 bp of homology was required for hybridization under the conditions used in this study. Combinations of Cy3- and Cy5-labeled DNAs can also be hybridized to the same chip, permitting further differentiation of amplified molecules in complex mixtures. In a more realistic test of the technology, several sets of multiplex PCR primers that collectively target influenza A and B virus strains were identified and were used to type and subtype several previously unsequenced influenza virus isolates. The results show that DNA microarray technology provides a useful supplement to PCR-based diagnostic methods. PMID:11158130

  3. New molecular phenotypes in the dst mutants of Arabidopsis revealed by DNA microarray analysis.

    PubMed

    Pérez-Amador, M A; Lidder, P; Johnson, M A; Landgraf, J; Wisman, E; Green, P J

    2001-12-01

    In this study, DNA microarray analysis was used to expand our understanding of the dst1 mutant of Arabidopsis. The dst (downstream) mutants were isolated originally as specifically increasing the steady state level and the half-life of DST-containing transcripts. As such, txhey offer a unique opportunity to study rapid sequence-specific mRNA decay pathways in eukaryotes. These mutants show a threefold to fourfold increase in mRNA abundance for two transgenes and an endogenous gene, all containing DST elements, when examined by RNA gel blot analysis; however, they show no visible aberrant phenotype. Here, we use DNA microarrays to identify genes with altered expression levels in dst1 compared with the parental plants. In addition to verifying the increase in the transgene mRNA levels, which were used to isolate these mutants, we were able to identify new genes with altered mRNA abundance in dst1. RNA gel blot analysis confirmed the microarray data for all genes tested and also was used to catalog the first molecular differences in gene expression between the dst1 and dst2 mutants. These differences revealed previously unknown molecular phenotypes for the dst mutants that will be helpful in future analyses. Cluster analysis of genes altered in dst1 revealed new coexpression patterns that prompt new hypotheses regarding the nature of the dst1 mutation and a possible role of the DST-mediated mRNA decay pathway in plants.

  4. Statistical analysis of efficient unbalanced factorial designs for two-color microarray experiments.

    PubMed

    Tempelman, Robert J

    2008-01-01

    Experimental designs that efficiently embed a fixed effects treatment structure within a random effects design structure typically require a mixed-model approach to data analyses. Although mixed model software tailored for the analysis of two-color microarray data is increasingly available, much of this software is generally not capable of correctly analyzing the elaborate incomplete block designs that are being increasingly proposed and used for factorial treatment structures. That is, optimized designs are generally unbalanced as it pertains to various treatment comparisons, with different specifications of experimental variability often required for different treatment factors. This paper uses a publicly available microarray dataset, as based upon an efficient experimental design, to demonstrate a proper mixed model analysis of a typical unbalanced factorial design characterized by incomplete blocks and hierarchical levels of variability.

  5. Statistical Analysis of Efficient Unbalanced Factorial Designs for Two-Color Microarray Experiments

    PubMed Central

    Tempelman, Robert J.

    2008-01-01

    Experimental designs that efficiently embed a fixed effects treatment structure within a random effects design structure typically require a mixed-model approach to data analyses. Although mixed model software tailored for the analysis of two-color microarray data is increasingly available, much of this software is generally not capable of correctly analyzing the elaborate incomplete block designs that are being increasingly proposed and used for factorial treatment structures. That is, optimized designs are generally unbalanced as it pertains to various treatment comparisons, with different specifications of experimental variability often required for different treatment factors. This paper uses a publicly available microarray dataset, as based upon an efficient experimental design, to demonstrate a proper mixed model analysis of a typical unbalanced factorial design characterized by incomplete blocks and hierarchical levels of variability. PMID:18584033

  6. Simple PCR-based DNA microarray system to identify human pathogenic fungi in skin.

    PubMed

    Sato, Tomotaka; Takayanagi, Atsushi; Nagao, Keisuke; Tomatsu, Nobuhiro; Fukui, Toshifumi; Kawaguchi, Masahiro; Kudoh, Jun; Amagai, Masayuki; Yamamoto, Nobuko; Shimizu, Nobuyoshi

    2010-07-01

    Fungal diseases in immunocompromised hosts pose significant threats to their prognoses. An accurate diagnosis and identification of the fungal pathogens causing the infection are critical to determine the proper therapeutic interventions, but these are often not achieved, due to difficulties with isolation and morphological identification. In an effort to ultimately carry out the simultaneous detection of all human pathogenic microbes, we developed a simple system to identify 26 clinically important fungi by using a combination of PCR amplification and DNA microarray assay (designated PCR-DM), in which PCR-amplified DNA from the internal transcribed spacer region of the rRNA gene was hybridized to a DNA microarray fabricated with species-specific probes sets using the Bubble Jet technology. PCR-DM reliably identified all 26 reference strains; hence, we applied it to cases of onychomycosis, taking advantage of the accessibility of tissue from skin. PCR-DM detected fungal DNA and identified pathogens in 92% of 106 microscopy-confirmed onychomycosis specimens. In contrast, culture was successful for only 36 specimens (34%), 3 of which had results inconsistent with the results of PCR-DM, but sequence analysis of the isolates proved that the PCR-DM result was correct. Thus, PCR-DM provides a powerful method to identify pathogenic fungi with high sensitivity and speed directly from tissue specimens, and this concept could be applied to other fungal or nonfungal infectious human diseases in less accessible anatomical sites.

  7. 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

  8. DNA microarray mediated transcriptional profiling of Nitrosomonas europaea in response to linear alkylbenzene sulfonates.

    PubMed

    Urakawa, Hidetoshi; Matsumoto, Junpei; Inaba, Kazuho; Tsuneda, Satoshi

    2008-05-01

    Linear alkylbenzene sulfonates (LAS) constitute, quantitatively, the most important group of synthetic surfactants used today. We studied the gene expression of Nitrosomonas europaea in response to LAS using a DNA microarray because ammonia-oxidizers are thought to be more sensitive to LAS than other microorganisms. Our objective was to elucidate which genes are expressed for N. europaea in response to LAS exposure. Microarray analysis and real-time PCR assay revealed that c. 30 genes were significantly expressed after LAS exposure, in particular genes associated with energy production and conversion. Our findings demonstrate that physical disruption of membrane structures, which contain enzymes associated with energy production and conversion, might be an important explanation for the high sensitivity of N. europaea to LAS exposure.

  9. 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

  10. 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.

  11. Surface ligation-based resonance light scattering analysis of methylated genomic DNA on a microarray platform.

    PubMed

    Ma, Lan; Lei, Zhen; Liu, Xia; Liu, Dianjun; Wang, Zhenxin

    2016-05-10

    DNA methylation is a crucial epigenetic modification and is closely related to tumorigenesis. Herein, a surface ligation-based high throughput method combined with bisulfite treatment is developed for analysis of methylated genomic DNA. In this method, a DNA microarray is employed as a reaction platform, and resonance light scattering (RLS) of nanoparticles is used as the detection principle. The specificity stems from allele-specific ligation of Taq DNA ligase, which is further enhanced by improving the fidelity of Taq DNA ligase in a heterogeneous reaction. Two amplification techniques, rolling circle amplification (RCA) and silver enhancement, are employed after the ligation reaction and a gold nanoparticle (GNP) labeling procedure is used to amplify the signal. As little as 0.01% methylated DNA (i.e. 2 pmol L(-1)) can be distinguished from the cocktail of methylated and unmethylated DNA by the proposed method. More importantly, this method shows good accuracy and sensitivity in profiling the methylation level of genomic DNA of three selected colonic cancer cell lines. This strategy provides a high throughput alternative with reasonable sensitivity and resolution for cancer study and diagnosis.

  12. 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.

  13. Scanning electrochemical microscopy of DNA hybridization on DNA microarrays enhanced by HRP-modified SiO2 nanoparticles.

    PubMed

    Fan, Huajun; Wang, Xiaolan; Jiao, Fang; Zhang, Fan; Wang, Qingjiang; He, Pingang; Fang, Yuzhi

    2013-07-02

    Imaging of localized hybridization of nucleic acids immobilized on a glass DNA microarray was performed by means of generation collection (GC) mode scanning electrochemical microscopy (SECM). Amine-tethered oligodeoxynucleotide probes, spotted on the glass surface, were hybridized with an unmodified target sequence and a biotinylated indicator probe via sandwich hybridization. Spots where sequence-specific hybridization had occurred were modified by streptavidin-horseradish-peroxidase-(HRP)-wrapped SiO2 nanoparticles through the biotin-streptavidin interaction. In the presence of H2O2, hydroquinone (H2Q) was oxidized to benzoquinone (BQ) at the modified spot surface through the HRP catalytic reaction, and the generated BQ corresponding to the amount of target DNA was reduced in solution by an SECM tip. With this DNA microarray, a number of genes could be detected simultaneously and selectively enough to discriminate between complementary sequences and those containing base mismatches. The DNA targets at prepared spots could be imaged in SECM GC mode over a wide concentration range (10(-7)-10(-12) M). This technique may find applications in genomic sequencing.

  14. Mapping the affinity landscape of Thrombin-binding aptamers on 2'F-ANA/DNA chimeric G-Quadruplex microarrays.

    PubMed

    Lietard, Jory; Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos; Somoza, Mark M; Damha, Masad J

    2017-01-18

    In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2'-Fluoroarabinonucleic acid (2'F-ANA) is a prime candidate for such use in microarrays. Indeed, 2'F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2'F-ANA and 2'F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2'F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2'F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2'F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2'F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.

  15. Enhancing the Sensitivity of DNA Microarray Using Dye-Doped Silica Nanoparticles: Detection of Human Papilloma Virus

    NASA Astrophysics Data System (ADS)

    Enrichi, F.; Riccò, R.; Meneghello, A.; Pierobon, R.; Canton, G.; Cretaio, E.

    2010-10-01

    DNA microarray is a high-throughput technology used for detection and quantification of nucleic acid molecules and others of biological interest. The analysis is based on the specific hybridization between probe sequences deposited in array and a target ss-DNA amplified by PCR and functionalized by a fluorescent dye. Organic labels have well known disadvantages like photobleaching and low signal intensities, which put a limitation to the lower amount of DNA material that can be detected. Therefore for trace analysis the development of more efficient biomarkers is required. With this aim we present in this paper the synthesis and application of alternative hybrid nanosystems obtained by incorporating standard fluorescent molecules into monodisperse silica nanoparticles. Efficient application to the detection of Human Papilloma Virus is demonstrated. This virus is associated to the formation of cervical cancer, a leading cause of death by cancer for women worldwide. It is shown that the use of the novel biomarkers increases the optical signal of about one order of magnitude with respect to the free dyes or quantum dots in conventional instruments. This is due to the high number of molecules that can be accommodated into each nanoparticle, to the reduced photobleaching and to the improved environmental protection of the dyes when encapsulated in the silica matrix. The cheap and easy synthesis of these luminescent particles, the stability in water, the surface functionalizability and bio-compatibility make them very promising for present and future bio-labeling and bio-imaging applications.

  16. Integration of clinical point-of-care requirements in a DNA microarray genotyping test.

    PubMed

    Van Dorst, Bieke; Cremers, Amelieke; Jans, Karolien; Van Domburg, Trees; Steegen, Kim; Huang, Chengjun; Dorrer, Christian; Lagae, Liesbet; Ferwerda, Gerben; Stuyver, Lieven J

    2014-11-15

    Various proof-of-concept studies have shown the potential of biosensors with a high multiplex detection capability for the readout of DNA microarrays in a lab-on-a-chip. This is particularly interesting for the development of point-of-care genotyping tests, to screen for multiple pathogens and/or antibiotic resistance patterns. In this paper, an assay workflow is presented, suited for the development of novel lab-on-a-chips with an integrated DNA microarray. Besides the description of the different assay steps (DNA purification, amplification and detection), a control strategy is presented according to recommendations of the US Food and Drug Administration (FDA). To use a lab-on-a-chip for diagnostic applications, the optimization and evaluation of the assay performance with clinical samples is very important. Therefore, appropriate quantification methods are described, which allow optimization and evaluation of the separate assay steps, as well as total assay performance. In order to demonstrate and evaluate the total workflow, blood samples spiked with Streptococcus pneumoniae were tested. All blood samples with ≥ 10(3)CFU S. pneumoniae per ml of human blood were successfully detected by this genotyping assay.

  17. Metatranscriptomic analysis of the response of river biofilms to pharmaceutical products, using anonymous DNA microarrays.

    PubMed

    Yergeau, Etienne; Lawrence, John R; Waiser, Marley J; Korber, Darren R; Greer, Charles W

    2010-08-01

    Pharmaceutical products are released at low concentrations into aquatic environments following domestic wastewater treatment. Such low concentrations have been shown to induce transcriptional responses in microorganisms, which could have consequences on aquatic ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two rivers of differing trophic statuses and subsequently treated with environmentally relevant doses (ng/liter to microg/liter range) of four pharmaceuticals (erythromycin [ER], gemfibrozil [GM], sulfamethazine [SN], and sulfamethoxazole [SL]). To monitor functional gene expression, we constructed a 9,600-feature anonymous DNA microarray platform onto which cDNA from the biofilms was hybridized. Pharmaceutical treatments induced both positive and negative transcriptional responses from biofilm microorganisms. For instance, ER induced the transcription of several stress, transcription, and replication genes, while GM, a lipid regulator, induced transcriptional responses from several genes involved in lipid metabolism. SN caused shifts in genes involved in energy production and conversion, and SL induced responses from a range of cell membrane and outer envelope genes, which in turn could affect biofilm formation. The results presented here demonstrate for the first time that low concentrations of small molecules can induce transcriptional changes in a complex microbial community. The relevance of these results also demonstrates the usefulness of anonymous DNA microarrays for large-scale metatranscriptomic studies of communities from differing aquatic ecosystems.

  18. Microarray-based DNA methylation study of Ewing's sarcoma of the bone.

    PubMed

    Park, Hye-Rim; Jung, Woon-Won; Kim, Hyun-Sook; Park, Yong-Koo

    2014-10-01

    Alterations in DNA methylation patterns are a hallmark of malignancy. However, the majority of epigenetic studies of Ewing's sarcoma have focused on the analysis of only a few candidate genes. Comprehensive studies are thus lacking and are required. The aim of the present study was to identify novel methylation markers in Ewing's sarcoma using microarray analysis. The current study reports the microarray-based DNA methylation study of 1,505 CpG sites of 807 cancer-related genes from 69 Ewing's sarcoma samples. The Illumina GoldenGate Methylation Cancer Panel I microarray was used, and with the appropriate controls (n=14), a total of 92 hypermethylated genes were identified in the Ewing's sarcoma samples. The majority of the hypermethylated genes were associated with cell adhesion, cell regulation, development and signal transduction. The overall methylation mean values were compared between patients who survived and those that did not. The overall methylation mean was significantly higher in the patients who did not survive (0.25±0.03) than in those who did (0.22±0.05) (P=0.0322). However, the overall methylation mean was not found to significantly correlate with age, gender or tumor location. GDF10, OSM, APC and HOXA11 were the most significant differentially-methylated genes, however, their methylation levels were not found to significantly correlate with the survival rate. The DNA methylation profile of Ewing's sarcoma was characterized and 92 genes that were significantly hypermethylated were detected. A trend towards a more aggressive behavior was identified in the methylated group. The results of this study indicated that methylation may be significant in the development of Ewing's sarcoma.

  19. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis

    PubMed Central

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  20. A High Phosphorus Diet Affects Lipid Metabolism in Rat Liver: A DNA Microarray Analysis.

    PubMed

    Chun, Sunwoo; Bamba, Takeshi; Suyama, Tatsuya; Ishijima, Tomoko; Fukusaki, Eiichiro; Abe, Keiko; Nakai, Yuji

    2016-01-01

    A high phosphorus (HP) diet causes disorders of renal function, bone metabolism, and vascular function. We previously demonstrated that DNA microarray analysis is an appropriate method to comprehensively evaluate the effects of a HP diet on kidney dysfunction such as calcification, fibrillization, and inflammation. We reported that type IIb sodium-dependent phosphate transporter is significantly up-regulated in this context. In the present study, we performed DNA microarray analysis to investigate the effects of a HP diet on the liver, which plays a pivotal role in energy metabolism. DNA microarray analysis was performed with total RNA isolated from the livers of rats fed a control diet (containing 0.3% phosphorus) or a HP diet (containing 1.2% phosphorus). Gene Ontology analysis of differentially expressed genes (DEGs) revealed that the HP diet induced down-regulation of genes involved in hepatic amino acid catabolism and lipogenesis, while genes related to fatty acid β-oxidation process were up-regulated. Although genes related to fatty acid biosynthesis were down-regulated in HP diet-fed rats, genes important for the elongation and desaturation reactions of omega-3 and -6 fatty acids were up-regulated. Concentrations of hepatic arachidonic acid and eicosapentaenoic acid were increased in HP diet-fed rats. These essential fatty acids activate peroxisome proliferator-activated receptor alpha (PPARα), a transcription factor for fatty acid β-oxidation. Evaluation of the upstream regulators of DEGs using Ingenuity Pathway Analysis indicated that PPARα was activated in the livers of HP diet-fed rats. Furthermore, the serum concentration of fibroblast growth factor 21, a hormone secreted from the liver that promotes fatty acid utilization in adipose tissue as a PPARα target gene, was higher (p = 0.054) in HP diet-fed rats than in control diet-fed rats. These data suggest that a HP diet enhances energy expenditure through the utilization of free fatty acids

  1. Characterization, validation and application of a DNA microarray for the detection of mandatory and other waterborne pathogens.

    PubMed

    Gomes, Maria; Vieira, Helena; Vale, Filipa F

    2015-11-01

    Culture methods for the detection of indicator bacteria are currently used for detection of waterborne bacteria. The need for an increased range of analyzed bacteria coupled with the obtainment of rapid and early results justify the development of a DNA microarray for the identification of waterborne pathogens. This DNA microarray has 16 implanted probes with a median size of 147 bases, targeting 12 different parameters, including all mandatory indicator microorganisms, such as Escherichia coli, Clostridium perfringens, Pseudomonas aeruginosa, Staphylococcus aureus, total and fecal coliforms and enterococci. The validation performed with DNA extracted from pure microbial cultures showed the suitability of the probes for detection of the target microorganism. To overcome the high dilution of water samples it was included either a prior culture step of bacterial contaminants retained after filtering 100 ml of water, or a 10-fold increase in the volume of filtered water, that resulted in the increase of the detected bacteria. The analysis of complex environmental water samples using culture methods and the DNA microarray revealed that the latter detected the same parameters plus other bacteria tested only in the DNA microarray. The results show that this DNA microarray may be a useful tool for water microbiological surveillance. © The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

  2. 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

  3. 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.

  4. Thymus and Myasthenia Gravis: what can we learn from DNA microarrays?

    PubMed

    Cizeron-Clairac, Géraldine; Le Panse, Rozen; Frenkian-Cuvelier, Mélinée; Meraouna, Amel; Truffault, Frédérique; Bismuth, Jacky; Mussot, Sacha; Kerlero de Rosbo, Nicole; Berrih-Aknin, Sonia

    2008-09-15

    This review is dedicated to John Newsom-Davis, who was an exceptional colleague and friend, always exchanging ideas with respect and consideration. We shall not forget his involvement and passion in search for the truth on the role of thymectomy in the management of Myasthenia Gravis (MG). In this short review, we shall summarize what we learnt from DNA microarrays applied to MG thymus. We shall focus on three main comparisons of the thymic transcriptomes: 1) highly hyperplastic MG patients versus non-MG adults; 2) corticosteroid-treated versus untreated seropositive MG patients; and 3) seronegative versus seropositive MG patients.

  5. DNA microarray-based detection of multiple pathogens: Mycoplasma spp. and Chlamydia spp.

    PubMed

    Schnee, Christiane; Sachse, Konrad

    2015-01-01

    Rapid detection of slow-growing or non-culturable microorganisms, such as Mycoplasma spp. and Chlamydia spp., is still a challenge to diagnosticians in the veterinary field. In addition, as epidemiological evidence on the frequency of mixed infections involving two and more bacterial species has been emerging, detection methods allowing simultaneous identification of different pathogens are required. In the present chapter, we describe DNA microarray-based procedures for the detection of 83 Mollicutes species (Mycoplasma assay) and 11 Chlamydia spp. (Chlamydia assay). The assays are suitable for use in a routine diagnostic environment, as well as in microbiological research.

  6. Array painting: a protocol for the rapid analysis of aberrant chromosomes using DNA microarrays

    PubMed Central

    Gribble, Susan M; Ng, Bee Ling; Prigmore, Elena; Fitzgerald, Tomas; Carter, Nigel P

    2012-01-01

    Aarray painting is a technique that uses microarray technology to rapidly map chromosome translocation breakpoints. previous methods to map translocation breakpoints have used fluorescence in situ hybridization (FIsH) and have consequently been labor-intensive, time-consuming and restricted to the low breakpoint resolution imposed by the use of metaphase chromosomes. array painting combines the isolation of derivative chromosomes (chromosomes with translocations) and high-resolution microarray analysis to refine the genomic location of translocation breakpoints in a single experiment. In this protocol, we describe array painting by isolation of derivative chromosomes using a MoFlo flow sorter, amplification of these derivatives using whole-genome amplification and hybridization onto commercially available oligonucleotide microarrays. although the sorting of derivative chromosomes is a specialized procedure requiring sophisticated equipment, the amplification, labeling and hybridization of Dna is straightforward, robust and can be completed within 1 week. the protocol described produces good quality data; however, array painting is equally achievable using any combination of the available alternative methodologies for chromosome isolation, amplification and hybridization. PMID:19893508

  7. Using DNA Microarrays To Identify Library-Independent Markers for Bacterial Source Tracking

    PubMed Central

    Soule, Marilyn; Kuhn, Edward; Loge, Frank; Gay, John; Call, Douglas R.

    2006-01-01

    Bacterial source tracking is used to apportion fecal pollution among putative sources. Within this context, library-independent markers are genetic or phenotypic traits that can be used to identify the host origin without a need for library-dependent classification functions. The objective of this project was to use mixed-genome Enterococcus microarrays to identify library-independent markers. Separate shotgun libraries were prepared for five host groups (cow, dog, elk/deer, human, and waterfowl), using genomic DNAs (gDNAs) from ca. 50 Enterococcus isolates for each library. Microarrays were constructed (864 probes per library), and 385 comparative genomic hybridizations were used to identify putative markers. PCR assays were used to screen 95 markers against gDNAs from isolates from known sources collected throughout the United States. This validation process narrowed the selection to 15 markers, with 7 having no recognized homologues and the remaining markers being related to genes involved in metabolic pathways and DNA replication. In most cases, each marker was exclusive to one of four Enterococcus species (Enterococcus casseliflavus, E. faecalis, E. hirae, or E. mundtii). Eight markers were highly specific to either cattle, humans, or elk/deer, while the remaining seven markers were positive for various combinations of hosts other than humans. Based on microarray hybridization data, the prevalence of host-specific markers ranged from 2% to 45% of isolates collected from their respective hosts. A 20-fold difference in prevalence could present challenges for the interpretation of library-independent markers. PMID:16517630

  8. Global gene expression analysis of two Streptococcus thermophilus bacteriophages using DNA microarray.

    PubMed

    Duplessis, Martin; Russell, W Michael; Romero, Dennis A; Moineau, Sylvain

    2005-09-30

    A custom microarray was developed to study the temporal gene expression of the two groups of phages infecting the Gram-positive lactic acid bacterium Streptococcus thermophilus. The complete genomic sequence of the virulent cos-type phage DT1 (34,815 bp) and the pac-type phage 2972 (34,704 bp) were used for the construction of the microarray. Gene expression was measured at nine time intervals (0, 2, 7, 12, 17, 22, 27, 32 and 37 min) during phage infection and an expression curve was determined for each gene. Each phage gene was then classified into one of the three traditional transcription classes and these data were used to generate the complete transcriptional map of DT1 and 2972. Phage DT1 possesses 18 early genes, 12 middle genes and 12 late-expressed genes whereas 2972 has 16 early, 11 middle and 14 late genes. The trends of the phage gene expression profiles were also confirmed by slot blot hybridizations. Significant differences were observed when comparing the transcriptional maps of DT1 and 2972 with those already available for the S. thermophilus phages Sfi19 and Sfi21. To our knowledge, this report presents the first complete transcription analysis of bacteriophages infecting Gram-positive bacteria using the DNA microarray technology.

  9. Use of DNA Microarrays for Rapid Genotyping of TEM Beta-Lactamases That Confer Resistance

    PubMed Central

    Grimm, Verena; Ezaki, Satoshi; Susa, Milorad; Knabbe, Cornelius; Schmid, Rolf. D.; Bachmann, Till T.

    2004-01-01

    Standard clinical procedures for pathogen resistance identification are laborious and usually require 2 days of cultivation before the resistance can be determined unequivocally. In contrast, clinicians and patients face increasing threats from antibiotic-resistant pathogenic bacteria in terms of their frequencies and levels of resistance. A major class of microbial resistance stems from the occurrence of beta-lactamases, which, if mutated, can cause the severe extended-spectrum beta-lactamase (ESBL) or inhibitor-resistant TEM (IRT) phenotype, which cause resistance to extended-spectrum cephalosporins, monobactams, and beta-lactamase inhibitors. We describe an oligonucleotide microarray for identification of the single nucleotide polymorphisms (SNPs) of 96% of the TEM beta-lactamase variants described to date which are related to the ESBL and/or IRT phenotype. The target DNA, originating from Escherichia coli, Enterobacter cloacae, and Klebsiella pneumoniae cells isolated from clinical samples, was amplified and fluorescently labeled by PCR with consensus primers in the presence of cyanine 5-labeled nucleotides. The total assay, including PCR, hybridization, and image analysis, could be performed in 3.5 h. The microarray results were validated by standard clinical procedures. The microarray outperformed the standard procedures in terms of assay time and the depth of information provided. In conclusion, this array offers an attractive option for the identification and epidemiologic monitoring of TEM beta-lactamases in the routine clinical diagnostic laboratory. PMID:15297528

  10. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation.

    PubMed

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi's individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  11. A Combinational Clustering Based Method for cDNA Microarray Image Segmentation

    PubMed Central

    Shao, Guifang; Li, Tiejun; Zuo, Wangda; Wu, Shunxiang; Liu, Tundong

    2015-01-01

    Microarray technology plays an important role in drawing useful biological conclusions by analyzing thousands of gene expressions simultaneously. Especially, image analysis is a key step in microarray analysis and its accuracy strongly depends on segmentation. The pioneering works of clustering based segmentation have shown that k-means clustering algorithm and moving k-means clustering algorithm are two commonly used methods in microarray image processing. However, they usually face unsatisfactory results because the real microarray image contains noise, artifacts and spots that vary in size, shape and contrast. To improve the segmentation accuracy, in this article we present a combination clustering based segmentation approach that may be more reliable and able to segment spots automatically. First, this new method starts with a very simple but effective contrast enhancement operation to improve the image quality. Then, an automatic gridding based on the maximum between-class variance is applied to separate the spots into independent areas. Next, among each spot region, the moving k-means clustering is first conducted to separate the spot from background and then the k-means clustering algorithms are combined for those spots failing to obtain the entire boundary. Finally, a refinement step is used to replace the false segmentation and the inseparable ones of missing spots. In addition, quantitative comparisons between the improved method and the other four segmentation algorithms--edge detection, thresholding, k-means clustering and moving k-means clustering--are carried out on cDNA microarray images from six different data sets. Experiments on six different data sets, 1) Stanford Microarray Database (SMD), 2) Gene Expression Omnibus (GEO), 3) Baylor College of Medicine (BCM), 4) Swiss Institute of Bioinformatics (SIB), 5) Joe DeRisi’s individual tiff files (DeRisi), and 6) University of California, San Francisco (UCSF), indicate that the improved approach is

  12. Optical and surface analysis of DNA microarrays to assess printed spot heterogeneity

    NASA Astrophysics Data System (ADS)

    Nagaraja Rao, Archana

    DNA microarrays have been plagued with analytical problems with quantitation, metrics, figures of merit, and reliability and reproducibility issues, hindering their acceptance in clinical and diagnostic settings. The main deficiency in the printed DNA format is the microspot heterogeneity occurring during array fabrication and further amplified during target hybridization. Work described in this dissertation focuses on assessment of DNA microarray spots generated with conventional pin-type contact printing of fluorescently labeled DNA probes, on industry-standard commercial polymer-coated array slides and their hybridization with complementary oligomer DNA target. Printing of probe DNA microspots shares many features of commonly reported droplet evaporation dynamics that lead to different drying patterns and spot morphologies. This study directly identifies and analyzes different DNA probe chemical and spatial microenvironments within spots, analyzed with high-resolution time-of-flight secondary ion mass spectrometry (TOF-SIMS) chemical imaging, confocal epifluorescence, and probe microscopy force imaging methods. Drying of DNA probe spots shows Marangoni flow effects with high densities of probe DNA-Cy3 located in spot centers and nonhomogeneous DNA distributed radially within printed spots with both TOF-SIMS imaging and epifluorescence microscopy. Target hybridization kinetics and duplex formation were assessed using real-time in situ confocal imaging, and confirmed radial hemispherical diffusion-mediated distribution of target capture from spot edge to its interior. Kinetic modeling indicates pseudo-first order kinetics due to transport limitations and local density-dependent probe interactions with diffusing target. Fluorescence resonance energy transfer (FRET) and photobleaching results show that the high- density probe overcrowding in spots facilitates a broad range of target binding interactions regardless of dye orientations. Moreover, lateral probe density

  13. Background adjustment of cDNA microarray images by Maximum Entropy distributions.

    PubMed

    Argyropoulos, Christos; Daskalakis, Antonis; Nikiforidis, George C; Sakellaropoulos, George C

    2010-08-01

    Many empirical studies have demonstrated the exquisite sensitivity of both traditional and novel statistical and machine intelligence algorithms to the method of background adjustment used to analyze microarray datasets. In this paper we develop a statistical framework that approaches background adjustment as a classic stochastic inverse problem, whose noise characteristics are given in terms of Maximum Entropy distributions. We derive analytic closed form approximations to the combined problem of estimating the magnitude of the background in microarray images and adjusting for its presence. The proposed method reduces standardized measures of log expression variability across replicates in situations of known differential and non-differential gene expression without increasing the bias. Additionally, it results in computationally efficient procedures for estimation and learning based on sufficient statistics and can filter out spot measures with intensities that are numerically close to the background level resulting in a noise reduction of about 7%.

  14. Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray.

    PubMed

    Viadas, C; Rodríguez, M C; García-Lobo, J M; Sangari, F J; López-Goñi, I

    2009-10-01

    The genus Brucella contains bacteria producing a zoonosis of large sanitary and economical impact. The complete nucleotide sequence of eight Brucella isolates is currently available. This information can be used for high throughput approaches to the biology of this genus such as the construction of comprehensive collections of ORF clones or ORFeomes. The ORFeome of Brucella melitensis was a first contribution to this goal. Using the Brucella ORFeome as starting material we have amplified each ORF and printed them in duplicate onto coated glass slides along with the appropriate positive and negative controls. Quality control of the microarray was performed by image analysis after ethidium bromide staining. This Brucella DNA microarray was used to determine the global transcriptional profile of Brucella abortus grown under laboratory conditions. Two sets of genes representing strongly and poorly expressed genes have been defined. The occurrence of several genes of the same operon in the same data set has been taken as additional proof of the significance of the results. The two sets have been validated by RT-PCR of retrotranscribed RNA. Among the more abundant transcripts we found ribosomal proteins, Krebs cycle and oxidative phosphorylation enzymes. virB, flagellar components and other genes related with virulence and intracellular growth were in the poorly transcribed set. This report demonstrated the usefulness of the ORFeome for the construction of a PCR product microarray for the analysis of global gene expression in Brucella and also applicable to other microorganisms. The results provided here represent a comprehensive description of the global transcriptional profile of B. abortus grown under laboratory conditions and, at the same time, validate the use of this Brucella microarray for the study of the biology and pathogenesis of Brucella through the analysis of gene expression under any experimental conditions.

  15. Massively multiplexed microbial identification using resequencing DNA microarrays for outbreak investigation

    NASA Astrophysics Data System (ADS)

    Leski, T. A.; Ansumana, R.; Jimmy, D. H.; Bangura, U.; Malanoski, A. P.; Lin, B.; Stenger, D. A.

    2011-06-01

    Multiplexed microbial diagnostic assays are a promising method for detection and identification of pathogens causing syndromes characterized by nonspecific symptoms in which traditional differential diagnosis is difficult. Also such assays can play an important role in outbreak investigations and environmental screening for intentional or accidental release of biothreat agents, which requires simultaneous testing for hundreds of potential pathogens. The resequencing pathogen microarray (RPM) is an emerging technological platform, relying on a combination of massively multiplex PCR and high-density DNA microarrays for rapid detection and high-resolution identification of hundreds of infectious agents simultaneously. The RPM diagnostic system was deployed in Sierra Leone, West Africa in collaboration with Njala University and Mercy Hospital Research Laboratory located in Bo. We used the RPM-Flu microarray designed for broad-range detection of human respiratory pathogens, to investigate a suspected outbreak of avian influenza in a number of poultry farms in which significant mortality of chickens was observed. The microarray results were additionally confirmed by influenza specific real-time PCR. The results of the study excluded the possibility that the outbreak was caused by influenza, but implicated Klebsiella pneumoniae as a possible pathogen. The outcome of this feasibility study confirms that application of broad-spectrum detection platforms for outbreak investigation in low-resource locations is possible and allows for rapid discovery of the responsible agents, even in cases when different agents are suspected. This strategy enables quick and cost effective detection of low probability events such as outbreak of a rare disease or intentional release of a biothreat agent.

  16. A generally applicable validation scheme for the assessment of factors involved in reproducibility and quality of DNA-microarray data

    PubMed Central

    van Hijum, Sacha AFT; de Jong, Anne; Baerends, Richard JS; Karsens, Harma A; Kramer, Naomi E; Larsen, Rasmus; den Hengst, Chris D; Albers, Casper J; Kok, Jan; Kuipers, Oscar P

    2005-01-01

    Background In research laboratories using DNA-microarrays, usually a number of researchers perform experiments, each generating possible sources of error. There is a need for a quick and robust method to assess data quality and sources of errors in DNA-microarray experiments. To this end, a novel and cost-effective validation scheme was devised, implemented, and employed. Results A number of validation experiments were performed on Lactococcus lactis IL1403 amplicon-based DNA-microarrays. Using the validation scheme and ANOVA, the factors contributing to the variance in normalized DNA-microarray data were estimated. Day-to-day as well as experimenter-dependent variances were shown to contribute strongly to the variance, while dye and culturing had a relatively modest contribution to the variance. Conclusion Even in cases where 90 % of the data were kept for analysis and the experiments were performed under challenging conditions (e.g. on different days), the CV was at an acceptable 25 %. Clustering experiments showed that trends can be reliably detected also from genes with very low expression levels. The validation scheme thus allows determining conditions that could be improved to yield even higher DNA-microarray data quality. PMID:15907200

  17. Concordance between RNA-sequencing data and DNA microarray data in transcriptome analysis of proliferative and quiescent fibroblasts

    PubMed Central

    Trost, Brett; Moir, Catherine A.; Gillespie, Zoe E.; Kusalik, Anthony; Mitchell, Jennifer A.; Eskiw, Christopher H.

    2015-01-01

    DNA microarrays and RNA sequencing (RNA-seq) are major technologies for performing high-throughput analysis of transcript abundance. Recently, concerns have been raised regarding the concordance of data derived from the two techniques. Using cDNA libraries derived from normal human foreskin fibroblasts, we measured changes in transcript abundance as cells transitioned from proliferative growth to quiescence using both DNA microarrays and RNA-seq. The internal reproducibility of the RNA-seq data was greater than that of the microarray data. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarray values were moderate. The two technologies had good agreement when considering probes with the largest (both positive and negative) fold change (FC) values. An independent technique, quantitative reverse-transcription PCR (qRT-PCR), was used to measure the FC of 76 genes between proliferative and quiescent samples, and a higher correlation was observed between the qRT-PCR data and the RNA-seq data than between the qRT-PCR data and the microarray data. PMID:26473061

  18. Fabrication of polyurethane molecular stamps for the synthesis of DNA microarray

    NASA Astrophysics Data System (ADS)

    Liu, Zhengchun; He, Quanguo; Xiao, Pengfeng; He, Nongyao; Lu, Zuhong; Bo, Liang

    2001-10-01

    Polyurethane based on polypropylene glycol (PPG) and Toluene diisocyanate (TDI) using 3,3'-dichloride-4,4'- methylenedianiline (MOCA) as the crosslinker is presented for the first time to fabricate molecular stamps (PU stamps) for the synthesis of DNA microarray with contact procedure. The predictability of the process is achieved by utilizing commercially available starting materials. SEM analysis of the morphology of PU stamps and master showed that PU elastometer could replicate subtly the motherboard's patterns with high fidelity. It was proved from the contact angle measurement that PU stamps surface has good affinity with acetonitrile, which guarantee the well-distribution of DNA monomers on patterned stamps. Laser confocal fluorescence microscopy images of oligonucleotide arrays confirmed polyurethane is an excellent material for molecular stamps.

  19. Probe classification of on-off type DNA microarray images with a nonlinear matching measure

    NASA Astrophysics Data System (ADS)

    Ryu, Munho; Kim, Jong Dae; Min, Byoung Goo; Kim, Jongwon; Kim, Y. Y.

    2006-01-01

    We propose a nonlinear matching measure, called counting measure, as a signal detection measure that is defined as the number of on pixels in the spot area. It is applied to classify probes for an on-off type DNA microarray, where each probe spot is classified as hybridized or not. The counting measure also incorporates the maximum response search method, where the expected signal is obtained by taking the maximum among the measured responses of the various positions and sizes of the spot template. The counting measure was compared to existing signal detection measures such as the normalized covariance and the median for 2390 patient samples tested on the human papillomavirus (HPV) DNA chip. The counting measure performed the best regardless of whether or not the maximum response search method was used. The experimental results showed that the counting measure combined with the positional search was the most preferable.

  20. Optimisation of a silicon/silicon dioxide substrate for a fluorescence DNA microarray.

    PubMed

    Bras, M; Dugas, V; Bessueille, F; Cloarec, J P; Martin, J R; Cabrera, M; Chauvet, J P; Souteyrand, E; Garrigues, M

    2004-11-01

    This paper presents a comprehensive theory and experimental characterisation of the modulation of the fluorescence intensity by the construction of optical interferences on oxidised silicon substrates used for DNA microarrays. The model predicts a 90-fold variation of the fluorescence signal depending on the oxide thickness. For a Cy3 dye, the signal is maximal for a 90 nm oxide thickness corresponding to a 7.5-fold enhancement with respect to a standard glass substrate. For experimental validation of the model, we have prepared Si/SiO2 substrates with different parallel steps of decreasing oxide thicknesses on the same sample using a buffered oxide etch (BOE) etching process after thermal oxidation. The SiO2 surface has been functionalized by a silane monolayer before in situ synthesis of L185 oligonucleotide probes. After hybridisation with complementary targets, the variations of the fluorescence intensity versus oxide thickness are in very good accordance with the theoretical model. The experimental comparison against a glass substrate shows a 10-fold enhancement of the detection sensitivity. Our results demonstrate that a Si/SiO2 substrate is an attractive alternative to standard glass slides for the realisation of fluorescence DNA microarrays whenever detection sensitivity is an important issue.

  1. Application of complementary DNA microarray technology to carcinogen identification, toxicology, and drug safety evaluation.

    PubMed

    Afshari, C A; Nuwaysir, E F; Barrett, J C

    1999-10-01

    One major challenge facing today's cancer researchers and toxicologists is the development of new approaches for the identification of carcinogens and other environmental hazards. Here, we describe the potential impact of emerging technologies for measuring gene expression profiles on carcinogen identification and on the general field of toxicology. An example of one of these technologies is the use of cDNA microarray chips. We provide an overview to the key questions that are confronting investigators charged with determining the relative safety of natural or synthetic chemicals to which humans are exposed, followed by a discussion of how cDNA microarray technology may be applied to these questions. Gene chip technology is still a relatively new technology, and only a handful of studies have demonstrated its utility. However, as the technical hurdles to development are passed, the use of this methodology in addressing the questions raised here will be critical to increase the sensitivity of detection of the potential toxic effects of environmental chemicals and to understand their risks to humans.

  2. Prospects for exploring the molecular-genomic foundations of therapeutic hypnosis with DNA microarrays.

    PubMed

    Rossi, Ernest Lawrence

    A new perspective on how therapeutic hypnosis and neuroscience may be integrated on the molecular-genomic level is offered as a guide for basic research and clinical applications. An update of Watson and Crick's original formulation of molecular biology is proposed to illustrate how psychosocial experiences modulate gene expression, protein synthesis, and brain plasticity during memory trace reactivation for the reorganization of neural networks that encode fear, stress, and traumatic symptoms. Examples of the scientific literature on DNA microarrays are used to explore how this new technology could integrate therapeutic hypnosis, neuroscience, and psychosocial genomics as a new foundation for mind-body medicine. Researchers and clinicians in therapeutic hypnosis need to partner with colleagues in neuroscience and molecular biology that utilize DNA microarray technology. It is recommended that hypnotic susceptibility scales of the future incorporate gene expression data to include the concept of "embodied imagination" and the "ideo-plastic faculty" on a molecular-genomic level as well as the psychological and behavioral level of ideomotor and ideosensory responses that are currently assessed.

  3. Differential gene expression profiling of vocal fold polyps and Reinke's edema by complementary DNA microarray.

    PubMed

    Duflo, Suzy M; Thibeault, Susan L; Li, Wenhua; Smith, Marshall E; Schade, Goetz; Hess, Markus M

    2006-09-01

    Our purpose was to determine whether complementary DNA (cDNA) microarray analysis (MA) can establish distinct gene expression profiles for 2 phenotypically similar vocal fold lesions: Reinke's edema (RE) and polyps. Established transcript profiles can provide insight into the molecular and cellular processes involved in these diseases. Eleven RE specimens and 17 polyps were analyzed with MA for 8,745 genes. Further MA profiling was attempted within each lesion group to identify molecular markers for reflux exposure and smoking. Prediction analysis was used to predict lesion classification for 2 unclassified samples. A real-time polymerase chain reaction was performed to corroborate MA transcript levels for selected significant genes. Sixty-five genes were found to differentiate RE and polyps (p = .0088). For RE, 19 genes were differentiated for reflux exposure (p = .016). No genes were found to differentiate smokers from nonsmokers. For polyps, no genes were found to differentiate for reflux (p = .16) and smoking (p = .565). Categorization of unclassified lesions was possible with a minimum of 13 genes. We demonstrate the feasibility of benign lesion classification based on MA. Microarray analysis is useful not only for improving diagnosis and classification of such lesions, but also for potentially generating prognostic indicators and targets for therapy.

  4. PMA-PhyloChip DNA Microarray to Elucidate Viable Microbial Community Structure

    NASA Technical Reports Server (NTRS)

    Venkateswaran, Kasthuri J.; Stam, Christina N.; Andersen, Gary L.; DeSantis, Todd

    2011-01-01

    Since the Viking missions in the mid-1970s, traditional culture-based methods have been used for microbial enumeration by various NASA programs. Viable microbes are of particular concern for spacecraft cleanliness, for forward contamination of extraterrestrial bodies (proliferation of microbes), and for crew health/safety (viable pathogenic microbes). However, a "true" estimation of viable microbial population and differentiation from their dead cells using the most sensitive molecular methods is a challenge, because of the stability of DNA from dead cells. The goal of this research is to evaluate a rapid and sensitive microbial detection concept that will selectively estimate viable microbes. Nucleic acid amplification approaches such as the polymerase chain reaction (PCR) have shown promise for reducing time to detection for a wide range of applications. The proposed method is based on the use of a fluorescent DNA intercalating agent, propidium monoazide (PMA), which can only penetrate the membrane of dead cells. The PMA-quenched reaction mixtures can be screened, where only the DNA from live cells will be available for subsequent PCR reaction and microarray detection, and be identified as part of the viable microbial community. An additional advantage of the proposed rapid method is that it will detect viable microbes and differentiate from dead cells in only a few hours, as opposed to less comprehensive culture-based assays, which take days to complete. This novel combination approach is called the PMA-Microarray method. DNA intercalating agents such as PMA have previously been used to selectively distinguish between viable and dead bacterial cells. Once in the cell, the dye intercalates with the DNA and, upon photolysis under visible light, produces stable DNA adducts. DNA cross-linked in this way is unavailable for PCR. Environmental samples suspected of containing a mixture of live and dead microbial cells/spores will be treated with PMA, and then incubated

  5. A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

    PubMed

    Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

    2016-01-01

    Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

  6. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis.

    PubMed

    Salonen, Anne; Nikkilä, Janne; Jalanka-Tuovinen, Jonna; Immonen, Outi; Rajilić-Stojanović, Mirjana; Kekkonen, Riina A; Palva, Airi; de Vos, Willem M

    2010-05-01

    Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories. Copyright 2010 Elsevier B.V. All rights reserved.

  7. Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays.

    PubMed

    Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

    2012-01-01

    PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes.

  8. Selective recognition of DNA from olive leaves and olive oil by PNA and modified-PNA microarrays

    PubMed Central

    Rossi, Stefano; Calabretta, Alessandro; Tedeschi, Tullia; Sforza, Stefano; Arcioni, Sergio; Baldoni, Luciana; Corradini, Roberto; Marchelli, Rosangela

    2012-01-01

    PNA probes for the specific detection of DNA from olive oil samples by microarray technology were developed. The presence of as low as 5% refined hazelnut (Corylus avellana) oil in extra-virgin olive oil (Olea europaea L.) could be detected by using a PNA microarray. A set of two single nucleotide polymorphisms (SNPs) from the Actin gene of Olive was chosen as a model for evaluating the ability of PNA probes for discriminating olive cultivars. Both unmodified and C2-modified PNAs bearing an arginine side-chain were used, the latter showing higher sequence specificity. DNA extracted from leaves of three different cultivars (Ogliarola leccese, Canino and Frantoio) could be easily discriminated using a microarray with unmodified PNA probes, whereas discrimination of DNA from oil samples was more challenging, and could be obtained only by using chiral PNA probes. PMID:22772038

  9. Systematic Spatial Bias in DNA Microarray Hybridization Is Caused by Probe Spot Position-Dependent Variability in Lateral Diffusion

    PubMed Central

    Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

    2011-01-01

    Background The hybridization of nucleic acid targets with surface-immobilized probes is a widely used assay for the parallel detection of multiple targets in medical and biological research. Despite its widespread application, DNA microarray technology still suffers from several biases and lack of reproducibility, stemming in part from an incomplete understanding of the processes governing surface hybridization. In particular, non-random spatial variations within individual microarray hybridizations are often observed, but the mechanisms underpinning this positional bias remain incompletely explained. Methodology/Principal Findings This study identifies and rationalizes a systematic spatial bias in the intensity of surface hybridization, characterized by markedly increased signal intensity of spots located at the boundaries of the spotted areas of the microarray slide. Combining observations from a simplified single-probe block array format with predictions from a mathematical model, the mechanism responsible for this bias is found to be a position-dependent variation in lateral diffusion of target molecules. Numerical simulations reveal a strong influence of microarray well geometry on the spatial bias. Conclusions Reciprocal adjustment of the size of the microarray hybridization chamber to the area of surface-bound probes is a simple and effective measure to minimize or eliminate the diffusion-based bias, resulting in increased uniformity and accuracy of quantitative DNA microarray hybridization. PMID:21858215

  10. DNA nanofilm thickness measurement on microarray in air and in liquid using an atomic force microscope.

    PubMed

    Legay, Guillaume; Finot, Eric; Meunier-Prest, Rita; Cherkaoui-Malki, Mustapha; Latruffe, Norbert; Dereux, Alain

    2005-10-15

    The measurement of the thickness of DNA films on microarray as a function of the medium (liquid, air) is gaining importance for understanding the signal response of biosensors. Thiol group has been used to attach DNA strands to gold micropads deposited on silicon surface. Atomic force microscopy (AFM) was employed in its height mode to measure the change in the pad thickness and in its force mode to measure the indentation depth of the nanofilm. A good coherence between the height and force modes is observed for the film thickness in air. The adhesion force was found to be an alternative way to measure the surface coverage of the biolayer at nanoscopic scale. However the force analysis (compression, steric and electrostatic) provides baseline information necessary to interpret the AFM height image in liquid. Analysis of the film thickness distribution shows that the height of the DNA strands depends on both the DNA strand length (15-35 base pairs) and the environment (air, liquid). In air, longer strands lay down onto gold surface whereas the charge reversal of gold in liquid causes a repulsion of longer strands, which stand up.

  11. Evaluation of normalization methods for cDNA microarray data by k-NN classification

    SciTech Connect

    Wu, Wei; Xing, Eric P; Myers, Connie; Mian, Saira; Bissell, Mina J

    2004-12-17

    Non-biological factors give rise to unwanted variations in cDNA microarray data. There are many normalization methods designed to remove such variations. However, to date there have been few published systematic evaluations of these techniques for removing variations arising from dye biases in the context of downstream, higher-order analytical tasks such as classification. Ten location normalization methods that adjust spatial- and/or intensity-dependent dye biases, and three scale methods that adjust scale differences were applied, individually and in combination, to five distinct, published, cancer biology-related cDNA microarray data sets. Leave-one-out cross-validation (LOOCV) classification error was employed as the quantitative end-point for assessing the effectiveness of a normalization method. In particular, a known classifier, k-nearest neighbor (k-NN), was estimated from data normalized using a given technique, and the LOOCV error rate of the ensuing model was computed. We found that k-NN classifiers are sensitive to dye biases in the data. Using NONRM and GMEDIAN as baseline methods, our results show that single-bias-removal techniques which remove either spatial-dependent dye bias (referred later as spatial effect) or intensity-dependent dye bias (referred later as intensity effect) moderately reduce LOOCV classification errors; whereas double-bias-removal techniques which remove both spatial- and intensity effect reduce LOOCV classification errors even further. Of the 41 different strategies examined, three two-step processes, IGLOESS-SLFILTERW7, ISTSPLINE-SLLOESS and IGLOESS-SLLOESS, all of which removed intensity effect globally and spatial effect locally, appear to reduce LOOCV classification errors most consistently and effectively across all data sets. We also found that the investigated scale normalization methods do not reduce LOOCV classification error. Using LOOCV error of k-NNs as the evaluation criterion, three double

  12. Gene expression profiling of in Moniezia expansa at different developmental proglottids using cDNA microarray.

    PubMed

    Bo, Xinwen; Zhao, Wenjuan; Zhang, Hui; Kang, Lichao; Wang, Xinhua

    2012-04-01

    Gene expression profiles of Moniezia expansa proglottids at varying developmental stages were analysed using cDNA microarray. A total of 4,056 spots, including full length and partial complementary DNAs that represent novel, known, and control genes, were studied. Results indicated an up-regulation of 55 genes in immature proglottids, 134 genes in mature proglottids and 103 genes in gravid proglottids were up-regulated, and a down-regulation of 7 genes in immature proglottids, 68 genes in mature proglottids and 78 genes in gravid proglottids compared to controls (scolex-neck proglottids). Many of these genes were identified as transcription factors and were involved in functions such as metabolism, transport, protein biosynthesis, apoptosis, cell differentiation, cell communication and nucleic acid binding. Expression level alterations in UBE2A, Cavβ, RAD51, DAZ, PKAc and 2 unknown genes were confirmed by real-time quantitative polymerase chain reaction (RT-PCR). The complete microarray data set has been deposited in the NCBI Gene Expression Omnibus, GEO Series accession number GSE13982. Results provide a gene expression profile at various development stages of M. expansa proglottids, which prove invaluable in understanding the pathogenesis of the tapeworm and studying the genes concerned with reproductive organ development.

  13. Enzymatic on-chip enhancement for high resolution genotyping DNA microarrays.

    PubMed

    Schulze, Holger; Barl, Timo; Vase, Hollie; Baier, Shiromi; Thomas, Peter; Giraud, Gerard; Crain, Jason; Bachmann, Till T

    2012-06-05

    Antibiotic resistance among pathogenic microorganisms is emerging as a major human healthcare concern. While there are a variety of resistance mechanisms, many can be related to single nucleotide polymorphisms and for which DNA microarrays have been widely deployed in bacterial genotyping. However, genotyping by means of allele-specific hybridization can suffer from the drawback that oligonucleotide probes with different nucleotide composition have varying thermodynamic parameters. This results in unpredictable hybridization behavior of mismatch probes. Consequently, the degree of discrimination between perfect match and mismatch probes is insufficient in some cases. We report here an on-chip enzymatic procedure to improve this discrimination in which false-positive hybrids are selectively digested. We find that the application of CEL1 Surveyor nuclease, a mismatch-specific endonuclease, significantly enhances the discrimination fidelity, as demonstrated here on a microarray for the identification of variants of carbapenem resistant Klebsiella pneumoniae carbapenemases and monitored by end point detection of fluorescence intensity. Further fundamental investigations applying total internal reflection fluorescence detection for kinetic real-time measurements confirmed the enzymatic enhancement for SNP discrimination.

  14. A comparison of parametric and nonparametric methods for normalising cDNA microarray data.

    PubMed

    Khondoker, Mizanur R; Glasbey, Chris A; Worton, Bruce J

    2007-12-01

    Normalisation is an essential first step in the analysis of most cDNA microarray data, to correct for effects arising from imperfections in the technology. Loess smoothing is commonly used to correct for trends in log-ratio data. However, parametric models, such as the additive plus multiplicative variance model, have been preferred for scale normalisation, though the variance structure of microarray data may be of a more complex nature than can be accommodated by a parametric model. We propose a new nonparametric approach that incorporates location and scale normalisation simultaneously using a Generalised Additive Model for Location, Scale and Shape (GAMLSS, Rigby and Stasinopoulos, 2005, Applied Statistics, 54, 507-554). We compare its performance in inferring differential expression with Huber et al.'s (2002, Bioinformatics, 18, 96-104) arsinh variance stabilising transformation (AVST) using real and simulated data. We show GAMLSS to be as powerful as AVST when the parametric model is correct, and more powerful when the model is wrong. (c) 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

  15. DNA microarray analysis on gene candidates possibly related to tetrodotoxin accumulation in pufferfish.

    PubMed

    Feroudj, Holger; Matsumoto, Takuya; Kurosu, Yohei; Kaneko, Gen; Ushio, Hideki; Suzuki, Katsuaki; Kondo, Hidehiro; Hirono, Ikuo; Nagashima, Yuji; Akimoto, Seiji; Usui, Kazushige; Kinoshita, Shigeharu; Asakawa, Shuichi; Kodama, Masaaki; Watabe, Shugo

    2014-01-01

    Pufferfish accumulate tetrodotoxin (TTX) at high levels in liver and ovary through the food chain. However, the mechanisms underlying TTX toxification in pufferfish have been poorly understood. In order to search gene candidates involved in TTX accumulation in the torafugu pufferfish Takifugu rubripes, a custom 4x44k oligonucleotide microarray slide was designed by the Agilent eArray program using oligonucleotide probes of 60 bp in length referring to 42,724 predicted transcripts in the publicly available Fugu genome database. DNA microarray analysis was performed with total RNA samples from the livers of two toxic wild specimens in comparison with those from a nontoxic wild specimen and two nontoxic cultured specimens. The mRNA levels of 1108 transcripts were more than 2-fold higher in the toxic specimens than in the nontoxic specimens. The levels of 613 transcripts were remarkably high, and 16 transcripts encoded by 9 genes were up-regulated more than 10-fold. These genes included those encoding forming structural filaments (keratins) and those related to vitamin D metabolism and immunity. It was also noted that the levels of the transcripts encoding serpin peptidase inhibitor clade C member 1, coagulation factor X precursor, complement C2, C3, C5, C8 precursors, and interleukin-6 receptor were high in the toxic liver samples. Copyright © 2013 Elsevier Ltd. All rights reserved.

  16. Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray

    PubMed Central

    Renn, Susan CP; Aubin-Horth, Nadia; Hofmann, Hans A

    2004-01-01

    Background Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits. Results Here we present a microarray constructed with ~4500 features, derived from a brain-specific cDNA library for the African cichlid fish Astatotilapia burtoni (Perciformes). Heterologous hybridization, targeting RNA to an array constructed for a different species, is used for eight different fish species. We quantified the concordance in gene expression profiles across these species (number of genes and fold-changes). Although most robust when target RNA is derived from closely related species (<10 MA divergence time), our results showed consistent profiles for other closely related taxa (~65 MA divergence time) and, to a lesser extent, even very distantly related species (>200 MA divergence time). Conclusion This strategy overcomes some of the restrictions imposed on model systems that are of importance for evolutionary and ecological studies, but for which only limited sequence information is available. Our work validates the use of expression profiling for functional genomics within a comparative framework and provides a foundation for the molecular and cellular analysis of complex traits in a wide range of organisms. PMID:15238158

  17. Administered chrysanthemum flower oil attenuates hyperuricemia: mechanism of action as revealed by DNA microarray analysis.

    PubMed

    Honda, Shinichi; Kawamoto, Seiji; Tanaka, Hozumi; Kishida, Hideyuki; Kitagawa, Masayasu; Nakai, Yuji; Abe, Keiko; Hirata, Dai

    2014-01-01

    We applied Chrysanthemum flower oil (CFO) to a hyperuricemia model by feeding rats a hyperuricemia-inducing diet (HID) and investigated its effect on serum uric acid (SUA) levels and its mode of action. CFO is the oily fraction that contains polyphenols derived from chrysanthemum flowers. Oral administration of CFO to HID-fed rats significantly decreased their SUA levels. It also inhibited xanthine oxidase activities in the liver and increased urine uric acid levels. The effects of CFO on the renal gene expressions that accompanied the induction of hyperuricemia were comprehensively confirmed by DNA microarray analysis. The analysis showed up-regulation of those genes for uric acid excretion by CFO administration. These results suggest that CFO suppresses the increase in SUA levels via two mechanisms: suppression of uric acid production by inhibition of xanthine oxidase in the liver and acceleration of its excretion by up-regulation of uric acid transporter genes in the kidney.

  18. Convergent evolution to an aptamer observed in small populations on DNA microarrays

    NASA Astrophysics Data System (ADS)

    Rowe, W.; Platt, M.; Wedge, D. C.; Day, P. J. R.; Kell, D. B.; Knowles, J. D.

    2010-09-01

    The development of aptamers on custom synthesized DNA microarrays, which has been demonstrated in recent publications, can facilitate detailed analyses of sequence and fitness relationships. Here we use the technique to observe the paths taken through sequence-fitness space by three different evolutionary regimes: asexual reproduction, recombination and model-based evolution. The different evolutionary runs are made on the same array chip in triplicate, each one starting from a small population initialized independently at random. When evolving to a common target protein, glucose-6-phosphate dehydrogenase (G6PD), these nine distinct evolutionary runs are observed to develop aptamers with high affinity and to converge on the same motif not present in any of the starting populations. Regime specific differences in the evolutions, such as speed of convergence, could also be observed.

  19. Fluorescence lifetime biosensing with DNA microarrays and a CMOS-SPAD imager

    PubMed Central

    Giraud, Gerard; Schulze, Holger; Li, Day-Uei; Bachmann, Till T.; Crain, Jason; Tyndall, David; Richardson, Justin; Walker, Richard; Stoppa, David; Charbon, Edoardo; Henderson, Robert; Arlt, Jochen

    2010-01-01

    Fluorescence lifetime of dye molecules is a sensitive reporter on local microenvironment which is generally independent of fluorophores concentration and can be used as a means of discrimination between molecules with spectrally overlapping emission. It is therefore a potentially powerful multiplexed detection modality in biosensing but requires extremely low light level operation typical of biological analyte concentrations, long data acquisition periods and on-chip processing capability to realize these advantages. We report here fluorescence lifetime data obtained using a CMOS-SPAD imager in conjunction with DNA microarrays and TIRF excitation geometry. This enables acquisition of single photon arrival time histograms for a 320 pixel FLIM map within less than 26 seconds exposure time. From this, we resolve distinct lifetime signatures corresponding to dye-labelled HCV and quantum-dot-labelled HCMV nucleic acid targets at concentrations as low as 10 nM. PMID:21258550

  20. DNA microarray unravels rapid changes in transcriptome of MK-801 treated rat brain

    PubMed Central

    Kobayashi, Yuka; Kulikova, Sofya P; Shibato, Junko; Rakwal, Randeep; Satoh, Hiroyuki; Pinault, Didier; Masuo, Yoshinori

    2015-01-01

    AIM: To investigate the impact of MK-801 on gene expression patterns genome wide in rat brain regions. METHODS: Rats were treated with an intraperitoneal injection of MK-801 [0.08 (low-dose) and 0.16 (high-dose) mg/kg] or NaCl (vehicle control). In a first series of experiment, the frontoparietal electrocorticogram was recorded 15 min before and 60 min after injection. In a second series of experiments, the whole brain of each animal was rapidly removed at 40 min post-injection, and different regions were separated: amygdala, cerebral cortex, hippocampus, hypothalamus, midbrain and ventral striatum on ice followed by DNA microarray (4 × 44 K whole rat genome chip) analysis. RESULTS: Spectral analysis revealed that a single systemic injection of MK-801 significantly and selectively augmented the power of baseline gamma frequency (30-80 Hz) oscillations in the frontoparietal electroencephalogram. DNA microarray analysis showed the largest number (up- and down- regulations) of gene expressions in the cerebral cortex (378), midbrain (376), hippocampus (375), ventral striatum (353), amygdala (301), and hypothalamus (201) under low-dose (0.08 mg/kg) of MK-801. Under high-dose (0.16 mg/kg), ventral striatum (811) showed the largest number of gene expression changes. Gene expression changes were functionally categorized to reveal expression of genes and function varies with each brain region. CONCLUSION: Acute MK-801 treatment increases synchrony of baseline gamma oscillations, and causes very early changes in gene expressions in six individual rat brain regions, a first report. PMID:26629322

  1. Comprehensive Census of Bacteria in Clean Rooms by Using DNA Microarray and Cloning Methods▿ †

    PubMed Central

    La Duc, Myron T.; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J. A.; Venkateswaran, Kasthuri

    2009-01-01

    A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments. PMID:19700540

  2. Comprehensive census of bacteria in clean rooms by using DNA microarray and cloning methods.

    PubMed

    La Duc, Myron T; Osman, Shariff; Vaishampayan, Parag; Piceno, Yvette; Andersen, Gary; Spry, J A; Venkateswaran, Kasthuri

    2009-10-01

    A census of clean room surface-associated bacterial populations was derived from the results of both the cloning and sequencing of 16S rRNA genes and DNA microarray (PhyloChip) analyses. Samples from the Lockheed Martin Aeronautics Multiple Testing Facility (LMA-MTF), the Kennedy Space Center Payload Hazard and Servicing Facility (KSC-PHSF), and the Jet Propulsion Laboratory Spacecraft Assembly Facility (JPL-SAF) clean rooms were collected during the various assembly phases of the Phoenix and Mars Science Laboratory (MSL) spacecraft. Clone library-derived analyses detected a larger bacterial diversity prior to the arrival of spacecraft hardware in these clean room facilities. PhyloChip results were in agreement with this trend but also unveiled the presence of anywhere from 9- to 70-fold more bacterial taxa than cloning approaches. Among the facilities sampled, the JPL-SAF (MSL mission) housed a significantly less diverse bacterial population than either the LMA-MTF or KSC-PHSF (Phoenix mission). Bacterial taxa known to thrive in arid conditions were frequently detected in MSL-associated JPL-SAF samples, whereas proteobacterial lineages dominated Phoenix-associated KSC-PHSF samples. Comprehensive bacterial censuses, such as that reported here, will help space-faring nations preemptively identify contaminant biomatter that may compromise extraterrestrial life detection experiments. The robust nature and high sensitivity of DNA microarray technologies should prove beneficial to a wide range of scientific, electronic, homeland security, medical, and pharmaceutical applications and to any other ventures with a vested interest in monitoring and controlling contamination in exceptionally clean environments.

  3. Automatic image analysis and spot classification for detection of pathogenic Escherichia coli on glass slide DNA microarrays

    USDA-ARS?s Scientific Manuscript database

    A computer algorithm was created to inspect scanned images from DNA microarray slides developed to rapidly detect and genotype E. Coli O157 virulent strains. The algorithm computes centroid locations for signal and background pixels in RGB space and defines a plane perpendicular to the line connect...

  4. 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.

  5. Using a DNA microarray to investigate the distribution of insect virulence factors in strains of photorhabdus bacteria.

    PubMed

    Marokhazi, Judit; Waterfield, Nicholas; LeGoff, Gaelle; Feil, Edward; Stabler, Richard; Hinds, Jason; Fodor, Andras; ffrench-Constant, Richard H

    2003-08-01

    Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.

  6. Using a DNA Microarray To Investigate the Distribution of Insect Virulence Factors in Strains of Photorhabdus Bacteria

    PubMed Central

    Marokhazi, Judit; Waterfield, Nicholas; LeGoff, Gaelle; Feil, Edward; Stabler, Richard; Hinds, Jason; Fodor, Andras; ffrench-Constant, Richard H.

    2003-01-01

    Photorhabdus is an insect-pathogenic bacterium in which oral toxicity to insects is found in two distinct taxonomic groups. Using a DNA microarray and comparative genomics, we show that oral toxicity is associated with toxin complex genes tcaABC and that this locus can be mobilized or deleted within different strains.   PMID:12867479

  7. Microfluidic chip for high efficiency DNA extraction.

    PubMed

    Chung, Yung-Chiang; Jan, Ming-Shiung; Lin, Yu-Cheng; Lin, Ju-Hwa; Cheng, Wang-Chin; Fan, Chia-Yu

    2004-04-01

    A high efficiency DNA extraction microchip was designed to extract DNA from lysed cells using immobilized beads and the solution flowing back and forth. This chip was able to increase the extraction efficiency by 2-fold when there was no serum. When serum existed in the solution, the extraction efficiency of immobilized beads was 88-fold higher than that of free beads. The extraction efficiency of the microchip was tested under different conditions and numbers of E. coli cells. When the number of E. coli cells was between 10(6) and 10(8) in 25 microl of whole blood, the extraction efficiency using immobilized beads was only slightly higher than that using free beads (10(0) to 10(1) fold). When the number of E. coli cells was in the range 10(4) to 10(6) in 25 microl of whole blood, the extraction efficiency of immobilized beads was greater than that of the free beads (10(1) to 10(2) fold). When the number of E. coli cells was lower, in the range 10(3) to 10(4) in 25 microl of whole blood, the extraction efficiency of immobilized beads was much higher than that of the free beads (10(2) to 10(3) fold). This study indicated that DNA could be efficiently extracted even when the number of bacterial cells was smaller (10(5) to 10(3)). This microfluidic extraction chip could find potential applications in rare sample genomic study.

  8. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays.

    PubMed

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T; Vinson, Charles

    2015-07-16

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS ((C)/GCGGAA GT: ) and CRE ( GT: GACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif ((C)/GCGGAA GT: GACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form (C)/GCGGA--CG-, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding. Copyright © 2015 He et al.

  9. GABPα Binding to Overlapping ETS and CRE DNA Motifs Is Enhanced by CREB1: Custom DNA Microarrays

    PubMed Central

    He, Ximiao; Syed, Khund Sayeed; Tillo, Desiree; Mann, Ishminder; Weirauch, Matthew T.; Vinson, Charles

    2015-01-01

    To achieve proper spatiotemporal control of gene expression, transcription factors cooperatively assemble onto specific DNA sequences. The ETS domain protein monomer of GABPα and the B-ZIP domain protein dimer of CREB1 cooperatively bind DNA only when the ETS (C/GCGGAAGT) and CRE (GTGACGTCAC) motifs overlap precisely, producing the ETS↔CRE motif (C/GCGGAAGTGACGTCAC). We designed a Protein Binding Microarray (PBM) with 60-bp DNAs containing four identical sectors, each with 177,440 features that explore the cooperative interactions between GABPα and CREB1 upon binding the ETS↔CRE motif. The DNA sequences include all 15-mers of the form C/GCGGA—–CG—, the ETS↔CRE motif, and all single nucleotide polymorphisms (SNPs), and occurrences in the human and mouse genomes. CREB1 enhanced GABPα binding to the canonical ETS↔CRE motif CCGGAAGT two-fold, and up to 23-fold for several SNPs at the beginning and end of the ETS motif, which is suggestive of two separate and distinct allosteric mechanisms of cooperative binding. We show that the ETS-CRE array data can be used to identify regions likely cooperatively bound by GABPα and CREB1 in vivo, and demonstrate their ability to identify human genetic variants that might inhibit cooperative binding. PMID:26185160

  10. Methods for strand-specific DNA detection with cationic conjugated polymers suitable for incorporation into DNA chips and microarrays.

    PubMed

    Liu, Bin; Bazan, Guillermo C

    2005-01-18

    A strand-specific DNA sensory method is described based on surface-bound peptide nucleic acids and water-soluble cationic conjugated polymers. The main transduction mechanism operates by taking advantage of the net increase in negative charge at the peptide nucleic acid surface that occurs upon single-stranded DNA hybridization. Electrostatic forces cause the oppositely charged cationic conjugated polymer to bind selectively to the "complementary" surfaces. This approach circumvents the current need to label the probe or target strands. The polymer used in these assays is poly[9,9'-bis(6''-N,N,N-trimethylammonium)hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide], which was specifically designed and synthesized to be compatible with excitation sources used in commonly used DNA microarray readers. Furthermore, the utility of poly[9,9'-bis(6''-N,N,N-trimethylammonium)-hexyl)fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide] has been demonstrated in homogenous and solid-state assays that involve fluorescence resonance energy transfer to a reporter dye (Cy5) and that can benefit from the light harvesting properties observed in water-soluble conjugated polymers.

  11. 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.

  12. 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.

  13. A proof-of-principle demonstration of a novel microarray-based method for quantifying DNA methylation levels.

    PubMed

    Zhang, Xiujuan; Zhou, Dongrui; Zhao, Ming; Luo, Yongqi; Zhang, Peng; Lu, Zuhong; Lu, Qianjin

    2010-11-01

    Demethylation of CD11a (ITGAL; GeneID:3683; HGNC: 6148) and CD70 (TNFSF7; GeneID:970; HGNC:11937) regulatory regions in CD4(+) T cells contributes to the development of autoreactivity and autoantibody overstimulation in systemic lupus erythematosus (SLE). In this study, we present a novel approach for measuring the methylation status of CD11a and CD70 promoter sequences. The procedure combines the standard method of bisulfite conversion of methylated CpG pairs with high-throughput oligonucleotide microarray-based technology that allows for rapid quantification of deoxycytosine and deoxymethylcytosine content in bisulfite-treated DNA samples. The microarrays were first used to generate a standard curve from fully methylated and fully unmethylated DNA samples using a one-dimensional linear regression equation that calculated fluorescence emission as a function of methylation levels. The methylation status of the CD70 and CD11a promoters in SLE and control CD4(+) T cell samples were measured, and the microarray prediction was found to be highly accurate when compared to bisulfite sequencing. Furthermore, the microarrays were able to detect differences in the methylation status between SLE patient and healthy control samples. These results indicate that our new microarray-based assay could prove to be a highly reliable, rapid, and cost effective diagnostic and prognostic test for SLE.

  14. Identification of target genes conferring ethanol stress tolerance to Saccharomyces cerevisiae based on DNA microarray data analysis.

    PubMed

    Hirasawa, Takashi; Yoshikawa, Katsunori; Nakakura, Yuki; Nagahisa, Keisuke; Furusawa, Chikara; Katakura, Yoshio; Shimizu, Hiroshi; Shioya, Suteaki

    2007-08-01

    During industrial production process using yeast, cells are exposed to the stress due to the accumulation of ethanol, which affects the cell growth activity and productivity of target products, thus, the ethanol stress-tolerant yeast strains are highly desired. To identify the target gene(s) for constructing ethanol stress tolerant yeast strains, we obtained the gene expression profiles of two strains of Saccharomyces cerevisiae, namely, a laboratory strain and a strain used for brewing Japanese rice wine (sake), in the presence of 5% (v/v) ethanol, using DNA microarray. For the selection of target genes for breeding ethanol stress tolerant strains, clustering of DNA microarray data was performed. For further selection, the ethanol sensitivity of the knockout mutants in each of which the gene selected by DNA microarray analysis is deleted, was also investigated. The integration of the DNA microarray data and the ethanol sensitivity data of knockout strains suggests that the enhancement of expression of genes related to tryptophan biosynthesis might confer the ethanol stress tolerance to yeast cells. Indeed, the strains overexpressing tryptophan biosynthesis genes showed a stress tolerance to 5% ethanol. Moreover, the addition of tryptophan to the culture medium and overexpression of tryptophan permease gene conferred ethanol stress tolerance to yeast cells. These results indicate that overexpression of the genes for trypophan biosynthesis increases the ethanol stress tolerance. Tryptophan supplementation to culture and overexpression of the tryptophan permease gene are also effective for the increase in ethanol stress tolerance. Our methodology for the selection of target genes for constructing ethanol stress tolerant strains, based on the data of DNA microarray analysis and phenotypes of knockout mutants, was validated.

  15. 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

  16. An improved K-means clustering method for cDNA microarray image segmentation.

    PubMed

    Wang, T N; Li, T J; Shao, G F; Wu, S X

    2015-07-14

    Microarray technology is a powerful tool for human genetic research and other biomedical applications. Numerous improvements to the standard K-means algorithm have been carried out to complete the image segmentation step. However, most of the previous studies classify the image into two clusters. In this paper, we propose a novel K-means algorithm, which first classifies the image into three clusters, and then one of the three clusters is divided as the background region and the other two clusters, as the foreground region. The proposed method was evaluated on six different data sets. The analyses of accuracy, efficiency, expression values, special gene spots, and noise images demonstrate the effectiveness of our method in improving the segmentation quality.

  17. 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

  18. Analysis of microarray leukemia data using an efficient MapReduce-based K-nearest-neighbor classifier.

    PubMed

    Kumar, Mukesh; Rath, Nitish Kumar; Rath, Santanu Kumar

    2016-04-01

    Microarray-based gene expression profiling has emerged as an efficient technique for classification, prognosis, diagnosis, and treatment of cancer. Frequent changes in the behavior of this disease generates an enormous volume of data. Microarray data satisfies both the veracity and velocity properties of big data, as it keeps changing with time. Therefore, the analysis of microarray datasets in a small amount of time is essential. They often contain a large amount of expression, but only a fraction of it comprises genes that are significantly expressed. The precise identification of genes of interest that are responsible for causing cancer are imperative in microarray data analysis. Most existing schemes employ a two-phase process such as feature selection/extraction followed by classification. In this paper, various statistical methods (tests) based on MapReduce are proposed for selecting relevant features. After feature selection, a MapReduce-based K-nearest neighbor (mrKNN) classifier is also employed to classify microarray data. These algorithms are successfully implemented in a Hadoop framework. A comparative analysis is done on these MapReduce-based models using microarray datasets of various dimensions. From the obtained results, it is observed that these models consume much less execution time than conventional models in processing big data.

  19. DNA polymerase preference determines PCR priming efficiency

    PubMed Central

    2014-01-01

    Background Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3’ hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Results Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3’ end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. Conclusions DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification

  20. A Comprehensive Microarray-Based DNA Methylation Study of 367 Hematological Neoplasms

    PubMed Central

    Bibikova, Marina; Wickham-Garcia, Eliza; Agirre, Xabier; Alvarez, Sara; Brüggemann, Monika; Bug, Stefanie; Calasanz, Maria J.; Deckert, Martina; Dreyling, Martin; Du, Ming Q.; Dürig, Jan; Dyer, Martin J. S.; Fan, Jian-Bing; Gesk, Stefan; Hansmann, Martin-Leo; Harder, Lana; Hartmann, Sylvia; Klapper, Wolfram; Küppers, Ralf; Montesinos-Rongen, Manuel; Nagel, Inga; Pott, Christiane; Richter, Julia; Román-Gómez, José; Seifert, Marc; Stein, Harald; Suela, Javier; Trümper, Lorenz; Vater, Inga; Prosper, Felipe; Haferlach, Claudia; Cigudosa, Juan Cruz; Siebert, Reiner

    2009-01-01

    Background Alterations in the DNA methylation pattern are a hallmark of leukemias and lymphomas. However, most epigenetic studies in hematologic neoplasms (HNs) have focused either on the analysis of few candidate genes or many genes and few HN entities, and comprehensive studies are required. Methodology/Principal Findings Here, we report for the first time a microarray-based DNA methylation study of 767 genes in 367 HNs diagnosed with 16 of the most representative B-cell (n = 203), T-cell (n = 30), and myeloid (n = 134) neoplasias, as well as 37 samples from different cell types of the hematopoietic system. Using appropriate controls of B-, T-, or myeloid cellular origin, we identified a total of 220 genes hypermethylated in at least one HN entity. In general, promoter hypermethylation was more frequent in lymphoid malignancies than in myeloid malignancies, being germinal center mature B-cell lymphomas as well as B and T precursor lymphoid neoplasias those entities with highest frequency of gene-associated DNA hypermethylation. We also observed a significant correlation between the number of hypermethylated and hypomethylated genes in several mature B-cell neoplasias, but not in precursor B- and T-cell leukemias. Most of the genes becoming hypermethylated contained promoters with high CpG content, and a significant fraction of them are targets of the polycomb repressor complex. Interestingly, T-cell prolymphocytic leukemias show low levels of DNA hypermethylation and a comparatively large number of hypomethylated genes, many of them showing an increased gene expression. Conclusions/Significance We have characterized the DNA methylation profile of a wide range of different HNs entities. As well as identifying genes showing aberrant DNA methylation in certain HN subtypes, we also detected six genes—DBC1, DIO3, FZD9, HS3ST2, MOS, and MYOD1—that were significantly hypermethylated in B-cell, T-cell, and myeloid malignancies. These might therefore play

  1. Semi-automated extraction of microbial DNA from feces for qPCR and phylogenetic microarray analysis.

    PubMed

    Nylund, Lotta; Heilig, Hans G H J; Salminen, Seppo; de Vos, Willem M; Satokari, Reetta

    2010-11-01

    The human gastrointestinal tract (GI-tract) harbors a complex microbial ecosystem, largely composed of so far uncultured species, which can be detected only by using techniques such as PCR and by different hybridization techniques including phylogenetic microarrays. Manual DNA extraction from feces is laborious and is one of the bottlenecks holding up the application of microarray and other DNA-based techniques in large cohort studies. In order to enhance the DNA extraction step we combined mechanical disruption of microbial cells by repeated bead-beating (RBB) with two automated DNA extraction methods, KingFisher with InviMag Stool DNA kit (KF) and NucliSENS easyMAG (NeM). The semi-automated DNA extraction methods, RBB combined with either KF or NeM, were compared to the manual extraction method currently considered the most suited method for fecal DNA extraction by assessing the yield of 16S rRNA gene copies by qPCR and total microbiota composition by the HITChip, a phylogenetic microarray. Parallel DNA extractions from infant fecal samples by using the three methods showed that the KF and manual methods gave comparable yields of 16S rRNA gene copies as assessed by qPCR, whereas NeM showed a significantly lower yield. All three methods showed highly similar microbiota profiles in HITChip. Both KF and NeM were found to be suitable methods for DNA extraction from fecal samples after the mechanical disruption of microbial cells by bead-beating. The semi-automated methods could be performed in half of the time required for the manual protocol, while being comparable to the manual method in terms of reagent costs. Copyright © 2010 Elsevier B.V. All rights reserved.

  2. Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology.

    PubMed

    Strauß, Lena; Ruffing, Ulla; Abdulla, Salim; Alabi, Abraham; Akulenko, Ruslan; Garrine, Marcelino; Germann, Anja; Grobusch, Martin Peter; Helms, Volkhard; Herrmann, Mathias; Kazimoto, Theckla; Kern, Winfried; Mandomando, Inácio; Peters, Georg; Schaumburg, Frieder; von Müller, Lutz; Mellmann, Alexander

    2016-04-01

    Staphylococcus aureusis a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed anin silicotyping scheme for the software SeqSphere(+)(Ridom GmbH, Münster, Germany). The implemented target genes (n= 182) correspond to those queried by the IdentibacS. aureusGenotyping DNA microarray (Alere Technologies, Jena, Germany). Thein silicoscheme was evaluated by comparing the typing results of microarray and of WGS for 154 humanS. aureusisolates. A total of 96.8% (n= 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosomemecelement types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences.

  3. Detecting Staphylococcus aureus Virulence and Resistance Genes: a Comparison of Whole-Genome Sequencing and DNA Microarray Technology

    PubMed Central

    Strauß, Lena; Ruffing, Ulla; Abdulla, Salim; Alabi, Abraham; Akulenko, Ruslan; Garrine, Marcelino; Germann, Anja; Grobusch, Martin Peter; Helms, Volkhard; Herrmann, Mathias; Kazimoto, Theckla; Kern, Winfried; Mandomando, Inácio; Peters, Georg; Schaumburg, Frieder; von Müller, Lutz

    2016-01-01

    Staphylococcus aureus is a major bacterial pathogen causing a variety of diseases ranging from wound infections to severe bacteremia or intoxications. Besides host factors, the course and severity of disease is also widely dependent on the genotype of the bacterium. Whole-genome sequencing (WGS), followed by bioinformatic sequence analysis, is currently the most extensive genotyping method available. To identify clinically relevant staphylococcal virulence and resistance genes in WGS data, we developed an in silico typing scheme for the software SeqSphere+ (Ridom GmbH, Münster, Germany). The implemented target genes (n = 182) correspond to those queried by the Identibac S. aureus Genotyping DNA microarray (Alere Technologies, Jena, Germany). The in silico scheme was evaluated by comparing the typing results of microarray and of WGS for 154 human S. aureus isolates. A total of 96.8% (n = 27,119) of all typing results were equally identified with microarray and WGS (40.6% present and 56.2% absent). Discrepancies (3.2% in total) were caused by WGS errors (1.7%), microarray hybridization failures (1.3%), wrong prediction of ambiguous microarray results (0.1%), or unknown causes (0.1%). Superior to the microarray, WGS enabled the distinction of allelic variants, which may be essential for the prediction of bacterial virulence and resistance phenotypes. Multilocus sequence typing clonal complexes and staphylococcal cassette chromosome mec element types inferred from microarray hybridization patterns were equally determined by WGS. In conclusion, WGS may substitute array-based methods due to its universal methodology, open and expandable nature, and rapid parallel analysis capacity for different characteristics in once-generated sequences. PMID:26818676

  4. Detection of Herpesviridae in whole blood by multiplex PCR DNA-based microarray analysis after hematopoietic stem cell transplantation.

    PubMed

    Debaugnies, France; Busson, Laurent; Ferster, Alina; Lewalle, Philippe; Azzi, Nadira; Aoun, Mickael; Verhaegen, Godelieve; Mahadeb, Bhavna; de Marchin, Jérôme; Vandenberg, Olivier; Hallin, Marie

    2014-07-01

    Viral infections are important causes of morbidity and mortality in patients after hematopoietic stem cell transplantation. The monitoring by PCR of Herpesviridae loads in blood samples has become a critical part of posttransplant follow-up, representing mounting costs for the laboratory. In this study, we assessed the clinical performance of the multiplex PCR DNA microarray Clart Entherpex kit for detection of cytomegalovirus (CMV), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6) as a screening test for virological follow-up. Two hundred fifty-five blood samples from 16 transplanted patients, prospectively tested by routine PCR assays, were analyzed by microarray. Routine PCR detected single or multiple viruses in 42% and 10% of the samples, respectively. Microarray detected single or multiple viruses in 34% and 18% of the samples, respectively. Microarray results correlated well with CMV and EBV detections by routine PCR (kappa tests = 0.79 and 0.78, respectively), whereas a weak correlation was observed with HHV-6 (0.43). HHV-7 was also detected in 48 samples by microarray. In conclusion, the microarray is a reliable screening assay for a posttransplant virological follow-up to detect CMV and EBV infections in blood. However, positive samples must be subsequently confirmed and viral loads must be quantified by PCR assays. Limitations were identified regarding HHV-6 detection. Although it is promising, is easy to use as a first-line test, and allows a reduction in the cost of analysis without undue delay in the reporting of the final quantitative result to the clinician, some characteristics of this microarray should be improved, particularly regarding quality control and the targeted virus panel, such that it could then be used as a routine test. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  5. Rapid quantification and taxonomic classification of environmentalDNA from both prokaryotic and eukaryotic origins using a microarray

    SciTech Connect

    DeSantis, Todd Z.; Stone, Carol E.; Murray, Sonya R.; Moberg,Jordan P.; Andersen, Gary L.

    2005-02-22

    A microarray has been designed using 62,358 probes matched to both prokaryotic and eukaryotic small-subunit ribosomal RNA genes. The array categorized environmental DNA to specific phylogenetic clusters in under 9 h. To a background of DNA generated from natural outdoor aerosols, known quantities of rRNA gene copies from distinct organisms were added producing corresponding hybridization intensity scores that correlated well with their concentrations (r=0.917). Reproducible differences in microbial community composition were observed by altering the genomic DNA extraction method. Notably, gentle extractions produced peak intensities for Mycoplasmatales and Burkholderiales, whereas a vigorous disruption produced peak intensities for Vibrionales,Clostridiales, and Bacillales.

  6. Genomic DNA Microarray Analysis: Identification of New Genes Regulated by Light Color in the Cyanobacterium Fremyella diplosiphon

    PubMed Central

    Stowe-Evans, Emily L.; Ford, James; Kehoe, David M.

    2004-01-01

    Many cyanobacteria use complementary chromatic adaptation to efficiently utilize energy from both green and red regions of the light spectrum during photosynthesis. Although previous studies have shown that acclimation to changing light wavelengths involves many physiological responses, research to date has focused primarily on the expression and regulation of genes that encode proteins of the major photosynthetic light-harvesting antennae, the phycobilisomes. We have used two-dimensional gel electrophoresis and genomic DNA microarrays to expand our understanding of the physiology of acclimation to light color in the cyanobacterium Fremyella diplosiphon. We found that the levels of nearly 80 proteins are altered in cells growing in green versus red light and have cloned and positively identified 17 genes not previously known to be regulated by light color in any species. Among these are homologs of genes present in many bacteria that encode well-studied proteins lacking clearly defined functions, such as tspO, which encodes a tryptophan-rich sensory protein, and homologs of genes encoding proteins of clearly defined function in many species, such as nblA and chlL, encoding phycobilisome degradation and chlorophyll biosynthesis proteins, respectively. Our results suggest novel roles for several of these gene products and highly specialized, unique uses for others. PMID:15205436

  7. Genomic DNA microarray analysis: identification of new genes regulated by light color in the cyanobacterium Fremyella diplosiphon.

    PubMed

    Stowe-Evans, Emily L; Ford, James; Kehoe, David M

    2004-07-01

    Many cyanobacteria use complementary chromatic adaptation to efficiently utilize energy from both green and red regions of the light spectrum during photosynthesis. Although previous studies have shown that acclimation to changing light wavelengths involves many physiological responses, research to date has focused primarily on the expression and regulation of genes that encode proteins of the major photosynthetic light-harvesting antennae, the phycobilisomes. We have used two-dimensional gel electrophoresis and genomic DNA microarrays to expand our understanding of the physiology of acclimation to light color in the cyanobacterium Fremyella diplosiphon. We found that the levels of nearly 80 proteins are altered in cells growing in green versus red light and have cloned and positively identified 17 genes not previously known to be regulated by light color in any species. Among these are homologs of genes present in many bacteria that encode well-studied proteins lacking clearly defined functions, such as tspO, which encodes a tryptophan-rich sensory protein, and homologs of genes encoding proteins of clearly defined function in many species, such as nblA and chlL, encoding phycobilisome degradation and chlorophyll biosynthesis proteins, respectively. Our results suggest novel roles for several of these gene products and highly specialized, unique uses for others.

  8. Annotated expressed sequence tags and cDNA microarrays for studies of brain and behavior in the honey bee.

    PubMed

    Whitfield, Charles W; Band, Mark R; Bonaldo, Maria F; Kumar, Charu G; Liu, Lei; Pardinas, Jose R; Robertson, Hugh M; Soares, M Bento; Robinson, Gene E

    2002-04-01

    To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708-BI517278. The sequences are also available at http://titan.biotec.uiuc.edu/bee/honeybee_project.htm.

  9. 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.

  10. DNA Microarray-Based Screening and Characterization of Traditional Chinese Medicine

    PubMed Central

    Kiyama, Ryoiti

    2017-01-01

    The application of DNA microarray assay (DMA) has entered a new era owing to recent innovations in omics technologies. This review summarizes recent applications of DMA-based gene expression profiling by focusing on the screening and characterization of traditional Chinese medicine. First, herbs, mushrooms, and dietary plants analyzed by DMA along with their effective components and their biological/physiological effects are summarized and discussed by examining their comprehensive list and a list of representative effective chemicals. Second, the mechanisms of action of traditional Chinese medicine are summarized by examining the genes and pathways responsible for the action, the cell functions involved in the action, and the activities found by DMA (silent estrogens). Third, applications of DMA for traditional Chinese medicine are discussed by examining reported examples and new protocols for its use in quality control. Further innovations in the signaling pathway-based evaluation of beneficial effects and the assessment of potential risks of traditional Chinese medicine are expected, just as are observed in other closely related fields, such as the therapeutic, environmental, nutritional, and pharmacological fields. PMID:28146102

  11. Modeling the temporal evolution of the Drosophila gene expression from DNA microarray time series

    NASA Astrophysics Data System (ADS)

    Haye, Alexandre; Dehouck, Yves; Kwasigroch, Jean Marc; Bogaerts, Philippe; Rooman, Marianne

    2009-03-01

    The time evolution of gene expression across the developmental stages of the host organism can be inferred from appropriate DNA microarray time series. Modeling this evolution aims eventually at improving the understanding and prediction of the complex phenomena that are the basis of life. We focus on the embryonic-to-adult development phases of Drosophila melanogaster, and chose to model the expression network with the help of a system of differential equations with constant coefficients, which are nonlinear in the transcript concentrations but linear in their logarithms. To reduce the dimensionality of the problem, genes having similar expression profiles are grouped into 17 clusters. We show that a simple linear model is able to reproduce the experimental data with very good precision, owing to the large number of parameters that represent the connections between the clusters. Remarkably, the parameter reduction allowed elimination of up to 80-85% of these connections while keeping fairly good precision. This result supports the low-connectivity hypothesis of gene expression networks, with about three connections per cluster, without introducing a priori hypotheses. The core of the network shows a few gene clusters with negative self-regulation, and some highly connected clusters involving proteins with crucial functions.

  12. A Hidden Markov model web application for analysing bacterial genomotyping DNA microarray experiments.

    PubMed

    Newton, Richard; Hinds, Jason; Wernisch, Lorenz

    2006-01-01

    Whole genome DNA microarray genomotyping experiments compare the gene content of different species or strains of bacteria. A statistical approach to analysing the results of these experiments was developed, based on a Hidden Markov model (HMM), which takes adjacency of genes along the genome into account when calling genes present or absent. The model was implemented in the statistical language R and applied to three datasets. The method is numerically stable with good convergence properties. Error rates are reduced compared with approaches that ignore spatial information. Moreover, the HMM circumvents a problem encountered in a conventional analysis: determining the cut-off value to use to classify a gene as absent. An Apache Struts web interface for the R script was created for the benefit of users unfamiliar with R. The application may be found at http://hmmgd.cryst.bbk.ac.uk/hmmgd. The source code illustrating how to run R scripts from an Apache Struts-based web application is available from the corresponding author on request. The application is also available for local installation if required.

  13. Genotyping and DNA microarray based characterization of Staphylococcus aureus isolates from rabbit carcasses.

    PubMed

    Merz, Axel; Stephan, Roger; Johler, Sophia

    2016-02-01

    Staphylococcus aureus can cause staphylococcal food poisoning. Although the organism is frequently detected on rabbit carcasses, little is known about the characteristics of S. aureus strains contaminating rabbit meat. In this study, 137 S. aureus isolates originating from 137 rabbit carcasses were spa typed and characterized by DNA microarray. The isolates were assigned to CC5, CC7, CC8, CC15, CC96, CC101, CC121, and ST890, and to 13 spa types (t056, t085, t091, t160, t179, t681, t741, t745, t1190, t1773, t4770, t8456, t14871). Enterotoxin genes detected included sea, sed, sej, and ser. In addition, the egc operon, encoding the newly described staphylococcal enterotoxins SEG/SEI/SElM/SElN/SElO/SElU, was found in all isolates except those of t091. While none of the examined isolates presented genes conferring methicillin, vancomycin, or aminoglycoside resistance, we frequently detected blaZ/I/R conferring resistance to penicillin. The isolates represented a heterogeneous group assigned to clonal lineages detected among humans and animals, with two spa types exclusively associated with rabbit meat (t4770, t8456).

  14. Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays.

    PubMed

    Sun, Xiaoli; Jia, Yu; Wei, Yuanyu; Liu, Shuai; Yue, Baohong

    2016-07-01

    Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53‑mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53‑independent NS pathway. NS expression was silenced using lentivirus‑mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse‑transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c‑Jun N‑terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54‑independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia.

  15. 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

  16. Rapid and accurate detection of RMP- and INH- resistant Mycobacterium tuberculosis in spinal tuberculosis specimens by CapitalBio™ DNA microarray: a prospective validation study.

    PubMed

    Zhang, Zehua; Li, Litao; Luo, Fei; Cheng, Peng; Wu, Feng; Wu, Zheng; Hou, Tianyong; Zhong, Min; Xu, Jianzhong

    2012-11-14

    DNA microarrays can detect tuberculosis and its multi-drug resistant form in M. tuberculosis isolates and sputum specimens with high sensitivity and specificity. However, no performance data currently exists for its use in spinal tuberculosis specimens. This study was aimed to assess the performance of the CapitalBio™ DNA microarray in the detection of isoniazid (INH) and rifampicin (RMP) resistance in spinal tuberculosis compared with the BACT/MGIT 960 system. From March 2009 to December 2011, 153 consecutive patients from Southwest Hospital, Chongqing with clinically and pathologically diagnosed spinal tuberculosis were enrolled into this study. Specimens collected during surgery from the tuberculosis patients were subjected to M. tuberculosis species identification and drug-resistance detection by the CapitalBio™ DNA microarray, and results were compared with those obtained from the absolute concentration drug susceptibility testing. The CapitalBio™ DNA microarray achieved 93.55% sensitivity for the correct M. tuberculosis species identification of the 93 specimens that tested positive for spinal tuberculosis through culture. In addition, twenty-seven additional patients (45.0%) were detected by the DNA microarray to be positive for M. tuberculosis among sixty spinal tuberculosis patients who were culture negative. Moreover, the DNA microarray had a sensitivity of 88.9% and a specificity of 90.7% for RMP resistance, and the microarray had a sensitivity of 80.0% and a specificity of 91.0% for INH resistance. The mean turn-around time of M. tuberculosis species identification and drug resistance detection using the DNA microarray was 5.8 (range, 4-9) hours. The CapitalBio™ DNA microarray is a feasible and accurate tool for the species identification of M. tuberculosis and for directly detecting RMP and INH resistance from spinal tuberculosis specimens in fewer than 9 hours.

  17. Gene profile in the spleen under massive partial hepatectomy using complementary DNA microarray and pathway analysis.

    PubMed

    Arakawa, Yusuke; Shimada, Mitsuo; Utsunomiya, Tohru; Imura, Satoru; Morine, Yuji; Ikemoto, Tetsuya; Mori, Hiroki; Kanamoto, Mami; Iwahashi, Shuichi; Saito, Yu; Takasu, Chie

    2014-08-01

    In general, the spleen is one of the abdominal organs connected by the portal system, and a splenectomy improves hepatic functions in the settings of partial hepatectomy (Hx) for portal hypertensive cases or living donor liver transplantation with excessive portal vein flow. Those precise mechanisms remain still unclear; therefore, we investigated the DNA expression profile in the spleen after 90% Hx in rats using complementary DNA microarray and pathway analysis. Messenger RNAs (mRNAs) were prepared from three rat spleens at each time point (0, 3, and 6 h after 90% Hx). Using the gene chip, mRNA was hybridized to Affymetrix GeneChip Rat Genome 230 2.0 Array (Affymetrix®) and pathway analysis was done with Ingenuity Pathway Analysis (IPA®). We determined the 3-h or 6-h/0-h ratio to assess the influence of Hx, and cut-off values were set at more than 2.0-fold or less than 1/2 (0.5)-fold. Chemokine activity-related genes including Cxcl1 (GRO1) and Cxcl2 (MIP-2) related pathway were upregulated in the spleen. Also, immediate early response genes including early growth response-1 (EGR1), FBJ murine osteosarcoma (FOS) and activating transcription factor 3 (ATF3) related pathway were upregulated in the spleen. We concluded that in the spleen the expression of numerous inflammatory-related genes would occur after 90% Hx. The spleen could take a harmful role and provide a negative impact during post Hx phase due to the induction of chemokine and transcription factors including GRO1 and EGR1. © 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.

  18. Combining suppressive subtractive hybridization and cDNA microarrays to identify dietary phosphorus-responsive genes of the rainbow trout (Oncorhynchus mykiss) kidney.

    PubMed

    Lake, Jennifer; Gravel, Catherine; Koko, Gabriel Koffi D; Robert, Claude; Vandenberg, Grant W

    2010-03-01

    Phosphorus (P)-responsive genes and how they regulate renal adaptation to phosphorous-deficient diets in animals, including fish, are not well understood. RNA abundance profiling using cDNA microarrays is an efficient approach to study nutrient-gene interactions and identify these dietary P-responsive genes. To test the hypothesis that dietary P-responsive genes are differentially expressed in fish fed varying P levels, rainbow trout were fed a practical high-P diet (R20: 0.96% P) or a low-P diet (R0: 0.38% P) for 7 weeks. The differentially-expressed genes between dietary groups were identified and compared from the kidney by combining suppressive subtractive hybridization (SSH) with cDNA microarray analysis. A number of genes were confirmed by real-time PCR, and correlated with plasma and bone P concentrations. Approximately 54 genes were identified as potential dietary P-responsive after 7 weeks on a diet deficient in P according to cDNA microarray analysis. Of 18 selected genes, 13 genes were confirmed to be P-responsive at 7 weeks by real-time PCR analysis, including: iNOS, cytochrome b, cytochrome c oxidase subunit II , alpha-globin I, beta-globin, ATP synthase, hyperosmotic protein 21, COL1A3, Nkef, NDPK, glucose phosphate isomerase 1, Na+/H+ exchange protein and GDP dissociation inhibitor 2. Many of these dietary P-responsive genes responded in a moderate way (R0/R20 ratio: <2-3 or >0.5) and in a transient manner to dietary P limitation. In summary, renal adaptation to dietary P deficiency in trout involves changes in the expression of several genes, suggesting a profile of metabolic stress, since many of these differentially-expressed candidates are associated with the cellular adaptative responses.

  19. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    NASA Technical Reports Server (NTRS)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  20. Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

    PubMed Central

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

  1. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

    NASA Technical Reports Server (NTRS)

    Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

    2002-01-01

    A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

  2. DNA microarray analysis reveals that antibiotic resistance-gene diversity in human gut microbiota is age related

    PubMed Central

    Lu, Na; Hu, Yongfei; Zhu, Liying; Yang, Xi; Yin, Yeshi; Lei, Fang; Zhu, Yongliang; Du, Qin; Wang, Xin; Meng, Zhiqi; Zhu, Baoli

    2014-01-01

    The human gut is a reservoir for antibiotic resistance genes. In this report, we used a DNA microarray chip covering 369 resistance types to investigate the relationship between antibiotic resistance-gene diversity and human age. Metagenomic DNA from fecal samples from 124 healthy volunteers of four different age groups (pre-school-aged children (CH), school-aged children (SC), high school students (HSS) and adults (AD)) were hybridized to the microarray chip. The results showed that 80 different gene types were recovered from the gut microbiota of the 124 individuals: 25 from CH, 37 from SC, 58 from HSS and 72 from AD. Further analysis indicated that the antibiotic resistance genes in the CH, SC and AD groups clustered independently, whereas the gene types in the HSS group were more divergent. Our results indicated that antibiotic resistance genes in the human gut microbiota accumulate from childhood to adulthood and become more complex with age. PMID:24618772

  3. Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays.

    PubMed

    El-Adawy, Hosny; Ahmed, Marwa; Hotzel, Helmut; Monecke, Stefan; Schulz, Jochen; Hartung, Joerg; Ehricht, Ralf; Neubauer, Heinrich; Hafez, Hafez M

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding

  4. Understanding the molecular aspects of oriental obesity pattern differentiation using DNA microarray.

    PubMed

    Hong, Sun Woo; Yoo, Jae-Wook; Bose, Shambhunath; Park, Jung-Hyun; Han, Kyungsun; Kim, Soyoun; Lim, Chi-Yeon; Kim, Hojun; Lee, Dong-Ki

    2015-10-19

    Human constitution, the fundamental basis of oriental medicine, is categorized into different patterns for a particular disease according to the physical, physiological, and clinical characteristics of the individuals. Obesity, a condition of metabolic disorder, is classified according to six patterns in oriental medicine, as follows: spleen deficiency syndrome, phlegm fluid syndrome, yang deficiency syndrome (YDS), food accumulation syndrome (FAS), liver depression syndrome (LDS), and blood stasis syndrome. In oriental medicine, identification of the disease pattern for individual obese patients is performed on the basis of differentiation in obesity syndrome index and, accordingly, personalized treatment is provided to the patients. The aim of the current study was to understand the obesity patterns in oriental medicine from the genomic point of view via determining the gene expression signature of obese patients using peripheral blood mononuclear cells as the samples. The study was conducted in 23 South Korean obese subjects (19 female and four male) with BMI ≥25 kg/m(2). Identification of oriental obesity pattern was based on the software-guided evaluation of the responses of the subjects to a questionnaire developed by the Korean Institute of Oriental Medicine. The expression profiles of genes were determined using DNA microarray and the level of transcription of genes of interest was further evaluated using quantitative real-time PCR (qRT-PCR). Gene clustering analysis of the microarray data from the FAS, LDS, and YDS subjects exhibited disease pattern-specific upregulation of expression of several genes in a particular cluster. Further analysis of transcription of selected genes using qRT-PCR led to identification of specific genes, including prostaglandin endoperoxide synthase 2, G0/G1 switch 2, carcinoembryonic antigen-related cell adhesion molecule 3, cystein-serine-rich nuclear protein 1, and interleukin 8 receptor, alpha which were highly expressed in

  5. Characterization of Methicillin-Resistant Staphylococcus aureus Isolated from Healthy Turkeys and Broilers Using DNA Microarrays

    PubMed Central

    El-Adawy, Hosny; Ahmed, Marwa; Hotzel, Helmut; Monecke, Stefan; Schulz, Jochen; Hartung, Joerg; Ehricht, Ralf; Neubauer, Heinrich; Hafez, Hafez M.

    2016-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA) is a major human health problem and recently, domestic animals are described as carriers and possible reservoirs. Twenty seven S. aureus isolates from five turkey farms (n = 18) and two broiler farms (n = 9) were obtained by culturing of choana and skin swabs from apparently healthy birds, identified by Taqman-based real-time duplex nuc-mecA-PCR and characterized by spa typing as well as by a DNA microarray based assay which covered, amongst others, a considerable number of antibiotic resistance genes, species controls, and virulence markers. The antimicrobial susceptibility profiles were tested by agar diffusion assays and genotypically confirmed by the microarray. Five different spa types (3 in turkeys and 2 in broilers) were detected. The majority of MRSA isolates (24/27) belonged to clonal complex 398-MRSA-V. The most frequently occurring spa types were accordingly t011, t034, and t899. A single CC5-MRSA-III isolated from turkey and CC398-MRSA with an unidentified/truncated SCCmec element in turkey and broiler were additionally detected. The phenotypic antimicrobial resistance profiles of S. aureus isolated from both turkeys and broilers against 14 different antimicrobials showed that all isolates were resistant to ampicillin, cefoxitin, oxacillin, doxycycline, and tetracycline. Moreover, all S. aureus isolated from broilers were resistant to erythromycin and azithromycin. All isolates were susceptible to gentamicin, chloramphenicol, sulphonamides, and fusidic acid. The resistance rate against ciprofloxacin was 55.6% in broiler isolates and 42.1% in turkey isolates. All tetracycline resistant isolates possessed genes tetK/M. All erythromycin-resistant broiler isolates carried ermA. Only one broiler isolate (11.1%) carried genes ermA, ermB, and ermC, while 55.6% of turkey isolates possessed ermA and ermB genes. Neither PVL genes (lukF/S-PV), animal-associated leukocidin (lukM and luk-P83) nor the gene encoding

  6. Use of DNA microarray chips for the rapid detection of Mycobacterium tuberculosis resistance to rifampicin and isoniazid.

    PubMed

    Tang, Peijun; Wang, Xiafang; Shen, Xinghua; Shi, Meihua; Zhu, Xuefeng; Yu, Xin; Liu, Jia; Ling, Chunhua; Wu, Meiying

    2017-05-01

    The objective of the present study was to evaluate the potential development of DNA microarray chips to detect rifampicin (RFP) and isoniazid (INH) resistance in Mycobacterium tuberculosis (MTB), using samples from clinical tuberculosis (TB) patients in Soochow City, China. The sputum samples of 42 patients with TB in the Affiliated Hospital of Infectious Diseases of Soochow University (Soochow, China) were collected. The conventional Lowenstein-Jensen culture medium (Gold Standard) was used to assess drug sensitivity using the absolute concentration method. GeeDom MTB drug detection kits were also used to create a DNA microarray chip and examine the RFP-resistance associated gene mutation points rpoB-RRDR 511, 513, 516, 526, 531 and 533, and the INH-resistance associated gene mutation points katG315 and inhA-15 of the sputum samples. Compared with the results from the absolute concentration method, the susceptibility and specificity of RFP sensitivity detected by the DNA microarray chip were 92.8 and 93.8%, respectively. The susceptibility and specificity of INH sensitivity detected were 66.7 and 81%, respectively. The rpoB-RRDR 526, 531 mutations were the primary causes of MTB RFP resistance and the katG315 mutation was the primary cause of INH resistance. The detection of rpoB and katG gene mutation points by a DNA microarray chip may be used as a rapid, accurate and bulk clinical detection method for RFP and INH resistance in MTB. This is very valuable for the control of TB epidemics.

  7. 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

  8. eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

    NASA Astrophysics Data System (ADS)

    Liu, Robin H.; Longiaru, Mathew

    2009-05-01

    DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

  9. DNA microarrays in the undergraduate microbiology lab: experimentation and handling large datasets in as few as six weeks.

    PubMed

    Kushner, David B

    2007-01-01

    DNA microarrays have significantly impacted the study of gene expression on a genome-wide level but also have forced a more global consideration of research questions. As such, it has become critical to introduce undergraduate students to genomics approaches to research. A challenge with performing a DNA microarray experiment in the teaching lab is determining the time required for the study and how to handle the voluminous data generated. At an unexpectedly low cost, a 6-week, project-based lab module has been developed that provides 3 weeks for wet lab (hands-on work with the DNA microarrays) and 3 weeks for dry lab (analyzing data, using databases to help with data analysis, and considering the meaning of data within the large dataset). Options exist for extending the number of weeks dedicated to the project, but 6 weeks is sufficient for providing an introduction to both experimental genomics and data analysis. Students indicate that being able to both perform array experiments and thoroughly analyze data enriches their understanding of genomics and the complexity of biological systems.

  10. High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

    PubMed

    Inoue, Daisuke; Hinoura, Takuji; Suzuki, Noriko; Pang, Junqin; Malla, Rabin; Shrestha, Sadhana; Chapagain, Saroj Kumar; Matsuzawa, Hiroaki; Nakamura, Takashi; Tanaka, Yasuhiro; Ike, Michihiko; Nishida, Kei; Sei, Kazunari

    2015-01-01

    Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1-37 pathogen species/groups, including 1-27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

  11. Sensitive immunoassay detection of multiple environmental chemicals on protein microarrays using DNA/dye conjugate as a fluorescent label.

    PubMed

    Fan, Ziyan; Keum, Young Soo; Li, Qing X; Shelver, Weilin L; Guo, Liang-Hong

    2012-05-01

    Indirect competitive immunoassays were developed on protein microarrays for the sensitive and simultaneous detection of multiple environmental chemicals in one sample. In this assay, a DNA/SYTOX Orange conjugate was employed as an antibody label to increase the fluorescence signal and sensitivity of the immunoassays. Epoxy-modified glass slides were selected as the substrate for the production of 4 × 4 coating antigen microarrays. With this signal-enhancing system, competition curves for 17β-estradiol (E2), benzo[a]pyrene (BaP) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) were obtained individually on the protein microarray. The IC(50) and calculated limit of detection (LOD) are 0.32 μg L(-1) and 0.022 μg L(-1) for E2, 37.2 μg L(-1) and 24.5 μg L(-1) for BaP, and 31.6 μg L(-1) and 2.8 μg L(-1) for BDE-47, respectively. LOD of E2 is 14-fold lower than the value reported in a previous study using Cy3 labeled antibody (Du et al., Clin. Chem, 2005, 51, 368-375). The results of the microarray immunoassay were within 15% of chromatographic analysis for all three pollutants in spiked river water samples, thus verifying the immunoassay. Simultaneous detection of E2, BaP and BDE-47 in one sample was demonstrated. There was no cross-reaction in the immunoassay between these three environmental chemicals. These results suggest that microarray-based immunoassays with DNA/dye conjugate labels are useful tools for the rapid, sensitive, and high throughput screening of multiple environmental contaminants.

  12. High-resolution genomic copy number profiling of glioblastoma multiforme by single nucleotide polymorphism DNA microarray.

    PubMed

    Yin, Dong; Ogawa, Seishi; Kawamata, Norihiko; Tunici, Patrizia; Finocchiaro, Gaetano; Eoli, Marica; Ruckert, Christian; Huynh, Thien; Liu, Gentao; Kato, Motohiro; Sanada, Masashi; Jauch, Anna; Dugas, Martin; Black, Keith L; Koeffler, H Phillip

    2009-05-01

    Glioblastoma multiforme (GBM) is an extremely malignant brain tumor. To identify new genomic alterations in GBM, genomic DNA of tumor tissue/explants from 55 individuals and 6 GBM cell lines were examined using single nucleotide polymorphism DNA microarray (SNP-Chip). Further gene expression analysis relied on an additional 56 GBM samples. SNP-Chip results were validated using several techniques, including quantitative PCR (Q-PCR), nucleotide sequencing, and a combination of Q-PCR and detection of microsatellite markers for loss of heterozygosity with normal copy number [acquired uniparental disomy (AUPD)]. Whole genomic DNA copy number in each GBM sample was profiled by SNP-Chip. Several signaling pathways were frequently abnormal. Either the p16(INK4A)/p15(INK4B)-CDK4/6-pRb or p14(ARF)-MDM2/4-p53 pathways were abnormal in 89% (49 of 55) of cases. Simultaneous abnormalities of both pathways occurred in 84% (46 of 55) samples. The phosphoinositide 3-kinase pathway was altered in 71% (39 of 55) GBMs either by deletion of PTEN or amplification of epidermal growth factor receptor and/or vascular endothelial growth factor receptor/platelet-derived growth factor receptor alpha. Deletion of chromosome 6q26-27 often occurred (16 of 55 samples). The minimum common deleted region included PARK2, PACRG, QKI, and PDE10A genes. Further reverse transcription Q-PCR studies showed that PARK2 expression was decreased in another collection of GBMs at a frequency of 61% (34 of 56) of samples. The 1p36.23 region was deleted in 35% (19 of 55) of samples. Notably, three samples had homozygous deletion encompassing this site. Also, a novel internal deletion of a putative tumor suppressor gene, LRP1B, was discovered causing an aberrant protein. AUPDs occurred in 58% (32 of 55) of the GBM samples and five of six GBM cell lines. A common AUPD was found at chromosome 17p13.3-12 (included p53 gene) in 13 of 61 samples and cell lines. Single-strand conformational polymorphism and nucleotide

  13. DNA microarray analysis reveals a role for lysophosphatidic acid in the regulation of anti-inflammatory genes in MC3T3-E1 cells

    SciTech Connect

    Waters, Katrina M.; Tan, Ruimin; Genetos, Damian C.; Verma, Seema; Yellowley, Clare E.; Karin, Norm J.

    2007-11-01

    DNA microarray analysis revealed that treatment of bone cells with a lipid growth factor led to extensive changes in gene expression. Particular relevance to fracture healing and inflammation was revealed.

  14. Efficient alignment-free DNA barcode analytics

    PubMed Central

    Kuksa, Pavel; Pavlovic, Vladimir

    2009-01-01

    Background In this work we consider barcode DNA analysis problems and address them using alternative, alignment-free methods and representations which model sequences as collections of short sequence fragments (features). The methods use fixed-length representations (spectrum) for barcode sequences to measure similarities or dissimilarities between sequences coming from the same or different species. The spectrum-based representation not only allows for accurate and computationally efficient species classification, but also opens possibility for accurate clustering analysis of putative species barcodes and identification of critical within-barcode loci distinguishing barcodes of different sample groups. Results New alignment-free methods provide highly accurate and fast DNA barcode-based identification and classification of species with substantial improvements in accuracy and speed over state-of-the-art barcode analysis methods. We evaluate our methods on problems of species classification and identification using barcodes, important and relevant analytical tasks in many practical applications (adverse species movement monitoring, sampling surveys for unknown or pathogenic species identification, biodiversity assessment, etc.) On several benchmark barcode datasets, including ACG, Astraptes, Hesperiidae, Fish larvae, and Birds of North America, proposed alignment-free methods considerably improve prediction accuracy compared to prior results. We also observe significant running time improvements over the state-of-the-art methods. Conclusion Our results show that newly developed alignment-free methods for DNA barcoding can efficiently and with high accuracy identify specimens by examining only few barcode features, resulting in increased scalability and interpretability of current computational approaches to barcoding. PMID:19900305

  15. Tissue tablet method: an efficient tissue banking procedure applicable to both molecular analysis and frozen tissue microarray.

    PubMed

    Torata, Nobuhiro; Ohuchida, Kenoki; Akagawa, Shin; Cui, Lin; Kozono, Shingo; Mizumoto, Kazuhiro; Aishima, Shinichi; Oda, Yoshinao; Tanaka, Masao

    2014-01-01

    Frozen human tissues are necessary for research purposes, but tissue banking methods have not changed for more than a decade. Many institutions use cryovial tubes or plastic molds with an optimal cutting temperature compound. However, these methods are associated with several problems, such as samples sticking to one another and the need for a larger storing space. We established an efficient tissue freezing and storing procedure ("tissue tablet method") applicable to both molecular analysis and frozen tissue microarray. Tissue samples were chopped into tiny fragments and embedded into tablet-shaped frozen optimal cutting temperature compound using our original tissue-freezing plate. These tablets can be sectioned and stored in cryovial tubes. We compared the tissue quality of tablet-shaped samples with that of conventional optimal cutting temperature blocks and found no significant difference between them. Tissue microarray is a key method to utilize tissue-banking specimens. However, most tissue microarrays require the coring out of cylindrically shaped tissues from formalin-fixed, paraffin-embedded tissue blocks. Antigenic changes and mRNA degradation are frequently observed with formalin-fixed, paraffin-embedded samples. Therefore, we have applied tablet-shaped samples to construct frozen tissue microarrays with our original mounting base. Constructed tissue microarray sections showed good morphology without obvious artifact and good immunohistochemistry and in situ hybridization results. These results suggest that the quality of arrayed samples was sufficiently appropriate for research purposes. In conclusion, the tissue tablet method and frozen tissue microarray procedure can save time, provides easy tissue handling and processing, and satisfies the demands of research methodologies and tissue banking. © 2013.

  16. 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.

  17. Development of a single nucleotide polymorphism DNA microarray for the detection and genotyping of the SARS coronavirus.

    PubMed

    Guo, Xi; Geng, Peng; Wang, Quan; Cao, Boyang; Liu, Bin

    2014-10-01

    Severe acute respiratory syndrome (SARS), a disease that spread widely in the world during late 2002 to 2004, severely threatened public health. Although there have been no reported infections since 2004, the extremely pathogenic SARS coronavirus (SARS-CoV), as the causative agent of SARS, has recently been identified in animals, showing the potential for the re-emergence of this disease. Previous studies showed that 27 single nucleotide polymorphism (SNP) mutations among the spike (S) gene of this virus are correlated closely with the SARS pathogenicity and epidemicity. We have developed a SNP DNA microarray in order to detect and genotype these SNPs, and to obtain related information on the pathogenicity and epidemicity of a given strain. The microarray was hybridized with PCR products amplified from cDNAs obtained from different SARS-CoV strains. We were able to detect 24 SNPs and determine the type of a given strain. The hybridization profile showed that 19 samples were detected and genotyped correctly by using our microarray, with 100% accuracy. Our microarray provides a novel method for the detection and epidemiological surveillance of SARS-CoV.

  18. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis

    PubMed Central

    Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

    2002-01-01

    Background T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Results Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3–3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. Conclusion T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays. PMID:12445333

  19. Optimization and evaluation of T7 based RNA linear amplification protocols for cDNA microarray analysis.

    PubMed

    Zhao, Hongjuan; Hastie, Trevor; Whitfield, Michael L; Børresen-Dale, Anne-Lise; Jeffrey, Stefanie S

    2002-10-30

    T7 based linear amplification of RNA is used to obtain sufficient antisense RNA for microarray expression profiling. We optimized and systematically evaluated the fidelity and reproducibility of different amplification protocols using total RNA obtained from primary human breast carcinomas and high-density cDNA microarrays. Using an optimized protocol, the average correlation coefficient of gene expression of 11,123 cDNA clones between amplified and unamplified samples is 0.82 (0.85 when a virtual array was created using repeatedly amplified samples to minimize experimental variation). Less than 4% of genes show changes in expression level by 2-fold or greater after amplification compared to unamplified samples. Most changes due to amplification are not systematic both within one tumor sample and between different tumors. Amplification appears to dampen the variation of gene expression for some genes when compared to unamplified poly(A)+ RNA. The reproducibility between repeatedly amplified samples is 0.97 when performed on the same day, but drops to 0.90 when performed weeks apart. The fidelity and reproducibility of amplification is not affected by decreasing the amount of input total RNA in the 0.3-3 micrograms range. Adding template-switching primer, DNA ligase, or column purification of double-stranded cDNA does not improve the fidelity of amplification. The correlation coefficient between amplified and unamplified samples is higher when total RNA is used as template for both experimental and reference RNA amplification. T7 based linear amplification reproducibly generates amplified RNA that closely approximates original sample for gene expression profiling using cDNA microarrays.

  20. DNA Microarray Platform for Detection and Surveillance of Viruses Transmitted by Small Mammals and Arthropods

    PubMed Central

    Khan, Mohd Jaseem; Trabuco, Amanda Cristina; Alfonso, Helda Liz; Figueiredo, Mario Luis; Batista, Weber Cheli; Badra, Soraya Jabur; Figueiredo, Luiz Tadeu; Lavrador, Marco Aurélio

    2016-01-01

    Viruses transmitted by small mammals and arthropods serve as global threats to humans. Most emergent and re-emergent viral agents are transmitted by these groups; therefore, the development of high-throughput screening methods for the detection and surveillance of such viruses is of great interest. In this study, we describe a DNA microarray platform that can be used for screening all viruses transmitted by small mammals and arthropods (SMAvirusChip) with nucleotide sequences that have been deposited in the GenBank. SMAvirusChip was designed with more than 15,000 oligonucleotide probes (60-mers), including viral and control probes. Two SMAvirusChip versions were designed: SMAvirusChip v1 contains 4209 viral probes for the detection of 409 viruses, while SMAvirusChip v2 contains 4943 probes for the detection of 416 viruses. SMAvirusChip was evaluated with 20 laboratory reference-strain viruses. These viruses could be specifically detected when alone in a sample or when artificially mixed within a single sample. The sensitivity of SMAvirusChip was evaluated using 10-fold serial dilutions of dengue virus (DENV). The results showed a detection limit as low as 2.6E3 RNA copies/mL. Additionally, the sensitivity was one log10 lower (2.6E2 RNA copies/mL) than quantitative real-time RT-PCR and sufficient to detect viral genomes in clinical samples. The detection of DENV in serum samples of DENV-infected patients (n = 6) and in a whole blood sample spiked with DENV confirmed the applicability of SMAvirusChip for the detection of viruses in clinical samples. In addition, in a pool of mosquito samples spiked with DENV, the virus was also detectable. SMAvirusChip was able to specifically detect viruses in cell cultures, serum samples, total blood samples and a pool of mosquitoes, confirming that cellular RNA/DNA did not interfere with the assay. Therefore, SMAvirusChip may represent an innovative surveillance method for the rapid identification of viruses transmitted by small

  1. DNA microarray fabricated on poly(acrylic acid) brushes-coated porous silicon by in situ rolling circle amplification.

    PubMed

    Wang, Cuie; Jia, Xue-Mei; Jiang, Chuan; Zhuang, Guang-Nan; Yan, Qin; Xiao, Shou-Jun

    2012-10-07

    Microarrays hold considerable promise in large-scale biology on account of their analytical, massive and parallel nature. In a step toward further enabling such a capability, we describe the application of rolling circle amplification (RCA) for a sensitive and multiplex detection of nucleic acid targets on oligonucleotide-conjugated polymer brushes covalently grown from porous silicon. Both RCA and polymer brushes have been taken to increase the loading quantity of target molecules and thus improve the detection sensitivity without loss of multiplexing. Besides, polymer brushes were employed to protect porous silicon and to provide biologically simulated environments, making the attached biomolecules maintain bioactivity. This approach can reach a detection limit of 0.1 nM target analytes and three orders of magnitude dynamic range of 0.1-100 nM, with a fluorescence scanner. A two-colour DNA microarray was achieved via RCA of two kinds of circular DNA targets on one chip simultaneously. The porous silicon chip-based RCA technique is promising for the multiplex detection of deoxynucleic acids on microarrays.

  2. Development of a DNA-based microarray for the detection of zoonotic pathogens in rodent species.

    PubMed

    Giles, Timothy; Yon, Lisa; Hannant, Duncan; Barrow, Paul; Abu-Median, Abu-Bakr

    2015-12-01

    The demand for diagnostic tools that allow simultaneous screening of samples for multiple pathogens is increasing because they overcome the limitations of other methods, which can only screen for a single or a few pathogens at a time. Microarrays offer the advantages of being capable to test a large number of samples simultaneously, screening for multiple pathogen types per sample and having comparable sensitivity to existing methods such as PCR. Array design is often considered the most important process in any microarray experiment and can be the deciding factor in the success of a study. There are currently no microarrays for simultaneous detection of rodent-borne pathogens. The aim of this report is to explicate the design, development and evaluation of a microarray platform for use as a screening tool that combines ease of use and rapid identification of a number of rodent-borne pathogens of zoonotic importance. Nucleic acid was amplified by multiplex biotinylation PCR prior to hybridisation onto microarrays. The array sensitivity was comparable to standard PCR, though less sensitive than real-time PCR. The array presented here is a prototype microarray identification system for zoonotic pathogens that can infect rodent species.

  3. Single-cell multiple gene expression analysis based on single-molecule-detection microarray assay for multi-DNA determination.

    PubMed

    Li, Lu; Wang, Xianwei; Zhang, Xiaoli; Wang, Jinxing; Jin, Wenrui

    2015-01-07

    We report a novel ultra-sensitive and high-selective single-molecule-detection microarray assay (SMA) for multiple DNA determination. In the SMA, a capture DNA (DNAc) microarray consisting of 10 subarrays with 9 spots for each subarray is fabricated on a silanized glass coverslip as the substrate. On the subarrays, the spot-to-spot spacing is 500 μm and each spot has a diameter of ∼300 μm. The sequence of the DNAcs on the 9 spots of a subarray is different, to determine 8 types of target DNAs (DNAts). Thus, 8 types of DNAts are captured to their complementary DNAcs at 8 spots of a subarray, respectively, and then labeled with quantum dots (QDs) attached to 8 types of detection DNAs (DNAds) with different sequences. The ninth spot is used to detect the blank value. In order to determine the same 8 types of DNAts in 10 samples, the 10 DNAc-modified subarrays on the microarray are identical. Fluorescence single-molecule images of the QD-labeled DNAts on each spot of the subarray are acquired using a home-made single-molecule microarray reader. The amounts of the DNAts are quantified by counting the bright dots from the QDs. For a microarray, 8 types of DNAts in 10 samples can be quantified in parallel. The limit of detection of the SMA for DNA determination is as low as 1.3×10(-16) mol L(-1). The SMA for multi-DNA determination can also be applied in single-cell multiple gene expression analysis through quantification of complementary DNAs (cDNAs) corresponding to multiple messenger RNAs (mRNAs) in single cells. To do so, total RNA in single cells is extracted and reversely transcribed into their cDNAs. Three types of cDNAs corresponding to beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase and ribosomal protein, large, P2 mRNAs in single human breast cancer cells and 5 random synthetic DNAts are simultaneously quantified to examine the SMA and SMA-based single-cell multiple gene expression analysis. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.

    PubMed

    Lohman, Gregory J S; Zhang, Yinhua; Zhelkovsky, Alexander M; Cantor, Eric J; Evans, Thomas C

    2014-02-01

    Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.

  5. Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

    PubMed

    Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

    2015-06-01

    White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

  6. Surface plasmon resonance imaging system with Mach-Zehnder phase-shift interferometry for DNA micro-array hybridization

    NASA Astrophysics Data System (ADS)

    Hsiu, Feng-Ming; Chen, Shean-Jen; Tsai, Chien-Hung; Tsou, Chia-Yuan; Su, Y.-D.; Lin, G.-Y.; Huang, K.-T.; Chyou, Jin-Jung; Ku, Wei-Chih; Chiu, S.-K.; Tzeng, C.-M.

    2002-09-01

    Surface plasmon resonance (SPR) imaging system is presented as a novel technique based on modified Mach-Zehnder phase-shifting interferometry (PSI) for biomolecular interaction analysis (BIA), which measures the spatial phase variation of a resonantly reflected light in biomolecular interaction. In this technique, the micro-array SPR biosensors with over a thousand probe NDA spots can be detected simultaneously. Owing to the feasible and swift measurements, the micro-array SPR biosensors can be extensively applied to the nonspecific adsorption of protein, the membrane/protein interactions, and DNA hybridization. The detection sensitivity of the SPR PSI imaging system is improved to about 1 pg/mm2 for each spot over the conventional SPR imaging systems. The SPR PSI imaging system and its SPR sensors have been successfully used to observe slightly index change in consequence of argon gas flow through the nitrogen in real time, with high sensitivity, and at high-throughout screening rates.

  7. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray

    PubMed Central

    Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

    2003-01-01

    AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators. PMID:12632483

  8. Analysis of gene expression profile induced by EMP-1 in esophageal cancer cells using cDNA Microarray.

    PubMed

    Wang, Hai-Tao; Kong, Jian-Ping; Ding, Fang; Wang, Xiu-Qin; Wang, Ming-Rong; Liu, Lian-Xin; Wu, Min; Liu, Zhi-Hua

    2003-03-01

    To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1. The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component. Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion. Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.

  9. Annotated Expressed Sequence Tags and cDNA Microarrays for Studies of Brain and Behavior in the Honey Bee

    PubMed Central

    Whitfield, Charles W.; Band, Mark R.; Bonaldo, Maria F.; Kumar, Charu G.; Liu, Lei; Pardinas, Jose R.; Robertson, Hugh M.; Soares, M. Bento; Robinson, Gene E.

    2002-01-01

    To accelerate the molecular analysis of behavior in the honey bee (Apis mellifera), we created expressed sequence tag (EST) and cDNA microarray resources for the bee brain. Over 20,000 cDNA clones were partially sequenced from a normalized (and subsequently subtracted) library generated from adult A. mellifera brains. These sequences were processed to identify 15,311 high-quality ESTs representing 8912 putative transcripts. Putative transcripts were functionally annotated (using the Gene Ontology classification system) based on matching gene sequences in Drosophila melanogaster. The brain ESTs represent a broad range of molecular functions and biological processes, with neurobiological classifications particularly well represented. Roughly half of Drosophila genes currently implicated in synaptic transmission and/or behavior are represented in the Apis EST set. Of Apis sequences with open reading frames of at least 450 bp, 24% are highly diverged with no matches to known protein sequences. Additionally, over 100 Apis transcript sequences conserved with other organisms appear to have been lost from the Drosophila genome. DNA microarrays were fabricated with over 7000 EST cDNA clones putatively representing different transcripts. Using probe derived from single bee brain mRNA, microarrays detected gene expression for 90% of Apis cDNAs two standard deviations greater than exogenous control cDNAs. [The sequence data described in this paper have been submitted to Genbank data library under accession nos. BI502708–BI517278. The sequences are also available at http://titan.biotec.uiuc.edu/bee/honeybee_project.htm.] PMID:11932240

  10. Differentially expressed transcripts in shell glands from low and high egg production strains of chickens using cDNA microarrays.

    PubMed

    Yang, Kuo-Tai; Lin, Chia-Yu; Liou, Jong-Shian; Fan, Yi-Hsing; Chiou, Shiow-Her; Huang, Chang-Wen; Wu, Chean-Ping; Lin, En-Chung; Chen, Chih-Feng; Lee, Yen-Pai; Lee, Wen-Chuan; Ding, Shih-Torng; Cheng, Winston Teng-Kuei; Huang, Mu-Chiou

    2007-09-01

    We have constructed a tissue-specific in-house cDNA microarray to identify differentially expressed transcripts in shell glands from low (B) and high (L2) egg production strains of Taiwanese country chickens during their egg-laying period. The shell gland cDNA library was constructed from the high egg production strain. cDNA clones (7680) were randomly selected and their 5'-end sequences characterized. After excluding overlapping sequences, an in-house cDNA microarray, representing 2743 non-redundant transcripts, was generated for functional genomic studies. Using our microarray, we have successfully identified 85 differentially expressed transcripts from the two different strains of chicken shell glands. In this study, 34 of these transcripts were associated with signal transduction, protein biosynthesis, cell adhesion, cellular metabolism, skeletal development, cell organization and biogenesis. We selected a number of the differentially expressed transcripts for further validation using semi-quantitative RT-PCR. These included elongation factor 2 (EEF2), ovocalyxin-32 (OCX-32) and annexin A2 (ANXA2) which were expressed at high levels in the chicken shell glands of the B strain and, in contrast, the coactosin-like protein (COTL1), transcription factor SOX18 and MX protein were more highly expressed in the L2 strain. Our results suggest that these differentially expressed transcripts may be suitable to use as molecular markers for high rates of egg production, and now need to be investigated further to assess whether they can be applied for use in breeding selection programs in Taiwanese country chickens.

  11. Fuzzy support vector machine: an efficient rule-based classification technique for microarrays.

    PubMed

    Hajiloo, Mohsen; Rabiee, Hamid R; Anooshahpour, Mahdi

    2013-01-01

    The abundance of gene expression microarray data has led to the development of machine learning algorithms applicable for tackling disease diagnosis, disease prognosis, and treatment selection problems. However, these algorithms often produce classifiers with weaknesses in terms of accuracy, robustness, and interpretability. This paper introduces fuzzy support vector machine which is a learning algorithm based on combination of fuzzy classifiers and kernel machines for microarray classification. Experimental results on public leukemia, prostate, and colon cancer datasets show that fuzzy support vector machine applied in combination with filter or wrapper feature selection methods develops a robust model with higher accuracy than the conventional microarray classification models such as support vector machine, artificial neural network, decision trees, k nearest neighbors, and diagonal linear discriminant analysis. Furthermore, the interpretable rule-base inferred from fuzzy support vector machine helps extracting biological knowledge from microarray data. Fuzzy support vector machine as a new classification model with high generalization power, robustness, and good interpretability seems to be a promising tool for gene expression microarray classification.

  12. Efficient monitoring of protein ubiquitylation levels using TUBEs-based microarrays.

    PubMed

    Serna, Sonia; Xolalpa, Wendy; Lang, Valérie; Aillet, Fabienne; England, Patrick; Reichardt, Niels; Rodriguez, Manuel S

    2016-08-01

    Analyzing protein ubiquitylation changes during physiological or pathological processes is challenging due to its high reversibility and dynamic turnover of modified targets. We have developed a protein microarray to assess endogenous ubiquitylation levels from cell cultures, employing tandem ubiquitin-binding entities (TUBEs) with three or four ubiquitin-associated (UBA) domains as capture probes. Adriamycin (ADR)-stimulated MCF7 cells were used to differentiate protein ubiquitylation levels between cells that are sensitive or resistant to ADR treatment. We show that TUBEs-based microarrays can be used for the analysis of cellular processes regulated by ubiquitylation and for the detection of pathologies with aberrant ubiquitylation levels.

  13. CometChip: a high-throughput 96-well platform for measuring DNA damage in microarrayed human cells.

    PubMed

    Ge, Jing; Prasongtanakij, Somsak; Wood, David K; Weingeist, David M; Fessler, Jessica; Navasummrit, Panida; Ruchirawat, Mathuros; Engelward, Bevin P

    2014-10-18

    DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The 'CometChip' is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

  14. Comparison of Alexa Fluor and CyDye for practical DNA microarray use.

    PubMed

    Ballard, Joanne L; Peeva, Violet K; deSilva, Christopher J S; Lynch, Jessica L; Swanson, Nigel R

    2007-07-01

    Microarrays are a powerful tool for comparison and understanding of gene expression levels in healthy and diseased states. The method relies upon the assumption that signals from microarray features are a reflection of relative gene expression levels of the cell types under investigation. It has previously been reported that the classical fluorescent dyes used for microarray technology, Cy3 and Cy5, are not ideal due to the decreased stability and fluorescence intensity of the Cy5 dye relative to the Cy3, such that dye bias is an accepted phenomena necessitating dye swap experimental protocols and analysis of differential dye affects. The incentive to find new fluorophores is based on alleviating the problem of dye bias through synonymous performance between counterpart dyes. Alexa Fluor 555 and Alexa Fluor 647 are increasingly promoted as replacements for CyDye in microarray experiments. Performance relates to the molecular and steric similarities, which will vary for each new pair of dyes as well as the spectral integrity for the specific application required. Comparative analysis of the performance of these two competitive dye pairs in practical microarray applications is warranted towards this end. The findings of our study showed that both dye pairs were comparable but that conventional CyDye resulted in significantly higher signal intensities (P < 0.05) and signal minus background levels (P < 0.05) with no significant difference in background values (P > 0.05). This translated to greater levels of differential gene expression with CyDye than with the Alexa Fluor counterparts. However, CyDye fluorophores and in particular Cy5, were found to be less photostable over time and following repeated scans in microarray experiments. These results suggest that precautions against potential dye affects will continue to be necessary and that no one dye pair negates this need.

  15. Genetic stock identification of chum salmon in the Bering Sea and North Pacific Ocean using mitochondrial DNA microarray.

    PubMed

    Moriya, Shogo; Sato, Shunpei; Azumaya, Tomonori; Suzuki, Osamu; Urawa, Shigehiko; Urano, Akihisa; Abe, Syuiti

    2007-01-01

    A newly developed DNA microarray was applied to identify mitochondrial (mt) DNA haplotypes of more than 2200 chum salmon in the Bering Sea and North Pacific Ocean in September 2002 and also 2003, when the majority of maturing fish were migrating toward their natal river. The distribution of haplotypes occurring in Asian and North American fish in the surveyed area was similar in the 2 years. A conditional maximum likelihood method for estimation of stock compositions indicated that the Japanese stocks were distributed mainly in the north central Bering Sea, whereas the Russian stocks were mainly in the western Bering Sea. The North American stocks were abundant in the North Pacific Ocean around the Aleutian Islands. These results indicate that the Asian and North American stocks of chum salmon are nonrandomly distributed in the Bering Sea and the North Pacific Ocean, and further the oligonuleotide DNA microarray developed by us has a high potential for identification of stocks among mixed ocean aggregates of high-seas chum salmon.

  16. Metatranscriptomic Analysis of the Response of River Biofilms to Pharmaceutical Products, Using Anonymous DNA Microarrays ▿ †

    PubMed Central

    Yergeau, Etienne; Lawrence, John R.; Waiser, Marley J.; Korber, Darren R.; Greer, Charles W.

    2010-01-01

    Pharmaceutical products are released at low concentrations into aquatic environments following domestic wastewater treatment. Such low concentrations have been shown to induce transcriptional responses in microorganisms, which could have consequences on aquatic ecosystem dynamics. In order to test if these transcriptional responses could also be observed in complex river microbial communities, biofilm reactors were inoculated with water from two rivers of differing trophic statuses and subsequently treated with environmentally relevant doses (ng/liter to μg/liter range) of four pharmaceuticals (erythromycin [ER], gemfibrozil [GM], sulfamethazine [SN], and sulfamethoxazole [SL]). To monitor functional gene expression, we constructed a 9,600-feature anonymous DNA microarray platform onto which cDNA from the biofilms was hybridized. Pharmaceutical treatments induced both positive and negative transcriptional responses from biofilm microorganisms. For instance, ER induced the transcription of several stress, transcription, and replication genes, while GM, a lipid regulator, induced transcriptional responses from several genes involved in lipid metabolism. SN caused shifts in genes involved in energy production and conversion, and SL induced responses from a range of cell membrane and outer envelope genes, which in turn could affect biofilm formation. The results presented here demonstrate for the first time that low concentrations of small molecules can induce transcriptional changes in a complex microbial community. The relevance of these results also demonstrates the usefulness of anonymous DNA microarrays for large-scale metatranscriptomic studies of communities from differing aquatic ecosystems. PMID:20562274

  17. Candidate genes involved in tanshinone biosynthesis in hairy roots of Salvia miltiorrhiza revealed by cDNA microarray.

    PubMed

    Cui, Guanghong; Huang, Luqi; Tang, Xiaojing; Zhao, Jingxue

    2011-04-01

    Salvia miltiorrhiza is a valuable Chinese herb (Danshen) that is widely used in traditional Chinese medicine. Diterpene quinones, known as tanshinones, are the main bioactive components of S. miltiorrhiza; however, there is only limited information regarding the molecular mechanisms underlying secondary metabolism in this plant. We used cDNA microarray analysis to identify changes in the gene expression profile at different stages of hairy root development in S. miltiorrhiza. A total of 203 genes were singled out from 4,354 cDNA clones on the microarray, and 114 unique differentially expressed cDNA clones were identified: six genes differentially expressed in 45-day hairy root compared with 30-day hairy root; 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root; and 12 genes unstably expressed at different stages. Among the 96 genes differentially expressed in 60-day hairy root compared with 30-day hairy root, a total of 57 genes were up-regulated, and 26 genes represent 29 metabolism-related enzymes. Copalyl diphosphate synthase, which catalyzes the conversion of the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate to copalyl diphosphate, was up-regulated 6.63 fold, and another six genes involved in tanshinone biosynthesis and eight candidate P450 genes were also differentially expressed. These data provide new insights for further identification of the enzymes involved in tanshinone biosynthesis.

  18. Investigation of luminescent dye-doped or rare-earth-doped monodisperse silica nanospheres for DNA microarray labelling

    NASA Astrophysics Data System (ADS)

    Enrichi, F.; Riccò, R.; Meneghello, A.; Pierobon, R.; Cretaio, E.; Marinello, F.; Schiavuta, P.; Parma, A.; Riello, P.; Benedetti, A.

    2010-10-01

    DNA microarray is a high-throughput technology used for detection and quantification of nucleic acid molecules and others of biological interest. The analysis is resulting by specific hybridization between probe sequences deposited in array and a target ss-DNA usually functionalized by a luminescent dye. These organic labels have well known disadvantages like photobleaching and limited sensitivity. Therefore in this work we investigate a different strategy, based on the use of inorganic silica nanospheres incorporating standard luminescent dyes or rare earth ions. The synthesis and characterization of these biomarkers is reported and their application to the DNA microarray technology in comparison to the use of standard molecular fluorophores or commercial quantum dots is discussed. We show that dye doped silica spheres provides a significant increase of the optical emission signal with respect to the use of free dyes, while rare earth doped silica spheres allow reducing or completely avoiding the background noise. These aspects, together with their cheap and easy synthesis, stability in water, easy surface functionalization and bio-compatibility makes them very promising for present and future applications in bio-labelling and bio-imaging.

  19. cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

    PubMed

    Antunes, Heliton S; Wajnberg, Gabriel; Pinho, Marcos B; Jorge, Natasha Andressa Nogueira; de Moraes, Joyce Luana Melo; Stefanoff, Claudio Gustavo; Herchenhorn, Daniel; Araújo, Carlos M M; Viégas, Celia Maria Pais; Rampini, Mariana P; Dias, Fernando L; de Araujo-Souza, Patricia Savio; Passetti, Fabio; Ferreira, Carlos G

    2017-08-24

    Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

  20. Living Cell Microarrays: An Overview of Concepts

    PubMed Central

    Jonczyk, Rebecca; Kurth, Tracy; Lavrentieva, Antonina; Walter, Johanna-Gabriela; Scheper, Thomas; Stahl, Frank

    2016-01-01

    Living cell microarrays are a highly efficient cellular screening system. Due to the low number of cells required per spot, cell microarrays enable the use of primary and stem cells and provide resolution close to the single-cell level. Apart from a variety of conventional static designs, microfluidic microarray systems have also been established. An alternative format is a microarray consisting of three-dimensional cell constructs ranging from cell spheroids to cells encapsulated in hydrogel. These systems provide an in vivo-like microenvironment and are preferably used for the investigation of cellular physiology, cytotoxicity, and drug screening. Thus, many different high-tech microarray platforms are currently available. Disadvantages of many systems include their high cost, the requirement of specialized equipment for their manufacture, and the poor comparability of results between different platforms. In this article, we provide an overview of static, microfluidic, and 3D cell microarrays. In addition, we describe a simple method for the printing of living cell microarrays on modified microscope glass slides using standard DNA microarray equipment available in most laboratories. Applications in research and diagnostics are discussed, e.g., the selective and sensitive detection of biomarkers. Finally, we highlight current limitations and the future prospects of living cell microarrays. PMID:27600077

  1. Evaluation of a field-portable DNA microarray platform and nucleic acid amplification strategies for the detection of arboviruses, arthropods, and bloodmeals.

    PubMed

    Grubaugh, Nathan D; Petz, Lawrence N; Melanson, Vanessa R; McMenamy, Scott S; Turell, Michael J; Long, Lewis S; Pisarcik, Sarah E; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L; Lee, John S

    2013-02-01

    Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors.

  2. Evaluation of a Field-Portable DNA Microarray Platform and Nucleic Acid Amplification Strategies for the Detection of Arboviruses, Arthropods, and Bloodmeals

    PubMed Central

    Grubaugh, Nathan D.; Petz, Lawrence N.; Melanson, Vanessa R.; McMenamy, Scott S.; Turell, Michael J.; Long, Lewis S.; Pisarcik, Sarah E.; Kengluecha, Ampornpan; Jaichapor, Boonsong; O'Guinn, Monica L.; Lee, John S.

    2013-01-01

    Highly multiplexed assays, such as microarrays, can benefit arbovirus surveillance by allowing researchers to screen for hundreds of targets at once. We evaluated amplification strategies and the practicality of a portable DNA microarray platform to analyze virus-infected mosquitoes. The prototype microarray design used here targeted the non-structural protein 5, ribosomal RNA, and cytochrome b genes for the detection of flaviviruses, mosquitoes, and bloodmeals, respectively. We identified 13 of 14 flaviviruses from virus inoculated mosquitoes and cultured cells. Additionally, we differentiated between four mosquito genera and eight whole blood samples. The microarray platform was field evaluated in Thailand and successfully identified flaviviruses (Culex flavivirus, dengue-3, and Japanese encephalitis viruses), differentiated between mosquito genera (Aedes, Armigeres, Culex, and Mansonia), and detected mammalian bloodmeals (human and dog). We showed that the microarray platform and amplification strategies described here can be used to discern specific information on a wide variety of viruses and their vectors. PMID:23249687

  3. Optimization and clinical validation of a pathogen detection microarray

    PubMed Central

    Wong, Christopher W; Heng, Charlie Lee Wah; Wan Yee, Leong; Soh, Shirlena WL; Kartasasmita, Cissy B; Simoes, Eric AF; Hibberd, Martin L; Sung, Wing-Kin; Miller, Lance D

    2007-01-01

    DNA microarrays used as 'genomic sensors' have great potential in clinical diagnostics. Biases inherent in random PCR-amplification, cross-hybridization effects, and inadequate microarray analysis, however, limit detection sensitivity and specificity. Here, we have studied the relationships between viral amplification efficiency, hybridization signal, and target-probe annealing specificity using a customized microarray platform. Novel features of this platform include the development of a robust algorithm that accurately predicts PCR bias during DNA amplification and can be used to improve PCR primer design, as well as a powerful statistical concept for inferring pathogen identity from probe recognition signatures. Compared to real-time PCR, the microarray platform identified pathogens with 94% accuracy (76% sensitivity and 100% specificity) in a panel of 36 patient specimens. Our findings show that microarrays can be used for the robust and accurate diagnosis of pathogens, and further substantiate the use of microarray technology in clinical diagnostics. PMID:17531104

  4. [DNA microarray-based gene expression profiling in diagnosis, assessing prognosis and predicting response to therapy in colorectal cancer].

    PubMed

    Kwiatkowski, Przemysław; Wierzbicki, Piotr; Kmieć, Andrzej; Godlewski, Janusz

    2012-06-11

     Colorectal cancer is the most common cancer of the gastrointestinal tract. It is considered as a biological model of a certain type of cancerogenesis process in which progression from an early to late stage adenoma and cancer is accompanied by distinct genetic alterations. Clinical and pathological parameters commonly used in clinical practice are often insufficient to determine groups of patients suitable for personalized treatment. Moreover, reliable molecular markers with high prognostic value have not yet been determined. Molecular studies using DNA-based microarrays have identified numerous genes involved in cell proliferation and differentiation during the process of cancerogenesis. Assessment of the genetic profile of colorectal cancer using the microarray technique might be a useful tool in determining the groups of patients with different clinical outcomes who would benefit from additional personalized treatment. The main objective of this study was to present the current state of knowledge on the practical application of gene profiling techniques using microarrays for determining diagnosis, prognosis and response to treatment in colorectal cancer.

  5. Development of a DNA Microarray for Enterococcal Species, Virulence, and Antibiotic Resistance Gene Determinations among Isolates from Poultry▿

    PubMed Central

    Champagne, J.; Diarra, M. S.; Rempel, H.; Topp, E.; Greer, C. W.; Harel, J.; Masson, L.

    2011-01-01

    A DNA microarray (Enteroarray) was designed with probes targeting four species-specific taxonomic identifiers to discriminate among 18 different enterococcal species, while other probes were designed to identify 18 virulence factors and 174 antibiotic resistance genes. In total, 262 genes were utilized for rapid species identification of enterococcal isolates, while characterizing their virulence potential through the simultaneous identification of endogenous antibiotic resistance and virulence genes. Enterococcal isolates from broiler chicken farms were initially identified by using the API 20 Strep system, and the results were compared to those obtained with the taxonomic genes atpA, recA, pheS, and ddl represented on our microarray. Among the 171 isolates studied, five different enterococcal species were identified by using the API 20 Strep system: Enterococcus faecium, E. faecalis, E. durans, E. gallinarum, and E. avium. The Enteroarray detected the same species as API 20 Strep, as well as two more: E. casseliflavus and E. hirae. Species comparisons resulted in 15% (27 isolates) disagreement between the two methods among the five API 20 Strep identifiable species and 24% (42 isolates) disagreement when considering the seven Enteroarray identified species. The species specificity of key antibiotic and virulence genes identified by the Enteroarray were consistent with the literature adding further robustness to the redundant taxonomic probe data. Sequencing of the cpn60 gene further confirmed the complete accuracy of the microarray results. The new Enteroarray should prove to be a useful tool to accurately genotype strains of enterococci and assess their virulence potential. PMID:21335389

  6. Oxidative stress response of European flounder (Platichthys flesus) to cadmium determined by a custom cDNA microarray.

    PubMed

    Sheader, Derek L; Williams, Timothy D; Lyons, Brett P; Chipman, J Kevin

    2006-07-01

    The monitoring of the impact of chemical pollutants upon marine ecosystems commonly employs a multi-biomarker approach. Functional genomics, using cDNA microarrays, allows for a comprehensive view of how an organism is responding to an exposure, with respect to changes in gene expression. Differentially expressed mRNAs were first isolated from livers of European flounder by means of suppressive, subtractive hybridisation. A clone set containing a total of 284 different potentially differentially expressed mRNAs was produced, of which 84 were tentatively identified. These were combined with previously cloned known stress genes isolated by degenerate PCR to produce a custom 500-clone microarray platform with each clone arrayed to four spots. Subsequent array experiments using cadmium-treated flounder detected up-regulation of 27 transcripts, including Cu/Zn superoxide dismutase, thioredoxin, a peroxiredoxin and a glutathione-S-transferase, reflecting oxidative stress in exposed flounder, while CYP1A expression was down-regulated. These changes were confirmed by real-time PCR. The array experiment highlighted a number of candidate genes for further analysis as potential novel biomarkers of cadmium exposure and demonstrated the applicability of the custom microarray approach in the study of the effects of toxicants.

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

    PubMed

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

    2016-07-01

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

  8. An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies.

    PubMed

    Martín, Verónica; Perales, Celia; Fernández-Algar, María; Dos Santos, Helena G; Garrido, Patricia; Pernas, María; Parro, Víctor; Moreno, Miguel; García-Pérez, Javier; Alcamí, José; Torán, José Luis; Abia, David; Domingo, Esteban; Briones, Carlos

    2016-01-01

    The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5-10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice.

  9. An Efficient Microarray-Based Genotyping Platform for the Identification of Drug-Resistance Mutations in Majority and Minority Subpopulations of HIV-1 Quasispecies

    PubMed Central

    Martín, Verónica; Perales, Celia; Fernández-Algar, María; Dos Santos, Helena G.; Garrido, Patricia; Pernas, María; Parro, Víctor; Moreno, Miguel; García-Pérez, Javier; Alcamí, José; Torán, José Luis; Abia, David; Domingo, Esteban

    2016-01-01

    The response of human immunodeficiency virus type 1 (HIV-1) quasispecies to antiretroviral therapy is influenced by the ensemble of mutants that composes the evolving population. Low-abundance subpopulations within HIV-1 quasispecies may determine the viral response to the administered drug combinations. However, routine sequencing assays available to clinical laboratories do not recognize HIV-1 minority variants representing less than 25% of the population. Although several alternative and more sensitive genotyping techniques have been developed, including next-generation sequencing (NGS) methods, they are usually very time consuming, expensive and require highly trained personnel, thus becoming unrealistic approaches in daily clinical practice. Here we describe the development and testing of a HIV-1 genotyping DNA microarray that detects and quantifies, in majority and minority viral subpopulations, relevant mutations and amino acid insertions in 42 codons of the pol gene associated with drug- and multidrug-resistance to protease (PR) and reverse transcriptase (RT) inhibitors. A customized bioinformatics protocol has been implemented to analyze the microarray hybridization data by including a new normalization procedure and a stepwise filtering algorithm, which resulted in the highly accurate (96.33%) detection of positive/negative signals. This microarray has been tested with 57 subtype B HIV-1 clinical samples extracted from multi-treated patients, showing an overall identification of 95.53% and 89.24% of the queried PR and RT codons, respectively, and enough sensitivity to detect minority subpopulations representing as low as 5–10% of the total quasispecies. The developed genotyping platform represents an efficient diagnostic and prognostic tool useful to personalize antiviral treatments in clinical practice. PMID:27959928

  10. A novel rapid DNA microarray assay enables identification of 37 Mycoplasma species and highlights multiple Mycoplasma infections.

    PubMed

    Schnee, Christiane; Schulsse, Samuel; Hotzel, Helmut; Ayling, Roger D; Nicholas, Robin A J; Schubert, Evelyn; Heller, Martin; Ehricht, Ralf; Sachse, Konrad

    2012-01-01

    Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus Mycoplasma alone contains more than 120 species at present, and new members are continuously being discovered. Therefore, it seems promising to use a single highly parallel detection assay rather than develop separate tests for each individual species. In this study, we have designed a DNA microarray carrying 70 oligonucleotide probes derived from the 23S rRNA gene and 86 probes from the tuf gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 Mycoplasma spp., as well as 3 Acholeplasma spp. and 3 Ureaplasma spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of Mollicutes spp. occurring in humans, animals or cell culture, from mono- and multiple infections, in a single run. The main advantages of the microarray assay include ease of operation, rapidity, high information content, and affordability. The new test's analytical sensitivity is equivalent to that of real-time PCR and allows examination of field samples without the need for culture. When 60 field samples from ruminants and birds previously analyzed by denaturing-gradient gel electrophoresis (DGGE) were tested by the microarray assay both tests identified the same agent in 98.3% of the cases. Notably, microarray testing revealed an unexpectedly high proportion (35%) of multiple mycoplasma infections, i.e., substantially more than DGGE (15%). Two of the samples were found to contain four different Mycoplasma spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment.

  11. A Novel Rapid DNA Microarray Assay Enables Identification of 37 Mycoplasma Species and Highlights Multiple Mycoplasma Infections

    PubMed Central

    Schnee, Christiane; Schulsse, Samuel; Hotzel, Helmut; Ayling, Roger D.; Nicholas, Robin A. J.; Schubert, Evelyn; Heller, Martin; Ehricht, Ralf; Sachse, Konrad

    2012-01-01

    Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus Mycoplasma alone contains more than 120 species at present, and new members are continuously being discovered. Therefore, it seems promising to use a single highly parallel detection assay rather than develop separate tests for each individual species. In this study, we have designed a DNA microarray carrying 70 oligonucleotide probes derived from the 23S rRNA gene and 86 probes from the tuf gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 Mycoplasma spp., as well as 3 Acholeplasma spp. and 3 Ureaplasma spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of Mollicutes spp. occurring in humans, animals or cell culture, from mono- and multiple infections, in a single run. The main advantages of the microarray assay include ease of operation, rapidity, high information content, and affordability. The new test's analytical sensitivity is equivalent to that of real-time PCR and allows examination of field samples without the need for culture. When 60 field samples from ruminants and birds previously analyzed by denaturing-gradient gel electrophoresis (DGGE) were tested by the microarray assay both tests identified the same agent in 98.3% of the cases. Notably, microarray testing revealed an unexpectedly high proportion (35%) of multiple mycoplasma infections, i.e., substantially more than DGGE (15%). Two of the samples were found to contain four different Mycoplasma spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment. PMID:22479374

  12. Development of an oligo DNA microarray for the European sea bass and its application to expression profiling of jaw deformity

    PubMed Central

    2010-01-01

    Background The European sea bass (Dicentrarchus labrax) is a marine fish of great importance for fisheries and aquaculture. Functional genomics offers the possibility to discover the molecular mechanisms underlying productive traits in farmed fish, and a step towards the application of marker assisted selection methods in this species. To this end, we report here on the development of an oligo DNA microarray for D. labrax. Results A database consisting of 19,048 unique transcripts was constructed, of which 12,008 (63%) could be annotated by similarity and 4,692 received a GO functional annotation. Two non-overlapping 60mer probes were designed for each unique transcript and in-situ synthesized on glass slides using Agilent SurePrint™ technology. Probe design was positively completed for 19,035 target clusters; the oligo microarray was then applied to profile gene expression in mandibles and whole-heads of fish affected by prognathism, a skeletal malformation that strongly affects sea bass production. Statistical analysis identified 242 transcripts that are significantly down-regulated in deformed individuals compared to normal fish, with a significant enrichment in genes related to nervous system development and functioning. A set of genes spanning a wide dynamic range in gene expression level were selected for quantitative RT-PCR validation. Fold change correlation between microarray and qPCR data was always significant. Conclusions The microarray platform developed for the European sea bass has a high level of flexibility, reliability, and reproducibility. Despite the well known limitations in achieving a proper functional annotation in non-model species, sufficient information was obtained to identify biological processes that are significantly enriched among differentially expressed genes. New insights were obtained on putative mechanisms involved on mandibular prognathism, suggesting that bone/nervous system development might play a role in this phenomenon

  13. Surface invasive cleavage assay on a maskless light-directed diamond DNA microarray for genome-wide human SNP mapping.

    PubMed

    Nie, Bei; Yang, Min; Fu, Weiling; Liang, Zhiqing

    2015-07-07

    The surface invasive cleavage assay, because of its innate accuracy and ability for self-signal amplification, provides a potential route for the mapping of hundreds of thousands of human SNP sites. However, its performance on a high density DNA array has not yet been established, due to the unusual "hairpin" probe design on the microarray and the lack of chemical stability of commercially available substrates. Here we present an applicable method to implement a nanocrystalline diamond thin film as an alternative substrate for fabricating an addressable DNA array using maskless light-directed photochemistry, producing the most chemically stable and biocompatible system for genetic analysis and enzymatic reactions. The surface invasive cleavage reaction, followed by degenerated primer ligation and post-rolling circle amplification is consecutively performed on the addressable diamond DNA array, accurately mapping SNP sites from PCR-amplified human genomic target DNA. Furthermore, a specially-designed DNA array containing dual probes in the same pixel is fabricated by following a reverse light-directed DNA synthesis protocol. This essentially enables us to decipher thousands of SNP alleles in a single-pot reaction by the simple addition of enzyme, target and reaction buffers.

  14. Effect of data normalization on fuzzy clustering of DNA microarray data

    PubMed Central

    Kim, Seo Young; Lee, Jae Won; Bae, Jong Sung

    2006-01-01

    Background Microarray technology has made it possible to simultaneously measure the expression levels of large numbers of genes in a short time. Gene expression data is information rich; however, extensive data mining is required to identify the patterns that characterize the underlying mechanisms of action. Clustering is an important tool for finding groups of genes with similar expression patterns in microarray data analysis. However, hard clustering methods, which assign each gene exactly to one cluster, are poorly suited to the analysis of microarray datasets because in such datasets the clusters of genes frequently overlap. Results In this study we applied the fuzzy partitional clustering method known as Fuzzy C-Means (FCM) to overcome the limitations of hard clustering. To identify the effect of data normalization, we used three normalization methods, the two common scale and location transformations and Lowess normalization methods, to normalize three microarray datasets and three simulated datasets. First we determined the optimal parameters for FCM clustering. We found that the optimal fuzzification parameter in the FCM analysis of a microarray dataset depended on the normalization method applied to the dataset during preprocessing. We additionally evaluated the effect of normalization of noisy datasets on the results obtained when hard clustering or FCM clustering was applied to those datasets. The effects of normalization were evaluated using both simulated datasets and microarray datasets. A comparative analysis showed that the clustering results depended on the normalization method used and the noisiness of the data. In particular, the selection of the fuzzification parameter value for the FCM method was sensitive to the normalization method used for datasets with large variations across samples. Conclusion Lowess normalization is more robust for clustering of genes from general microarray data than the two common scale and location adjustment methods

  15. cDNA Microarrays as a Tool for Identification of Biomineralization Proteins in the Coccolithophorid Emiliania huxleyi (Haptophyta)

    PubMed Central

    Quinn, Patrick; Bowers, Robert M.; Zhang, Xiaoyu; Wahlund, Thomas M.; Fanelli, Michael A.; Olszova, Daniela; Read, Betsy A.

    2006-01-01

    Marine unicellular coccolithophore algae produce species-specific calcite scales otherwise known as coccoliths. While the coccoliths and their elaborate architecture have attracted the attention of investigators from various scientific disciplines, our knowledge of the underpinnings of the process of biomineralization in this alga is still in its infancy. The processes of calcification and coccolithogenesis are highly regulated and likely to be complex, requiring coordinated expression of many genes and pathways. In this study, we have employed cDNA microarrays to investigate changes in gene expression associated with biomineralization in the most abundant coccolithophorid, Emiliania huxleyi. Expression profiling of cultures grown under calcifying and noncalcifying conditions has been carried out using cDNA microarrays corresponding to approximately 2,300 expressed sequence tags. A total of 127 significantly up- or down-regulated transcripts were identified using a P value of 0.01 and a change of >2.0-fold. Real-time reverse transcriptase PCR was used to test the overall validity of the microarray data, as well as the relevance of many of the proteins predicted to be associated with biomineralization, including a novel gamma-class carbonic anhydrase (A. R. Soto, H. Zheng, D. Shoemaker, J. Rodriguez, B. A. Read, and T. M. Wahlund, Appl. Environ. Microbiol. 72:5500-5511, 2006). Differentially regulated genes include those related to cellular metabolism, ion channels, transport proteins, vesicular trafficking, and cell signaling. The putative function of the vast majority of candidate transcripts could not be defined. Nonetheless, the data described herein represent profiles of the transcription changes associated with biomineralization-related pathways in E. huxleyi and have identified novel and potentially useful targets for more detailed analysis. PMID:16885305

  16. SVD identifies transcript length distribution functions from DNA microarray data and reveals evolutionary forces globally affecting GBM metabolism.

    PubMed

    Bertagnolli, Nicolas M; Drake, Justin A; Tennessen, Jason M; Alter, Orly

    2013-01-01

    To search for evolutionary forces that might act upon transcript length, we use the singular value decomposition (SVD) to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as "asymmetric generalized coherent states" from the DNA microarray data and with no a-priori assumptions. Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM) or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity. These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length-dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.

  17. Development of a cDNA microarray of zebra mussel (Dreissena polymorpha) foot and its use in understanding the early stage of underwater adhesion.

    PubMed

    Xu, Wei; Faisal, Mohamed

    2009-05-01

    experiment. Our findings demonstrated that the zebra mussel byssus cDNA microarray is an efficient tool for the studies of differential gene expression in different byssogenesis states, thereby revealing important details of the underwater adhesion.

  18. Efficient Assembly of DNA Using Yeast Homologous Recombination (YHR).

    PubMed

    Chandran, Sunil; Shapland, Elaine

    2017-01-01

    The assembly of multiple DNA parts into a larger DNA construct is a requirement in most synthetic biology laboratories. Here we describe a method for the efficient, high-throughput, assembly of DNA utilizing the yeast homologous recombination (YHR). The YHR method utilizes overlapping DNA parts that are assembled together by Saccharomyces cerevisiae via homologous recombination between designed overlapping regions. Using this method, we have successfully assembled up to 12 DNA parts in a single reaction.

  19. 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.

  20. Differential modulation of Bordetella pertussis virulence genes as evidenced by DNA microarray analysis.

    PubMed

    Hot, D; Antoine, R; Renauld-Mongénie, G; Caro, V; Hennuy, B; Levillain, E; Huot, L; Wittmann, G; Poncet, D; Jacob-Dubuisson, F; Guyard, C; Rimlinger, F; Aujame, L; Godfroid, E; Guiso, N; Quentin-Millet, M-J; Lemoine, Y; Locht, C

    2003-07-01

    The production of most factors involved in Bordetella pertussis virulence is controlled by a two-component regulatory system termed BvgA/S. In the Bvg+ phase virulence-activated genes (vags) are expressed, and virulence-repressed genes (vrgs) are down-regulated. The expression of these genes can also be modulated by MgSO(4) or nicotinic acid. In this study we used microarrays to analyse the influence of BvgA/S or modulation on the expression of nearly 200 selected genes. With the exception of one vrg, all previously known vags and vrgs were correctly assigned as such, and the microarray analyses identified several new vags and vrgs, including genes coding for putative autotransporters, two-component systems, extracellular sigma factors, the adenylate cyclase accessory genes cyaBDE, and two genes coding for components of a type III secretion system. For most of the new vrgs and vags the results of the microarray analyses were confirmed by RT-PCR analysis and/or lacZfusions. The degree of regulation and modulation varied between genes, and showed a continuum from strongly BvgA/S-activated genes to strongly BvgA/S-repressed genes. The microarray analyses also led to the identification of a subset of vags and vrgs that are differentially regulated and modulated by MgSO(4) or nicotinic acid, indicating that these genes may be targets for multiple regulatory circuits. For example, the expression of bilA, a gene predicted to encode an intimin-like protein, was found to be activated by BvgA/S and up-modulated by nicotinic acid. Furthermore, surprisingly, in the strain analysed here, which produces only type 2 fimbriae, the fim3 gene was identified as a vrg, while fim2 was confirmed to be a vag.

  1. Identification and removal of contaminating fluorescence from commercial and in-house printed DNA microarrays

    PubMed Central

    Martinez, M. Juanita; Aragon, Anthony D.; Rodriguez, Angelina L.; Weber, Jose M.; Timlin, Jerilyn A.; Sinclair, Michael B.; Haaland, David M.; Werner-Washburne, Margaret

    2003-01-01

    Microarray analysis is a critically important technology for genome-enabled biology, therefore it is essential that the data obtained be reliable. Current software and normalization techniques for microarray analysis rely on the assumption that fluorescent background within spots is essentially the same throughout the glass slide and can be measured by fluorescence surrounding the spots. This assumption is not valid if background fluorescence is spot-localized. Inaccurate estimates of background fluorescence under the spot create a source of error, especially for low expressed genes. We have identified spot-localized, contaminating fluorescence in the Cy3 channel on several commercial and in-house printed microarray slides. We determined through mock hybridizations (without labeled target) that pre-hybridization scans could not be used to predict the contribution of this contaminating fluorescence after hybridization because the change in spot-to-spot fluorescence after hybridization was too variable. Two solutions to this problem were identified. First, allowing 4 h of exposure to air prior to printing on to Corning UltraGAPS slides significantly reduced contaminating fluorescence intensities to approximately the value of the surrounding glass. Alternatively, application of a novel, hyperspectral imaging scanner and multivariate curve resolution algorithms, allowed the spectral contributions of Cy3 signal, glass, and contaminating fluorescence to be distinguished and quantified after hybridization. PMID:12582263

  2. Efficient Sleeping Beauty DNA Transposition From DNA Minicircles

    PubMed Central

    Sharma, Nynne; Cai, Yujia; Bak, Rasmus O; Jakobsen, Martin R; Schrøder, Lisbeth Dahl; Mikkelsen, Jacob Giehm

    2013-01-01

    DNA transposon-based vectors have emerged as new potential delivery tools in therapeutic gene transfer. Such vectors are now showing promise in hematopoietic stem cells and primary human T cells, and clinical trials with transposon-engineered cells are on the way. However, the use of plasmid DNA as a carrier of the vector raises safety concerns due to the undesirable administration of bacterial sequences. To optimize vectors based on the Sleeping Beauty (SB) DNA transposon for clinical use, we examine here SB transposition from DNA minicircles (MCs) devoid of the bacterial plasmid backbone. Potent DNA transposition, directed by the hyperactive SB100X transposase, is demonstrated from MC donors, and the stable transfection rate is significantly enhanced by expressing the SB100X transposase from MCs. The stable transfection rate is inversely related to the size of circular donor, suggesting that a MC-based SB transposition system benefits primarily from an increased cellular uptake and/or enhanced expression which can be observed with DNA MCs. DNA transposon and transposase MCs are easily produced, are favorable in size, do not carry irrelevant DNA, and are robust substrates for DNA transposition. In accordance, DNA MCs should become a standard source of DNA transposons not only in therapeutic settings but also in the daily use of the SB system. PMID:23443502

  3. The methylation status of plant genomic DNA influences PCR efficiency.

    PubMed

    Kiselev, K V; Dubrovina, A S; Tyunin, A P

    2015-03-01

    During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs. Copyright © 2014 Elsevier GmbH. All rights reserved.

  4. A parallel and incremental algorithm for efficient unique signature discovery on DNA databases.

    PubMed

    Lee, Hsiao Ping; Sheu, Tzu-Fang; Tang, Chuan Yi

    2010-03-16

    DNA signatures are distinct short nucleotide sequences that provide valuable information that is used for various purposes, such as the design of Polymerase Chain Reaction primers and microarray experiments. Biologists usually use a discovery algorithm to find unique signatures from DNA databases, and then apply the signatures to microarray experiments. Such discovery algorithms require to set some input factors, such as signature length l and mismatch tolerance d, which affect the discovery results. However, suggestions about how to select proper factor values are rare, especially when an unfamiliar DNA database is used. In most cases, biologists typically select factor values based on experience, or even by guessing. If the discovered result is unsatisfactory, biologists change the input factors of the algorithm to obtain a new result. This process is repeated until a proper result is obtained. Implicit signatures under the discovery condition (l, d) are defined as the signatures of length < or = l with mismatch tolerance > or = d. A discovery algorithm that could discover all implicit signatures, such that those that meet the requirements concerning the results, would be more helpful than one that depends on trial and error. However, existing discovery algorithms do not address the need to discover all implicit signatures. This work proposes two discovery algorithms - the consecutive multiple discovery (CMD) algorithm and the parallel and incremental signature discovery (PISD) algorithm. The PISD algorithm is designed for efficiently discovering signatures under a certain discovery condition. The algorithm finds new results by using previously discovered results as candidates, rather than by using the whole database. The PISD algorithm further increases discovery efficiency by applying parallel computing. The CMD algorithm is designed to discover implicit signatures efficiently. It uses the PISD algorithm as a kernel routine to discover implicit signatures

  5. Prevalence, identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia.

    PubMed

    Hmaïed, F; Helel, S; Le Berre, V; François, J-M; Leclercq, A; Lecuit, M; Smaoui, H; Kechrid, A; Boudabous, A; Barkallah, I

    2014-02-01

    We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates. Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus. The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases. We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  6. Quantitative Monitoring of Gene Expression Patterns with a Complementary DNA Microarray

    NASA Astrophysics Data System (ADS)

    Schena, Mark; Shalon, Dari; Davis, Ronald W.; Brown, Patrick O.

    1995-10-01

    A high-capacity system was developed to monitor the expression of many genes in parallel. Microarrays prepared by high-speed robotic printing of complementary DNAs on glass were used for quantitative expression measurements of the corresponding genes. Because of the small format and high density of the arrays, hybridization volumes of 2 microliters could be used that enabled detection of rare transcripts in probe mixtures derived from 2 micrograms of total cellular messenger RNA. Differential expression measurements of 45 Arabidopsis genes were made by means of simultaneous, two-color fluorescence hybridization.

  7. A simple and robust method for preparation of cDNA nylon microarrays.

    PubMed

    Yamakawa, Hisashi; Yokoyama, Satoru; Hirano, Takeo; Kitamura, Hiroshi; Ohara, Osamu

    2004-10-31

    DNA array technology has made remarkable progress in recent years and has become an indispensable tool in molecular biology research. However, preparing high-quality custom-made DNA arrays at a reasonable cost is still an important concern because we cannot abandon the use of DNA array systems designed for specific purposes. To address these problems, we here report the use of rolling circle amplification products of cDNA plasmids dissolved in 80% formamide as DNA probes immobilized on a nylon membrane. First, because formamide is practically non-volatile under ambient conditions and nucleic acids are easily dissolved in it, the use of formamide as a DNA solvent ensures that the DNA concentration of the solution will not change during arraying, which often takes several hours to a day depending on the number of DNA spots and arrays to produce. Secondly, the use of rolling circle amplification technology greatly reduced the labor needed to prepare the spotted DNA. The results in this study demonstrate that the introduction of these two modifications in preparation of nylon DNA array greatly improved its quality.

  8. Profiling Ethylene-Responsive Genes Expressed in the Latex of the Mature Virgin Rubber Trees Using cDNA Microarray.

    PubMed

    Nie, Zhiyi; Kang, Guijuan; Duan, Cuifang; Li, Yu; Dai, Longjun; Zeng, Rizhong

    2016-01-01

    Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2,973 unique genes (probes) was first developed and used to analyze the gene expression changes in the latex of the mature virgin rubber trees after ethephon treatment at three different time-points: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ -2 (q-value < 0.05) in ethephon-treated rubber trees compared with control trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. The 163 ethylene-responsive genes were involved in several biological processes including organic substance metabolism, cellular metabolism, primary metabolism, biosynthetic process, cellular response to stimulus and stress. The presented data suggest that the laticifer water circulation, production and scavenging of reactive oxygen species, sugar metabolism, and assembly and depolymerization of the latex actin cytoskeleton might play important roles in ethylene-induced increase of latex production. The results may provide useful insights into understanding the molecular mechanism underlying the effect of ethylene on latex metabolism of H. brasiliensis.

  9. DNA microarray-based experimental strategy for trustworthy expression profiling of the hippocampal genes by astaxanthin supplementation in adult mouse

    PubMed Central

    Yook, Jang Soo; Shibato, Junko; Rakwal, Randeep; Soya, Hideaki

    2015-01-01

    Naturally occurring astaxantin (ASX) is one of the noticeable carotenoid and dietary supplement, which has strong antioxidant and anti-inflammatory properties, and neuroprotective effects in the brain through crossing the blood–brain barrier. Specially, we are interested in the role of ASX as a brain food. Although ASX has been suggested to have potential benefit to the brain function, the underlying molecular mechanisms and events mediating such effect remain unknown. Here we examined molecular factors in the hippocampus of adult mouse fed ASX diets (0.1% and 0.5% doses) using DNA microarray (Agilent 4 × 44 K whole mouse genome chip) analysis. In this study, we described in detail our experimental workflow and protocol, and validated quality controls with the housekeeping gene expression (Gapdh and Beta-actin) on the dye-swap based approach to advocate our microarray data, which have been uploaded to Gene Expression Omnibus (accession number GSE62197) as a gene resource for the scientific community. This data will also form an important basis for further detailed experiments and bioinformatics analysis with an aim to unravel the potential molecular pathways or mechanisms underlying the positive effects of ASX supplementation on the brain, in particular the hippocampus. PMID:26981356

  10. Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments.

    PubMed

    Akkiprik, Mustafa; Peker, İrem; Özmen, Tolga; Amuran, Gökçe Güllü; Güllüoğlu, Bahadır M; Kaya, Handan; Özer, Ayşe

    2015-11-10

    IGFBP5 is an important regulatory protein in breast cancer progression. We tried to identify differentially expressed genes (DEGs) between breast tumor tissues with IGFBP5 overexpression and their adjacent normal tissues. In this study, thirty-eight breast cancer and adjacent normal breast tissue samples were used to determine IGFBP5 expression by qPCR. cDNA microarrays were applied to the highest IGFBP5 overexpressed tumor samples compared to their adjacent normal breast tissue. Microarray analysis revealed that a total of 186 genes were differentially expressed in breast cancer compared with normal breast tissues. Of the 186 genes, 169 genes were downregulated and 17 genes were upregulated in the tumor samples. KEGG pathway analyses showed that protein digestion and absorption, focal adhesion, salivary secretion, drug metabolism-cytochrome P450, and phenylalanine metabolism pathways are involved. Among these DEGs, the prominent top two genes (MMP11 and COL1A1) which potentially correlated with IGFBP5 were selected for validation using real time RT-qPCR. Only COL1A1 expression showed a consistent upregulation with IGFBP5 expression and COL1A1 and MMP11 were significantly positively correlated. We concluded that the discovery of coordinately expressed genes related with IGFBP5 might contribute to understanding of the molecular mechanism of the function of IGFBP5 in breast cancer. Further functional studies on DEGs and association with IGFBP5 may identify novel biomarkers for clinical applications in breast cancer.

  11. Mechanisms Underlying the Antiproliferative and Prodifferentiative Effects of Psoralen on Adult Neural Stem Cells via DNA Microarray

    PubMed Central

    Ning, You; Huang, Jian-Hua; Xia, Shi-Jin; Bian, Qin; Chen, Yang; Zhang, Xin-Min; Dong, Jing-Cheng; Shen, Zi-Yin

    2013-01-01

    Adult neural stem cells (NSCs) persist throughout life to replace mature cells that are lost during turnover, disease, or injury. The investigation of NSC creates novel treatments for central nervous system (CNS) injuries and neurodegenerative disorders. The plasticity and reparative potential of NSC are regulated by different factors, which are critical for neurological regenerative medicine research. We investigated the effects of Psoralen, which is the mature fruit of Psoralea corylifolia L., on NSC behaviors and the underlying mechanisms. The self-renewal and proliferation of NSC were examined. We detected neuron- and/or astrocyte-specific markers using immunofluorescence and Western blotting, which could evaluate NSC differentiation. Psoralen treatment significantly inhibited neurosphere formation in a dose-dependent manner. Psoralen treatment increased the expression of the astrocyte-specific marker but decreased neuron-specific marker expression. These results suggested that Psoralen was a differentiation inducer in astrocyte. Differential gene expression following Psoralen treatment was screened using DNA microarray and confirmed by quantitative real-time PCR. Our microarray study demonstrated that Psoralen could effectively regulate the specific gene expression profile of NSC. The genes involved in the classification of cellular differentiation, proliferation, and metabolism, the transcription factors belonging to Ets family, and the hedgehog pathway may be closely related to the regulation. PMID:23983781

  12. Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

    PubMed Central

    Petersen, Jesper; Poulsen, Lena; Petronis, Sarunas; Birgens, Henrik; Dufva, Martin

    2008-01-01

    DNA microarrays are generally operated at a single condition, which severely limits the freedom of designing probes for allele-specific hybridization assays. Here, we demonstrate a fluidic device for multi-stringency posthybridization washing of microarrays on microscope slides. This device is called a multi-thermal array washer (MTAW), and it has eight individually controlled heating zones, each of which corresponds to the location of a subarray on a slide. Allele-specific oligonucleotide probes for nine mutations in the beta-globin gene were spotted in eight identical subarrays at positions corresponding to the temperature zones of the MTAW. After hybridization with amplified patient material, the slides were mounted in the MTAW, and each subarray was exposed to different temperatures ranging from 22 to 40°C. When processed in the MTAW, probes selected without considering melting temperature resulted in improved genotyping compared with probes selected according to theoretical melting temperature and run under one condition. In conclusion, the MTAW is a versatile tool that can facilitate screening of a large number of probes for genotyping assays and can also enhance the performance of diagnostic arrays. PMID:18063568

  13. Identification of differentially expressed genes in shrimp (Penaeus stylirostris) infected with White spot syndrome virus by cDNA microarrays.

    PubMed

    Dhar, A K; Dettori, A; Roux, M M; Klimpel, K R; Read, B

    2003-12-01

    White spot syndrome virus (WSSV) is currently the most important viral pathogen infecting penaeid shrimp worldwide. Although considerable progress has been made in characterizing the WSSV genome and developing detection methods, information pertaining to host genes involved in WSSV pathogenesis is limited. We examined the potential of cDNA microarray analysis to study gene expression in WSSV-infected shrimp. Shrimp cDNAs were printed as low-density arrays on glass slides and were hybridized with Cy3/Cy5 labeled probes derived from RNA isolated from healthy and WSSV-infected shrimp. Genes that code for proteins that are relevant to crustacean immunity, structural proteins, as well as proteins of unknown function were among those whose mRNA expression was altered upon WSSV infection. To validate the microarray data, the temporal expression of three differentially expressed genes, an immune gene (C-type lectin-1), a structural gene (40S ribosomal protein), and a gene involved in lipid metabolism (fatty acid binding protein) was measured in healthy and WSSV-infected shrimp by real-time RT-PCR. The data suggest that WSSV infection alters the expression of a wide array of cellular genes, and provides a framework for further studies aimed at identifying genes whose function may provide insight into the mechanism of WSSV infection in shrimp.

  14. BioconductorBuntu: a Linux distribution that implements a web-based DNA microarray analysis server.

    PubMed

    Geeleher, Paul; Morris, Dermot; Hinde, John P; Golden, Aaron

    2009-06-01

    BioconductorBuntu is a custom distribution of Ubuntu Linux that automatically installs a server-side microarray processing environment, providing a user-friendly web-based GUI to many of the tools developed by the Bioconductor Project, accessible locally or across a network. System installation is via booting off a CD image or by using a Debian package provided to upgrade an existing Ubuntu installation. In its current version, several microarray analysis pipelines are supported including oligonucleotide, dual-or single-dye experiments, including post-processing with Gene Set Enrichment Analysis. BioconductorBuntu is designed to be extensible, by server-side integration of further relevant Bioconductor modules as required, facilitated by its straightforward underlying Python-based infrastructure. BioconductorBuntu offers an ideal environment for the development of processing procedures to facilitate the analysis of next-generation sequencing datasets. BioconductorBuntu is available for download under a creative commons license along with additional documentation and a tutorial from (http://bioinf.nuigalway.ie).

  15. cDNA Microarray Analysis of Serially Sampled Cervical Cancer Specimens From Patients Treated With Thermochemoradiotherapy

    SciTech Connect

    Borkamo, Erling Dahl; Schem, Baard-Christian; Fluge, Oystein; Bruland, Ove; Dahl, Olav; Mella, Olav

    2009-12-01

    Purpose: To elucidate changes in gene expression after treatment with regional thermochemoradiotherapy in locally advanced squamous cell cervical cancer. Methods and Materials: Tru-Cut biopsy specimens were serially collected from 16 patients. Microarray gene expression levels before and 24 h after the first and second trimodality treatment sessions were compared. Pathway and network analyses were conducted by use of Ingenuity Pathways Analysis (IPA; Ingenuity Systems, Redwood City, CA). Single gene expressions were analyzed by quantitative real-time reverse transcription-polymerase chain reaction. Results: We detected 53 annotated genes that were differentially expressed after trimodality treatment. Central in the three top networks detected by IPA were interferon alfa, interferon beta, and interferon gamma receptor; nuclear factor kappaB; and tumor necrosis factor, respectively. These genes encode proteins that are important in regulation cell signaling, proliferation, gene expression, and immune stimulation. Biological processes over-represented among the 53 genes were fibrosis, tumorigenesis, and immune response. Conclusions: Microarrays showed minor changes in gene expression after thermochemoradiotherapy in locally advanced cervical cancer. We detected 53 differentially expressed genes, mainly involved in fibrosis, tumorigenesis, and immune response. A limitation with the use of serial biopsy specimens was low quality of ribonucleic acid from tumors that respond to highly effective therapy. Another 'key limitation' is timing of the post-treatment biopsy, because 24 h may be too late to adequately assess the impact of hyperthermia on gene expression.

  16. Functional analysis of differentially expressed genes associated with glaucoma from DNA microarray data.

    PubMed

    Wu, Y; Zang, W D; Jiang, W

    2014-11-11

    Microarray data of astrocytes extracted from the optic nerves of donors with and without glaucoma were analyzed to screen for differentially expressed genes (DEGs). Functional exploration with bioinformatic tools was then used to understand the roles of the identified DEGs in glaucoma. Microarray data were downloaded from the Gene Expression Omnibus (GEO) database, which contains 13 astrocyte samples, 6 from healthy subjects and 7 from patients suffering from glaucoma. Data were pre-processed, and DEGs were screened out using R software packages. Interactions between DEGs were identified, and networks were built using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING). GENECODIS was utilized for the functional analysis of the DEGs, and GOTM was used for module division, for which functional annotation was conducted with the Database for Annotation, Visualization, and Integrated Discovery (DAVID). A total of 371 DEGs were identified between glaucoma-associated samples and normal samples. Three modules included in the PPID database were generated with 11, 12, and 2 significant functional annotations, including immune system processes, inflammatory responses, and synaptic vesicle endocytosis, respectively. We found that the most significantly enriched functions for each module were associated with immune function. Several genes that play interesting roles in the development of glaucoma are described; these genes may be potential biomarkers for glaucoma diagnosis or treatment.

  17. Genes regulated by thyrotropin and iodide in cultured human thyroid follicles: analysis by cDNA microarray.

    PubMed

    Yamazaki, Kazuko; Yamada, Emiko; Kanaji, Yoshio; Yanagisawa, Tetsuo; Kato, Yoshiyuki; Takano, Kazue; Obara, Takao; Sato, Kanji

    2003-02-01

    Thyrotropin (TSH) regulates a number of genes in thyrocytes, leading to iodide uptake, de novo synthesis and release of thyroid hormones, and cell proliferation, accompanied by increased blood flow. At higher doses of iodide, however, the TSH-induced increases in thyroid hormone release and blood flow are downregulated, and high iodide intake occasionally worsens autoimmune thyroiditis. To elucidate the genes involved in such effects, we cultured human thyrocytes and examined genes modulated by TSH and iodide, using a cDNA microarray study, which can analyze 2400 genes in each run. When thyroid follicles were cultured with TSH for 2 days, more than 100 genes were upregulated. These genes included those for enzymes involved in carbohydrate and lipid metabolism, adenylate and guanylate cyclases, and enzyme involved in cell proliferation. When thyroid follicles were cultured with high iodide concentrations (10(-5) M) for 24 hours, more than 100 genes were upregulated. Interesting genes were interleukin-8, IFP53, 90-kd heat shock protein, osteopontin, and intercellular adhesion molecule-1. These results were confirmed by reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot hybridization. In summary, TSH upregulated a number of genes regulating thyroid functions. It is intriguing that thyroid follicles cultured with a high iodide concentration (10(-5) M) increased the expression levels of genes capable of modulating lymphocyte functions, even though immunocompetent cells were extensively removed by the present experimental culture conditions. Although we have analyzed only approximately 6%-8% of all human genes, the cDNA microarray study is a powerful tool to elucidate the effects of TSH and iodide on thyroid function.

  18. Targeted cellular process profiling approach for uterine leiomyoma using cDNA microarray, proteomics and gene ontology analysis

    PubMed Central

    Ahn, Woong Shick; Kim, Ko-Woon; Bae, Su Mi; Yoon, Joo Hee; Lee, Joon Mo; Namkoong, Sung Eun; Kim, Jin Hong; Kim, Chong Kook; Lee, Young Joo; Kim, Yong-Wan

    2003-01-01

    This study utilized both cDNA microarray and two-dimensional protein gel electrophoresis technology to investigate the multiple interactions of genes and proteins involved in uterine leiomyoma pathophysiology. Also, the gene ontology analysis was used to systematically characterize the global expression profiles at cellular process levels. We profiled differentially expressed transcriptome and proteome in six-paired leiomyoma and normal myometrium. Screening up to 17 000 genes identified 21 upregulated and 50 downregulated genes. The gene-expression profiles were classified into mutually dependent 420 functional sets, resulting in 611 cellular processes according to the gene ontology. Also, protein analysis using two-dimensional gel electrophoresis identified 33 proteins (17 upregulated and 16 downregulated) of more than 500 total spots, which was classified into 302 cellular processes. Of these functional profilings, downregulations of transcriptomes and proteoms were shown in cell adhesion, cell motility, organogenesis, enzyme regulator, structural molecule activity and response to external stimulus functional activities that are supposed to play important roles in pathophysiology. In contrast, the upregulation was only shown in nucleic acid-binding activity. Taken together, potentially significant pathogenetic cellular processes were identified and showed that the downregulated functional profiling has a significant impact on the discovery of pathogenic pathway in leiomyoma. Also, the gene ontology analysis can overcome the complexity of expression profiles of cDNA microarray and two-dimensional protein analysis via its cellular process-level approach. Therefore, a valuable prognostic candidate gene with relevance to disease-specific pathogenesis can be found at cellular process levels. PMID:14748746

  19. Genomics of hepatitis B virus-related hepatocellular carcinoma and adjacent noncancerous tissues with cDNA microarray.

    PubMed

    Huang, Yu-kun; Fan, Xue-gong; Qiu, Fu; Wang, Zhi-ming

    2011-07-05

    Hepatocellular carcinoma (HCC) is a common primary cancer frequently associated with hepatitis B virus (HBV) infection. However, whether these identified genes are particularly associated with HBV-related HCC remains unknown. The aim of this study was to investigate the differential gene expression between HBV-related HCC tissues and adjacent noncancerous tissues. cDNA microarray was used to detect the differential gene expression profile in the HBV-related HCC tissues and adjacent noncancerous tissues, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the differential expression of candidate genes obtained from cDNA microarray experiment. In this study, 1369 genes or expressed sequence tags (ESTs) including 121 genes or ESTs with at least two-fold expression alterations between cancerous and noncancerous tissues were identified. Special AT-rich sequence binding protein 1 (SATB-1) expression was positive in 73% (16/22) of cancerous tissues and negative (0/22) in all noncancerous tissues of HBV-related HCC patients. Transmembrane 4 superfamily member 1 (TM4SF-1) expression was positive in 86% (19/22) of cancerous tissues and negative (0/22) in all noncancerous tissues. Suppression of tumorigenicity 14 (ST-14) expression was positive in 73% (16/22) of noncancerous tissues in patients with HBV-related HCC and negative in all HCC tissues (0/22). This study provided the gene expression profile of HBV-related HCC and presented differential expression patterns of SATB-1, TM4SF-1 and ST-14 between cancerous and noncancerous tissues in patients with HBV-related HCC.

  20. Development of a cDNA microarray for the measurement of gene expression in the sheep scab mite Psoroptes ovis

    PubMed Central

    2012-01-01

    Background Sheep scab is caused by the ectoparasitic mite Psoroptes ovis which initiates a profound cutaneous inflammatory response, leading to the development of the skin lesions which are characteristic of the disease. Existing control strategies rely upon injectable endectocides and acaricidal dips but concerns over residues, eco-toxicity and the development of acaricide resistance limit the sustainability of this approach. In order to identify alternative means of disease control, a deeper understanding of both the parasite and its interaction with the host are required. Methods Herein we describe the development and utilisation of an annotated P. ovis cDNA microarray containing 3,456 elements for the measurement of gene expression in this economically important ectoparasite. The array consists of 981 P. ovis EST sequences printed in triplicate along with 513 control elements. Array performance was validated through the analysis of gene expression differences between fed and starved P. ovis mites. Results Sequences represented on the array include homologues of major house dust mite allergens and tick salivary proteins, along with factors potentially involved in mite reproduction and xenobiotic metabolism. In order to validate the performance of this unique resource under biological conditions we used the array to analyse gene expression differences between fed and starved P. ovis mites. These analyses identified a number of house dust mite allergen homologues up-regulated in fed mites and P. ovis transcripts involved in stress responses, autophagy and chemosensory perception up-regulated in starved mites. Conclusion The P. ovis cDNA microarray described here has been shown to be both robust and reproducible and will enable future studies to analyse gene expression in this important ectoparasite. PMID:22316180

  1. Effect of Lactobacillus brevis KB290 on the cell-mediated cytotoxic activity of mouse splenocytes: a DNA microarray analysis.

    PubMed

    Fukui, Yuichiro; Sasaki, Erika; Fuke, Nobuo; Nakai, Yuji; Ishijima, Tomoko; Abe, Keiko; Yajima, Nobuhiro

    2013-11-14

    Lactic acid bacteria confer a variety of health benefits. Here, we investigate the mechanisms by which Lactobacillus brevis KB290 (KB290) enhances cell-mediated cytotoxic activity. Female BALB/c mice aged 9 weeks were fed a diet containing KB290 (3 × 10(9) colony-forming units/g) or starch for 1 d. The resulting cytotoxic activity of splenocytes against YAC-1 cells was measured using flow cytometry and analysed for gene expression using DNA microarray technology. KB290 enhanced the cell-mediated cytotoxic activity of splenocytes. DNA microarray analysis identified 327 up-regulated and 347 down-regulated genes that characterised the KB290 diet group. The up-regulated genes were significantly enriched in Gene Ontology terms related to immunity, and, especially, a positive regulation of T-cell-mediated cytotoxicity existed among these terms. Almost all the genes included in the term encoded major histocompatibility complex (MHC) class I molecules involved in the presentation of antigen to CD8(+) cytotoxic T cells. Marco and Signr1 specific to marginal zone macrophages (MZM), antigen-presenting cells, were also up-regulated. Flow cytometric analysis confirmed that the proportion of MZM was significantly increased by KB290 ingestion. Additionally, the over-represented Kyoto Encyclopedia of Genes and Genomes pathways among the up-regulated genes were those for natural killer (NK) cell-mediated cytotoxicity and antigen processing and presentation. The results for the selected genes associated with NK cells and CD8(+) cytotoxic T cells were confirmed by quantitative RT-PCR. These results suggest that enhanced cytotoxic activity could be caused by the activation of NK cells and/or of CD8(+) cytotoxic T cells stimulated via MHC class I presentation.

  2. A genome-wide study of preferential amplification/hybridization in microarray-based pooled DNA experiments

    PubMed Central

    Yang, H.-C.; Liang, Y.-J.; Huang, M.-C.; Li, L.-H.; Lin, C.-H.; Wu, J.-Y.; Chen, Y.-T.; Fann, C.S.J.

    2006-01-01

    Microarray-based pooled DNA methods overcome the cost bottleneck of simultaneously genotyping more than 100 000 markers for numerous study individuals. The success of such methods relies on the proper adjustment of preferential amplification/hybridization to ensure accurate and reliable allele frequency estimation. We performed a hybridization-based genome-wide single nucleotide polymorphisms (SNPs) genotyping analysis to dissect preferential amplification/hybridization. The majority of SNPs had less than 2-fold signal amplification or suppression, and the lognormal distributions adequately modeled preferential amplification/hybridization across the human genome. Comparative analyses suggested that the distributions of preferential amplification/hybridization differed among genotypes and the GC content. Patterns among different ethnic populations were similar; nevertheless, there were striking differences for a small proportion of SNPs, and a slight ethnic heterogeneity was observed. To fulfill appropriate and gratuitous adjustments, databases of preferential amplification/hybridization for African Americans, Caucasians and Asians were constructed based on the Affymetrix GeneChip Human Mapping 100 K Set. The robustness of allele frequency estimation using this database was validated by a pooled DNA experiment. This study provides a genome-wide investigation of preferential amplification/hybridization and suggests guidance for the reliable use of the database. Our results constitute an objective foundation for theoretical development of preferential amplification/hybridization and provide important information for future pooled DNA analyses. PMID:16931491

  3. Whole mitochondrial genome screening in maternally inherited non-syndromic hearing impairment using a microarray resequencing mitochondrial DNA chip.

    PubMed

    Lévêque, Marianne; Marlin, Sandrine; Jonard, Laurence; Procaccio, Vincent; Reynier, Pascal; Amati-Bonneau, Patrizia; Baulande, Sylvain; Pierron, Denis; Lacombe, Didier; Duriez, Françoise; Francannet, Christine; Mom, Thierry; Journel, Hubert; Catros, Hélène; Drouin-Garraud, Valérie; Obstoy, Marie-Françoise; Dollfus, Hélène; Eliot, Marie-Madeleine; Faivre, Laurence; Duvillard, Christian; Couderc, Remy; Garabedian, Eréa-Noël; Petit, Christine; Feldmann, Delphine; Denoyelle, Françoise

    2007-11-01

    Mitochondrial DNA (mtDNA) mutations have been implicated in non-syndromic hearing loss either as primary or as predisposing factors. As only a part of the mitochondrial genome is usually explored in deafness, its prevalence is probably under-estimated. Among 1350 families with non-syndromic sensorineural hearing loss collected through a French collaborative network, we selected 29 large families with a clear maternal lineage and screened them for known mtDNA mutations in 12S rRNA, tRNASer(UCN) and tRNALeu(UUR) genes. When no mutation could be identified, a whole mitochondrial genome screening was performed, using a microarray resequencing chip: the MitoChip version 2.0 developed by Affymetrix Inc. Known mtDNA mutations was found in nine of the 29 families, which are described in the article: five with A1555G, two with the T7511C, one with 7472insC and one with A3243G mutation. In the remaining 20 families, the resequencing Mitochip detected 258 mitochondrial homoplasmic variants and 107 potentially heteroplasmic variants. Controls were made by direct sequencing on selected fragments and showed a high sensibility of the MitoChip but a low specificity, especially for heteroplasmic variations. An original analysis on the basis of species conservation, frequency and phylogenetic investigation was performed to select the more probably pathogenic variants. The entire genome analysis allowed us to identify five additional families with a putatively pathogenic mitochondrial variant: T669C, C1537T, G8078A, G12236A and G15077A. These results indicate that the new MitoChip platform is a rapid and valuable tool for identification of new mtDNA mutations in deafness.

  4. Component retention in principal component analysis with application to cDNA microarray data

    PubMed Central

    Cangelosi, Richard; Goriely, Alain

    2007-01-01

    Shannon entropy is used to provide an estimate of the number of interpretable components in a principal component analysis. In addition, several ad hoc stopping rules for dimension determination are reviewed and a modification of the broken stick model is presented. The modification incorporates a test for the presence of an "effective degeneracy" among the subspaces spanned by the eigenvectors of the correlation matrix of the data set then allocates the total variance among subspaces. A summary of the performance of the methods applied to both published microarray data sets and to simulated data is given. This article was reviewed by Orly Alter, John Spouge (nominated by Eugene Koonin), David Horn and Roy Varshavsky (both nominated by O. Alter). PMID:17229320

  5. Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

    NASA Astrophysics Data System (ADS)

    Xie, Xin; Zhang, Xu; Yu, Bingbin; Gao, Huafang; Zhang, Huan; Fei, Weiyang

    2004-09-01

    A series of simplified protocols are developed for extracting genomic DNA from saliva by using the magnetic nanobeads as absorbents. In these protocols, both the enrichment of the target cells and the adsorption of DNA can be achieved simultaneously by our functionally modified magnetic beads in one step, and the DNA-nanobeads complex can be used as PCR templates. HLA typing based on an oligonucleotide array was conducted by hybridization with the PCR products. The result shows that the protocols are robust and sensitive.

  6. Knowledge-based image processing for on-off type DNA microarray

    NASA Astrophysics Data System (ADS)

    Kim, Jong D.; Kim, Seo K.; Cho, Jeong S.; Kim, Jongwon

    2002-06-01

    This paper addresses the image processing technique for discriminating whether the probes are hybrized with target DNA in the Human Papilloma Virus (HPV) DNA Chip designed for genotyping HPV. In addition to the probes, the HPV DNA chip has markers that always react with the sample DNA. The positions of probe-dots in the final scanned image are fixed relative to the marker-dot locations with a small variation according to the accuracy of the dotter and the scanner. The probes are duplicated 4 times for the diagnostic stability. The prior knowledges such as the maker relative distance and the duplication information of probes is integrated into the template matching technique with the normalized correlation measure. Results show that the employment of both of the prior knowledges is to simply average the template matching measures over the positions of the markers and probes. The eventual proposed scheme yields stable marker locating and probe classification.

  7. Efficient preparation of shuffled DNA libraries through recombination (Gateway) cloning.

    PubMed

    Lehtonen, Soili I; Taskinen, Barbara; Ojala, Elina; Kukkurainen, Sampo; Rahikainen, Rolle; Riihimäki, Tiina A; Laitinen, Olli H; Kulomaa, Markku S; Hytönen, Vesa P

    2015-01-01

    Efficient and robust subcloning is essential for the construction of high-diversity DNA libraries in the field of directed evolution. We have developed a more efficient method for the subcloning of DNA-shuffled libraries by employing recombination cloning (Gateway). The Gateway cloning procedure was performed directly after the gene reassembly reaction, without additional purification and amplification steps, thus simplifying the conventional DNA shuffling protocols. Recombination-based cloning, directly from the heterologous reassembly reaction, conserved the high quality of the library and reduced the time required for the library construction. The described method is generally compatible for the construction of DNA-shuffled gene libraries.

  8. Use of human cDNA microarrays for identification of differentially expressed genes in Atlantic salmon liver during Aeromonas salmonicida infection.

    PubMed

    Tsoi, Stephen C M; Cale, Jacqueline M; Bird, Ian M; Ewart, Vanya; Brown, Laura L; Douglas, Susan

    2003-01-01

    Commercially available human complementary DNA microarrays were used to compare differential expression in the livers of Atlantic salmon ( Salmo salar) infected with Aeromonas salmonicida and of healthy fish. Complementary DNA probes were prepared from total RNA isolated from livers of control salmon and infected salmon by reverse transcription in the presence of (33)P-dCTP and independently hybridized to human GENE-FILTERS GF211 microarrays. Of the 4131 known genes on the microarray, 241 spots gave clearly detectable signals using labeled RNA from the control salmon liver. Of these, 4 spots were consistently found to have a greater than 2-fold increase in infected salmon compared with controls when using the same pair of filters to generate hybridization data from triplicates. These up-regulated genes were ADP/ATP translocase (AAT2), Na(+)/K(+) ATPase, acyloxyacyl hydrolase (AOAH), and platelet-derived growth factor (PDFG-A). A BlastN search revealed an AAT2 homolog from Atlantic salmon, and a reverse transcriptase polymerase chain reaction assay using primers based on this sequence confirmed its up-regulation (approx. 1.8-fold) during early infection. This work demonstrates the feasibility of using human microarrays to facilitate the discovery of differentially expressed genes in Atlantic salmon, for which no homologous microarrays are available.

  9. Evaluation of applicability of DNA microarray-based characterization of bovine Shiga toxin-producing Escherichia coli isolates using whole genome sequence analysis.

    PubMed

    Barth, Stefanie A; Menge, Christian; Eichhorn, Inga; Semmler, Torsten; Pickard, Derek; Geue, Lutz

    2017-09-01

    We assessed the ability of a commercial DNA microarray to characterize bovine Shiga toxin-producing Escherichia coli (STEC) isolates and evaluated the results using in silico hybridization of the microarray probes within whole genome sequencing scaffolds. From a total of 69,954 reactions (393 probes with 178 isolates), 68,706 (98.2%) gave identical results by DNA microarray and in silico probe hybridization. Results were more congruent when detecting the genoserotype (209 differing results from 19,758 in total; 1.1%) or antimicrobial resistance genes (AMRGs; 141 of 26,878; 0.5%) than when detecting virulence-associated genes (VAGs; 876 of 22,072; 4.0%). Owing to the limited coverage of O-antigens by the microarray, only 37.2% of the isolates could be genoserotyped. However, the microarray proved suitable to rapidly screen bovine STEC strains for the occurrence of high numbers of VAGs and AMRGs and is suitable for molecular surveillance workflows.

  10. [An efficient method for isolation of mitochondrial DNA in wheat].

    PubMed

    Li, Wen-Qiang; Zhang, Gai-Sheng; Wang, Kui; Niu, Na; Pan, Dong-Liang

    2007-06-01

    An efficient method for isolation of mitochondrial DNA (mtDNA) from etiolated tissues of wheat was developed. The protocol consists of mitochondria isolation with differential centrifugation, Dnase I treatment, lysis with SDS and proteinase K, removing protein by TE-saturated phenol/chloroform extraction and a final RNase A treatment for obtaining mtDNA. The mtDNA samples were tested using spectrophotometry and agarose gel electrophoresis. It was proved that the mtDNA isolated by this method not only have the high yield but also structural complete, and contains no impurities, such as nuclear DNA, RNA and protein. The result showed that this high quality mtDNA can be successfully used in PCR and other genetic studies. In addition, it was found that adjusting the lysis temperature has a noticeable effect on the mtDNA yield.

  11. Massive Collection of Full-Length Complementary DNA Clones and Microarray Analyses:. Keys to Rice Transcriptome Analysis

    NASA Astrophysics Data System (ADS)

    Kikuchi, Shoshi

    2009-02-01

    Completion of the high-precision genome sequence analysis of rice led to the collection of about 35,000 full-length cDNA clones and the determination of their complete sequences. Mapping of these full-length cDNA sequences has given us information on (1) the number of genes expressed in the rice genome; (2) the start and end positions and exon-intron structures of rice genes; (3) alternative transcripts; (4) possible encoded proteins; (5) non-protein-coding (np) RNAs; (6) the density of gene localization on the chromosome; (7) setting the parameters of gene prediction programs; and (8) the construction of a microarray system that monitors global gene expression. Manual curation for rice gene annotation by using mapping information on full-length cDNA and EST assemblies has revealed about 32,000 expressed genes in the rice genome. Analysis of major gene families, such as those encoding membrane transport proteins (pumps, ion channels, and secondary transporters), along with the evolution from bacteria to higher animals and plants, reveals how gene numbers have increased through adaptation to circumstances. Family-based gene annotation also gives us a new way of comparing organisms. Massive amounts of data on gene expression under many kinds of physiological conditions are being accumulated in rice oligoarrays (22K and 44K) based on full-length cDNA sequences. Cluster analyses of genes that have the same promoter cis-elements, that have similar expression profiles, or that encode enzymes in the same metabolic pathways or signal transduction cascades give us clues to understanding the networks of gene expression in rice. As a tool for that purpose, we recently developed "RiCES", a tool for searching for cis-elements in the promoter regions of clustered genes.

  12. 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

  13. 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

  14. DNA barcoding, microarrays and next generation sequencing: recent tools for genetic diversity estimation and authentication of medicinal plants.

    PubMed

    Sarwat, Maryam; Yamdagni, Manu Mayank

    2016-01-01

    DNA barcoding, microarray technology and next generation sequencing have emerged as promising tools for the elucidation of plant genetic diversity and its conservation. They are proving to be immensely helpful in authenticating the useful medicinal plants for herbal drug preparations. These newer versions of molecular markers utilize short genetic markers in the genome to characterize the organism to a particular species. This has the potential not only to classify the known and yet unknown species but also has a promising future to link the medicinally important plants according to their properties. The newer trends being followed in DNA chips and barcoding pave the way for a future with many different possibilities. Several of these possibilities might be: characterization of unknown species in a considerably less time than usual, identification of newer medicinal properties possessed by the species and also updating the data of the already existing but unnoticed properties. This can assist us to cure many different diseases and will also generate novel opportunities in medicinal drug delivery and targeting.

  15. A novel brassinolide-enhanced gene identified by cDNA microarray is involved in the growth of rice.

    PubMed

    Yang, Guangxiao; Matsuoka, Makoto; Iwasaki, Yukimoto; Komatsu, Setsuko

    2003-07-01

    Brassinosteroids (BRs) are growth-promoting natural substances required for normal plant growth and development. To understand the molecular mechanism of BR action, a cDNA microarray containing 1265 rice genes was analyzed for expression differences in rice lamina joint treated with brassinolide (BL). A novel BL-enhanced gene, designated OsBLE2, was identified and cloned. The full-length cDNA is 3243 bp long, encoding a predicted polypeptide of 761 amino acid residues and nine possible transmembrane regions. OsBLE2 expression was most responsive to BL in the lamina joint and leaf sheath in rice seedlings. Besides, auxin and gibberellins also increased its expression. OsBLE2 expressed more, as revealed by in situ hybridization, in vascular bundles and root primordia, where the cells are actively undergoing division, elongation, and differentiation. Transgenic rice expressing antisense OsBLE2 exhibits various degrees of repressed growth. BL could not enhance its expression in transgenic rice expressing antisense BRI1, a BR receptor, indicating that BR signaling to the enhanced expression of OsBLE2 is through BRI1 . BL effect in the d1 mutant rice was much weaker than that in its wild-type control, indicating that heterotrimeric G protein may be a component of BRs signaling. These results suggest that OsBLE2 is involved in BL-regulated growth and development processes in rice.

  16. Investigating the Biological Significance of Metallointercalators with cDNA Microarrays

    NASA Astrophysics Data System (ADS)

    Wright, Elise P.; Lyons, Victoria; Wang, Shaoyu; Higgins, Vincent J.

    The double helix coded sequence of nucleotide bases with its protective sugar phosphate backbone forms deoxyribonucleic acid (DNA) which is the genetic blueprint of all living things. All the information required for the development, operation and maintenance of cells is contained in a sequence of adenine (A), thymine (T), cytosine (C) and guanine (G) bases, where adenine is paired with thymine and cytosine is paired with guanine [1]. The DNA sequence is made usable by transcription of the nucleotide sequence into single stranded messenger RNA (mRNA).. This means that the four member nucleic acid base code sequence is converted into a 22 member amino acid code [2].

  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. DNA Microarray for Genotyping Antibiotic Resistance Determinants in Acinetobacter baumannii Clinical Isolates

    PubMed Central

    Dally, Simon; Lemuth, Karin; Kaase, Martin; Rupp, Steffen; Knabbe, Cornelius

    2013-01-01

    In recent decades, Acinetobacter baumannii has emerged as an organism of great concern due to its ability to accumulate antibiotic resistance. In order to improve the diagnosis of resistance determinants in A. baumannii in terms of lead time and accuracy, we developed a microarray that can be used to detect 91 target sequences associated with antibiotic resistance within 4 h from bacterial culture to result. The array was validated with 60 multidrug-resistant strains of A. baumannii in a blinded, prospective study. The results were compared to phenotype results determined by the automated susceptibility testing system VITEK2. Antibiotics considered were piperacillin-tazobactam, ceftazidime, imipenem, meropenem, trimethoprim-sulfamethoxazole, amikacin, gentamicin, tobramycin, ciprofloxacin, and tigecycline. The average positive predictive value, negative predictive value, sensitivity, and specificity were 98, 98, 99, and 94%, respectively. For carbapenemase genes, the array results were compared to singleplex PCR results provided by the German National Reference Center for Gram-Negative Pathogens, and results were in complete concordance. The presented array is able to detect all relevant resistance determinants of A. baumannii in parallel. The short handling time of 4 h from culture to result helps to provide fast results in order to initiate adequate anti-infective therapy for critically ill patients. Another application would be data acquisition for epidemiologic surveillance. PMID:23856783

  19. Probing plant-pathogen interactions and downstream defense signaling using DNA microarrays.

    PubMed

    Wan, Jinrong; Dunning, F Mark; Bent, Andrew F

    2002-11-01

    The interaction between a plant and a pathogen activates a wide variety of defense responses. The recent development of microarray-based expression profiling methods, together with the availability of genomic and/or EST (expressed sequence tag) sequence data for some plant species, has allowed significant progress in the characterization of plant pathogenesis-related responses. The small number of expression profiling studies completed to date have already identified an amazing number of genes that had not previously been implicated in plant defense. Some of these genes can be associated with defense signal transduction or antimicrobial action, but the functional contribution of many others remains uncertain. Initial expression profiling work has also revealed similarities and distinctions between different defense signaling pathways, and cross-talk (both overlap and interference) between pathogenesis-related responses and plant responses to other stresses. Potential transcriptional cis-regulatory elements upstream of co-regulated genes can also be identified. Whole-genome arrays are only now becoming available, and many interactions remain to be studied (e.g. different pathogen species, plant genotypes, mutants, time-points after infection). Expression profiling technologies, in combination with other genomic tools, will have a substantial impact on our understanding of plant-pathogen interactions and defense signaling pathways.

  20. Novel light-regulated genes in Trichoderma atroviride: a dissection by cDNA microarrays.

    PubMed

    Rosales-Saavedra, T; Esquivel-Naranjo, E U; Casas-Flores, S; Martínez-Hernández, P; Ibarra-Laclette, E; Cortes-Penagos, C; Herrera-Estrella, A

    2006-11-01

    The influence of light on living organisms is critical, not only because of its importance as the main source of energy for the biosphere, but also due to its capacity to induce changes in the behaviour and morphology of nearly all forms of life. The common soil fungus Trichoderma atroviride responds to blue light in a synchronized manner, in time and space, by forming a ring of green conidia at what had been the colony perimeter at the time of exposure (photoconidiation). A putative complex formed by the BLR-1 and BLR-2 proteins in T. atroviride appears to play an essential role as a sensor and transcriptional regulator in photoconidiation. Expression analyses using microarrays containing 1438 unigenes were carried out in order to identify early light response genes. It was found that 2.8 % of the genes were light responsive: 2 % induced and 0.8 % repressed. Expression analysis in blr deletion mutants allowed the demonstration of the occurrence of two types of light responses, a blr-independent response in addition to the expected blr-dependent one, as well as a new role of the BLR proteins in repression of transcription. Exposure of T. atroviride to continuous light helped to establish that the light-responsive genes are subject to photoadaptation. Finally, evidence is provided of red-light-regulated gene expression and a possible crosstalk between the blue and red light signalling pathways.

  1. Whole-Genome DNA Microarray Analysis of a Hyperthermophile and an Archaeon: Pyrococcus furiosus Grown on Carbohydrates or Peptides

    PubMed Central

    Schut, Gerrit J.; Brehm, Scott D.; Datta, Susmita; Adams, Michael W. W.

    2003-01-01

    The first complete-genome DNA microarray was constructed for a hyperthermophile or a nonhalophilic archaeon by using the 2,065 open reading frames (ORFs) that have been annotated in the genome of Pyrococcus furiosus (optimal growth temperature, 100°C). This was used to determine relative transcript levels in cells grown at 95°C with either peptides or a carbohydrate (maltose) used as the primary carbon source. Approximately 20% (398 of 2065) of the ORFs did not appear to be significantly expressed under either growth condition. Of the remaining 1,667 ORFs, the expression of 125 of them (8%) differed by more than fivefold between the two cultures, and 82 of the 125 (65%) appear to be part of operons, indicating extensive coordinate regulation. Of the 27 operons that are regulated, 5 of them encode (conserved) hypothetical proteins. A total of 18 operons are up-regulated (greater than fivefold) in maltose-grown cells, including those responsible for maltose transport and for the biosynthesis of 12 amino acids, of ornithine, and of citric acid cycle intermediate products. A total of nine operons are up-regulated (greater than fivefold) in peptide-grown cells, including those encoding enzymes involved in the production of acyl and aryl acids and 2-ketoacids, which are used for energy conservation. Analyses of the spent growth media confirmed the production of branched-chain and aromatic acids during growth on peptides. In addition, six nonlinked enzymes in the pathways of sugar metabolism were regulated more than fivefold—three in maltose-grown cells that are unique to the unusual glycolytic pathway and three in peptide-grown cells that are unique to gluconeogenesis. The catalytic activities of 16 metabolic enzymes whose expression appeared to be highly regulated in the two cell types correlated very well with the microarray data. The degree of coordinate regulation revealed by the microarray data was unanticipated and shows that P. furiosus can readily adapt to a

  2. Profiling Ethylene-Responsive Genes Expressed in the Latex of the Mature Virgin Rubber Trees Using cDNA Microarray

    PubMed Central

    Nie, Zhiyi; Kang, Guijuan; Duan, Cuifang; Li, Yu; Dai, Longjun; Zeng, Rizhong

    2016-01-01

    Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2,973 unique genes (probes) was first developed and used to analyze the gene expression changes in the latex of the mature virgin rubber trees after ethephon treatment at three different time-points: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ –2 (q-value < 0.05) in ethephon-treated rubber trees compared with control trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. The 163 ethylene-responsive genes were involved in several biological processes including organic substance metabolism, cellular metabolism, primary metabolism, biosynthetic process, cellular response to stimulus and stress. The presented data suggest that the laticifer water circulation, production and scavenging of reactive oxygen species, sugar metabolism, and assembly and depolymerization of the latex actin cytoskeleton might play important roles in ethylene-induced increase of latex production. The results may provide useful insights into understanding the molecular mechanism underlying the effect of ethylene on latex metabolism of H. brasiliensis. PMID:26985821

  3. Sex-Related Differences in Rat Choroid Plexus and Cerebrospinal Fluid: A cDNA Microarray and Proteomic Analysis.

    PubMed

    Quintela, T; Marcelino, H; Deery, M J; Feret, R; Howard, J; Lilley, K S; Albuquerque, T; Gonçalves, I; Duarte, A C; Santos, C R A

    2016-01-01

    The choroid plexus (CP) epithelium is a unique structure in the brain that forms an interface between the peripheral blood and the cerebrospinal fluid (CSF), which is mostly produced by the CP itself. Because the CP transcriptome is regulated by the sex hormone background, the present study compared gene/protein expression profiles in the CP and CSF from male and female rats aiming to better understand sex-related differences in CP functions and brain physiology. We used data previously obtained by cDNA microarrays to compare the CP transcriptome between male and female rats, and complemented these data with the proteomic analysis of the CSF of castrated and sham-operated males and females. Microarray analysis showed that 17 128 and 17 002 genes are expressed in the male and female CP, which allowed the functional annotation of 141 and 134 pathways, respectively. Among the most expressed genes, canonical pathways associated with mitochondrial dysfunctions and oxidative phosphorylation were the most prominent, whereas the most relevant molecular and cellular functions annotated were protein synthesis, cellular growth and proliferation, cell death and survival, molecular transport, and protein trafficking. No significant differences were found between males and females regarding these pathways. Seminal functions of the CP differentially regulated between sexes were circadian rhythm signalling, as well as several canonical pathways related to stem cell differentiation, metabolism and the barrier function of the CP. The proteomic analysis identified five down-regulated proteins in the CSF samples from male rats compared to females and seven proteins exhibiting marked variation in the CSF of gonadectomised males compared to sham animals, whereas no differences were found between sham and ovariectomised females. These data clearly show sex-related differences in CP gene expression and CSF protein composition that may impact upon neurological diseases.

  4. Visualization and Curve-Parameter Estimation Strategies for Efficient Exploration of Phenotype Microarray Kinetics

    PubMed Central

    Vaas, Lea A. I.; Sikorski, Johannes; Michael, Victoria; Göker, Markus; Klenk, Hans-Peter

    2012-01-01

    Background The Phenotype MicroArray (OmniLog® PM) system is able to simultaneously capture a large number of phenotypes by recording an organism's respiration over time on distinct substrates. This technique targets the object of natural selection itself, the phenotype, whereas previously addressed ‘-omics’ techniques merely study components that finally contribute to it. The recording of respiration over time, however, adds a longitudinal dimension to the data. To optimally exploit this information, it must be extracted from the shapes of the recorded curves and displayed in analogy to conventional growth curves. Methodology The free software environment R was explored for both visualizing and fitting of PM respiration curves. Approaches using either a model fit (and commonly applied growth models) or a smoothing spline were evaluated. Their reliability in inferring curve parameters and confidence intervals was compared to the native OmniLog® PM analysis software. We consider the post-processing of the estimated parameters, the optimal classification of curve shapes and the detection of significant differences between them, as well as practically relevant questions such as detecting the impact of cultivation times and the minimum required number of experimental repeats. Conclusions We provide a comprehensive framework for data visualization and parameter estimation according to user choices. A flexible graphical representation strategy for displaying the results is proposed, including 95% confidence intervals for the estimated parameters. The spline approach is less prone to irregular curve shapes than fitting any of the considered models or using the native PM software for calculating both point estimates and confidence intervals. These can serve as a starting point for the automated post-processing of PM data, providing much more information than the strict dichotomization into positive and negative reactions. Our results form the basis for a freely

  5. Comparison of DNA Microarray, Loop-Mediated Isothermal Amplification (LAMP) and Real-Time PCR with DNA Sequencing for Identification of Fusarium spp. Obtained from Patients with Hematologic Malignancies.

    PubMed

    de Souza, Marcela; Matsuzawa, Tetsuhiro; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Busso-Lopes, Ariane Fidelis; Levin, Anna Sara Shafferman; Schreiber, Angélica Zaninelli; Mikami, Yuzuru; Gonoi, Tohoru; Kamei, Katsuhiko; Moretti, Maria Luiza; Trabasso, Plínio

    2017-03-21

    The performance of three molecular biology techniques, i.e., DNA microarray, loop-mediated isothermal amplification (LAMP), and real-time PCR were compared with DNA sequencing for properly identification of 20 isolates of Fusarium spp. obtained from blood stream as etiologic agent of invasive infections in patients with hematologic malignancies. DNA microarray, LAMP and real-time PCR identified 16 (80%) out of 20 samples as Fusarium solani species complex (FSSC) and four (20%) as Fusarium spp. The agreement among the techniques was 100%. LAMP exhibited 100% specificity, while DNA microarray, LAMP and real-time PCR showed 100% sensitivity. The three techniques had 100% agreement with DNA sequencing. Sixteen isolates were identified as FSSC by sequencing, being five Fusarium keratoplasticum, nine Fusarium petroliphilum and two Fusarium solani. On the other hand, sequencing identified four isolates as Fusarium non-solani species complex (FNSSC), being three isolates as Fusarium napiforme and one isolate as Fusarium oxysporum. Finally, LAMP proved to be faster and more accessible than DNA microarray and real-time PCR, since it does not require a thermocycler. Therefore, LAMP signalizes as emerging and promising methodology to be used in routine identification of Fusarium spp. among cases of invasive fungal infections.

  6. DNA Microarray Analysis of Anaerobic Methanosarcina Barkeri Reveals Responses to Heat Shock and Air Exposure

    SciTech Connect

    Zhang, Weiwen; Culley, David E.; Nie, Lei; Brockman, Fred J.

    2006-04-08

    Summary Methanosarcina barkeri can grow only under strictly anoxic conditions because enzymes in methane formation pathways of are very oxygen sensitive. However, it has been determined that M. barkeri can survive oxidative stress. To obtain further knowledge of cellular changes in M. barkeri in responsive to oxidative and other environmental stress, a first whole-genome M. barkeri oligonucleotide microarray was constructed according to the draft genome sequence that contains 5072 open reading frames (ORFs) and was used to investigate the global transcriptomic response of M. barkeri to oxidative stress and heat shock. The result showed that 552 genes in the M. barkeri genome were responsive to oxidative stress, while 177 genes responsive to heat-shock, respectively using a cut off of 2.5 fold change. Among them, 101 genes were commonly responsive to both environmental stimuli. In addition to various house-keeping genes, large number of functionally unknown genes (38-57% of total responsive genes) was regulated by both stress conditions. The result showed that the Hsp60 (GroEL) system, which was previously thought not present in archaea, was up-regulated and may play important roles in protein biogenesis in responsive to heat shock in M. barkeri. No gene encoding superoxide dismutase, catalase, nonspecific peroxidases or thioredoxin reductase was differentially expressed when subjected to oxidative stress. Instead, significant downregulation of house-keeping genes and up-regulation of genes encoding transposase was found in responsive to oxidative stress, suggesting that M. barkeri may be adopting a passive protective mechanism by slowing down cellular activities to survive the stress rather than activating a means against oxidative stress.

  7. cDNA Microarray Analysis Revealing Candidate Biomineralization Genes of the Pearl Oyster, Pinctada fucata martensii.

    PubMed

    Shi, Yaohua; Zheng, Xing; Zhan, Xin; Wang, Aimin; Gu, Zhifeng

    2016-06-01

    Biomineralization is a common biological phenomenon resulting in strong tissue, such as bone, tooth, and shell. Pinctada fucata martensii is an ideal animal for the study of biomineralization. Here, microarray technique was used to identify biomineralization gene in mantle edge (ME), mantle center (MC), and both ME and MC (ME-MC) for this pearl oyster. Results revealed that 804, 306, and 1127 contigs expressed at least three times higher in ME, MC, and ME-MC as those in other tissues. Blast against non-redundant database showed that 130 contigs (16.17 %), 53 contigs (17.32 %), and 248 contigs (22.01 %) hit reference genes (E ≤ -10), among which 91 contigs, 48 contigs, and 168 contigs could be assigned to 32, 26, and 63 biomineralization genes in tissue of ME, MC, and ME-MC at a threshold of 3 times upregulated expression level. The ratios of biomineralization contigs to homologous contigs were similar at 3 times, 10 times, and 100 times of upregulated expression level in either ME, MC, or ME-MC. Moreover, the ratio of biomineralization contigs was highest in MC. Although mRNA distribution characters were similar to those in other studies for eight biomineralization genes of PFMG3, Pif, nacrein, MSI7, mantle gene 6, Pfty1, prismin, and the shematrin, most biomineralization genes presented different expression profiles from existing reports. These results provided massive fundamental information for further study of biomineralization gene function, and it may be helpful for revealing gene nets of biomineralization and the molecular mechanisms underlining formation of shell and pearl for the oyster.

  8. Screening for key genes associated with atopic dermatitis with DNA microarrays.

    PubMed

    Zhang, Zhong-Kui; Yang, Yong; Bai, Shu-Rong; Zhang, Gui-Zhen; Liu, Tai-Hua; Zhou, Zhou; Wang, Chun-Mei; Tang, Li-Jun; Wang, Jun; He, Si-Xian

    2014-03-01

    The aim of the present study was to identify key genes associated with atopic dermatitis (AD) using microarray data and bioinformatic analyses. The dataset GSE6012, downloaded from the Gene Expression Omnibus (GEO) database, contains gene expression data from 10 AD skin samples and 10 healthy skin samples. Following data preprocessing, differentially expressed genes (DEGs) were identified using the limma package of the R project. Interaction networks were constructed comprising DEGs that showed a degree of node of >3, >5 and >10, using the Osprey software. Functional enrichment and pathway enrichment analysis of the network comprising all DEGs and of the network comprising DEGs with a high degree of node, were performed with the DAVID and WebGestalt toolkits, respectively. A total of 337 DEGs were identified. The functional enrichment analysis revealed that the list of DEGs was significantly enriched for proteins related to epidermis development (P=2.95E-07), including loricrin (LOR), keratin 17 (KRT17), small proline-rich repeat proteins (SPRRs) and involucrin (IVL). The chemokine signaling pathway was the most significantly enriched pathway (P=0.0490978) in the network of all DEGs and in the network consisting of high degree‑node DEGs (>10), which comprised the genes coding for chemokine receptor 7 (CCR7), chemokine ligand (CCL19), signal transducer and activator of transcription 1 (STAT1), and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1). In conclusion, the list of AD-associated proteins identified in this study, including LOR, KRT17, SPRRs, IVL, CCR7, CCL19, PIK3R1 and STAT1 may prove useful for the development of methods to treat AD. From these proteins, PIK3R1 and KRT17 are novel and promising targets for AD therapy.

  9. DNA Microarray Analysis of the Hyperthermophilic Archaeon Pyrococcus furiosus: Evidence for a New Type of Sulfur-Reducing Enzyme Complex

    PubMed Central

    Schut, Gerrit J.; Zhou, Jizhong; Adams, Michael W. W.

    2001-01-01

    DNA microarrays were constructed by using 271 open reading frame (ORFs) from the genome of the archaeon Pyrococcus furiosus. They were used to investigate the effects of elemental sulfur (S°) on the levels of gene expression in cells grown at 95°C with maltose as the carbon source. The ORFs included those that are proposed to encode proteins mainly involved in the pathways of sugar and peptide catabolism, in the metabolism of metals, and in the biosynthesis of various cofactors, amino acids, and nucleotides. The expression of 21 ORFs decreased by more than fivefold when cells were grown with S° and, of these, 18 encode subunits associated with three different hydrogenase systems. The remaining three ORFs encode homologs of ornithine carbamoyltransferase and HypF, both of which appear to be involved in hydrogenase biosynthesis, as well as a conserved hypothetical protein. The expression of two previously uncharacterized ORFs increased by more than 25-fold when cells were grown with S°. Their products, termed SipA and SipB (for sulfur-induced proteins), are proposed to be part of a novel S°-reducing, membrane-associated, iron-sulfur cluster-containing complex. Two other previously uncharacterized ORFs encoding a putative flavoprotein and a second FeS protein were upregulated more than sixfold in S°-grown cells, and these are also thought be involved in S° reduction. Four ORFs that encode homologs of proteins involved in amino acid metabolism were similarly upregulated in S°-grown cells, a finding consistent with the fact that growth on peptides is a S°-dependent process. An ORF encoding a homolog of the eukaryotic rRNA processing protein, fibrillarin, was also upregulated sixfold in the presence of S°, although the reason for this is as yet unknown. Of the 20 S°-independent ORFs that are the most highly expressed (at more than 20 times the detection limit), 12 of them represent enzymes purified from P. furiosus, but none of the products of the 34 S

  10. A whole genome SNP genotyping by DNA microarray and candidate gene association study for kidney stone disease.

    PubMed

    Rungroj, Nanyawan; Nettuwakul, Choochai; Sudtachat, Nirinya; Praditsap, Oranud; Sawasdee, Nunghathai; Sritippayawan, Suchai; Chuawattana, Duangporn; Yenchitsomanus, Pa-Thai

    2014-05-02

    Kidney stone disease (KSD) is a complex disorder with unknown etiology in majority of the patients. Genetic and environmental factors may cause the disease. In the present study, we used DNA microarray to genotype single nucleotide polymorphisms (SNP) and performed candidate gene association analysis to determine genetic variations associated with the disease. A whole genome SNP genotyping by DNA microarray was initially conducted in 101 patients and 105 control subjects. A set of 104 candidate genes reported to be involved in KSD, gathered from public databases and candidate gene association study databases, were evaluated for their variations associated with KSD. Altogether 82 SNPs distributed within 22 candidate gene regions showed significant differences in SNP allele frequencies between the patient and control groups (P < 0.05). Of these, 4 genes including BGLAP, AHSG, CD44, and HAO1, encoding osteocalcin, fetuin-A, CD44-molecule and glycolate oxidase 1, respectively, were further assessed for their associations with the disease because they carried high proportion of SNPs with statistical differences of allele frequencies between the patient and control groups within the gene. The total of 26 SNPs showed significant differences of allele frequencies between the patient and control groups and haplotypes associated with disease risk were identified. The SNP rs759330 located 144 bp downstream of BGLAP where it is a predicted microRNA binding site at 3'UTR of PAQR6 - a gene encoding progestin and adipoQ receptor family member VI, was genotyped in 216 patients and 216 control subjects and found to have significant differences in its genotype and allele frequencies (P = 0.0007, OR 2.02 and P = 0.0001, OR 2.02, respectively). Our results suggest that these candidate genes are associated with KSD and PAQR6 comes into our view as the most potent candidate since associated SNP rs759330 is located in the miRNA binding site and may affect mRNA expression

  11. Photoresponsive Supramolecular Complexes as Efficient DNA Regulator

    PubMed Central

    Cheng, Hong-Bo; Zhang, Ying-Ming; Xu, Chao; Liu, Yu

    2014-01-01

    Two supramolecular complexes of trans-1⊂CB[8] and trans-2⊂CB[8] were successfully achieved by the controlled selective complexation process of cucurbit[8]uril (CB[8]) with hetero-guest pair containing azobenzene and bispyridinium moieties in aqueous solution, exhibiting the reversibly light-driven movements of CB[8] upon the photocontrollable isomerization of azophenyl axle components. Significantly, the obtained bistable supramolecular complexes and their corresponding [2]pseudorotaxanes could act as a promising concentrator and cleavage agent to regulate the binding behaviors with DNA molecules. PMID:24572680

  12. CD14 and Complement Crosstalk and Largely Mediate the Transcriptional Response to Escherichia coli in Human Whole Blood as Revealed by DNA Microarray

    PubMed Central

    Lau, Corinna; Nygård, Ståle; Fure, Hilde; Olstad, Ole Kristoffer; Holden, Marit; Lappegård, Knut Tore; Brekke, Ole-Lars; Espevik, Terje; Hovig, Eivind; Mollnes, Tom Eirik

    2015-01-01

    Systemic inflammation like in sepsis is still lacking specific diagnostic markers and effective therapeutics. The first line of defense against intruding pathogens and endogenous damage signals is pattern recognition by e.g., complement and Toll-like receptors (TLR). Combined inhibition of a key complement component (C3 and C5) and TLR-co-receptor CD14 has been shown to attenuate certain systemic inflammatory responses. Using DNA microarray and gene annotation analyses, we aimed to decipher the effect of combined inhibition of C3 and CD14 on the transcriptional response to bacterial challenge in human whole blood. Importantly, combined inhibition reversed the transcriptional changes of 70% of the 2335 genes which significantly responded to heat-inactivated Escherichia coli by on average 80%. Single inhibition was less efficient (p<0.001) but revealed a suppressive effect of C3 on 21% of the responding genes which was partially counteracted by CD14. Furthermore, CD14 dependency of the Escherichia coli-induced response was increased in C5-deficient compared to C5-sufficient blood. The observed crucial distinct and synergistic roles for complement and CD14 on the transcriptional level correspond to their broad impact on the inflammatory response in human blood, and their combined inhibition may become inevitable in the early treatment of acute systemic inflammation. PMID:25706641

  13. Development and utility of the FDA 'GutProbe' DNA microarray for identification, genotyping and metagenomic analysis of commercially available probiotics.

    PubMed

    Patro, J N; Ramachandran, P; Lewis, J L; Mammel, M K; Barnaba, T; Pfeiler, E A; Elkins, C A

    2015-06-01

    Lactic acid bacteria are beneficial microbes added to many food products and dietary supplements for their purported health benefits. Proper identification of bacteria is important to assess safety as well as proper product labelling. A custom microarray (FDA GutProbe) was developed to verify accurate labelling in commercial dietary supplements. Strain-specific attribution was achieved with GutProbe array which contains genes from the most commonly found species in probiotic supplements and food ingredients. Applied utility of the array was assessed with direct from product DNA hybridization to determine (i) if identification of multiple strains in one sample can be conducted and (ii) if any lot-to-lot variations exist with eight probiotics found on the US market. GutProbe is a useful tool in identifying a mixture of microbials in probiotics and did reveal some product variations. In addition, the array is able to identify lot-to-lot differences in these products. These strain level attribution may be useful for routine monitoring of batch variation as part of a 'Good Manufacturing Practices' process. The FDA GutProbe is an efficient and reliable platform to identify the presence of microbial ingredients and determining microbe differences in dietary supplements. The GutProbe is a fast, rapid method for direct community profiling or food matrix sampling. Published 2015. This article is a U.S. Government work and is in the public domain in the USA.

  14. The influence of substrate on DNA transfer and extraction efficiency.

    PubMed

    Verdon, Timothy J; Mitchell, R John; van Oorschot, Roland A H<