WorldWideScience

Sample records for dna microarrays evidence

  1. DNA Microarray Technology

    Science.gov (United States)

    Skip to main content DNA Microarray Technology Enter Search Term(s): Español Research Funding An Overview Bioinformatics Current Grants Education and Training Funding Extramural Research News Features Funding Divisions Funding ...

  2. DNA Microarray Technology; TOPICAL

    International Nuclear Information System (INIS)

    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

  3. DNA microarrays : a molecular cloning manual

    National Research Council Canada - National Science Library

    Sambrook, Joseph; Bowtell, David

    2002-01-01

    .... DNA Microarrays provides authoritative, detailed instruction on the design, construction, and applications of microarrays, as well as comprehensive descriptions of the software tools and strategies...

  4. Microarrays (DNA Chips) for the Classroom Laboratory

    Science.gov (United States)

    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…

  5. Radioactive cDNA microarray in neurospsychiatry

    International Nuclear Information System (INIS)

    Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon

    2003-01-01

    Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

  6. Radioactive cDNA microarray in neurospsychiatry

    Energy Technology Data Exchange (ETDEWEB)

    Choe, Jae Gol; Shin, Kyung Ho; Lee, Min Soo; Kim, Meyoung Kon [Korea University Medical School, Seoul (Korea, Republic of)

    2003-02-01

    Microarray technology allows the simultaneous analysis of gene expression patterns of thousands of genes, in a systematic fashion, under a similar set of experimental conditions, thus making the data highly comparable. In some cases arrays are used simply as a primary screen leading to downstream molecular characterization of individual gene candidates. In other cases, the goal of expression profiling is to begin to identify complex regulatory networks underlying developmental processes and disease states. Microarrays were originally used with cell lines or other simple model systems. More recently, microarrays have been used in the analysis of more complex biological tissues including neural systems and the brain. The application of cDNA arrays in neuropsychiatry has lagged behind other fields for a number of reasons. These include a requirement for a large amount of input probe RNA in fluorescent-glass based array systems and the cellular complexity introduced by multicellular brain and neural tissues. An additional factor that impacts the general use of microarrays in neuropsychiatry is the lack of availability of sequenced clone sets from model systems. While human cDNA clones have been widely available, high quality rat, mouse, and drosophilae, among others are just becoming widely available. A final factor in the application of cDNA microarrays in neuropsychiatry is cost of commercial arrays. As academic microarray facilitates become more commonplace custom made arrays will become more widely available at a lower cost allowing more widespread applications. In summary, microarray technology is rapidly having an impact on many areas of biomedical research. Radioisotope-nylon based microarrays offer alternatives that may in some cases be more sensitive, flexible, inexpensive, and universal as compared to other array formats, such as fluorescent-glass arrays. In some situations of limited RNA or exotic species, radioactive membrane microarrays may be the most

  7. DNA Microarrays in Comparative Genomics and Transcriptomics

    DEFF Research Database (Denmark)

    Willenbrock, Hanni

    2007-01-01

    at identifying the exact breakpoints where DNA has been gained or lost. In this thesis, three popular methods are compared and a realistic simulation model is presented for generating artificial data with known breakpoints and known DNA copy number. By using simulated data, we obtain a realistic evaluation......During the past few years, innovations in the DNA sequencing technology has led to an explosion in available DNA sequence information. This has revolutionized biological research and promoted the development of high throughput analysis methods that can take advantage of the vast amount of sequence...... data. For this, the DNA microarray technology has gained enormous popularity due to its ability to measure the presence or the activity of thousands of genes simultaneously. Microarrays for high throughput data analyses are not limited to a few organisms but may be applied to everything from bacteria...

  8. Identifying Fishes through DNA Barcodes and Microarrays.

    Directory of Open Access Journals (Sweden)

    Marc Kochzius

    2010-09-01

    Full Text Available 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.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.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.

  9. Gene Expression Analysis Using Agilent DNA Microarrays

    DEFF Research Database (Denmark)

    Stangegaard, Michael

    2009-01-01

    Hybridization of labeled cDNA to microarrays is an intuitively simple and a vastly underestimated process. If it is not performed, optimized, and standardized with the same attention to detail as e.g., RNA amplification, information may be overlooked or even lost. Careful balancing of the amount ...

  10. Comparing transformation methods for DNA microarray data

    NARCIS (Netherlands)

    Thygesen, Helene H.; Zwinderman, Aeilko H.

    2004-01-01

    Background: When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include

  11. Discovery of distinctive gene expression profiles in rheumatoid synovium using cDNA microarray technology: evidence for the existence of multiple pathways of tissue destruction and repair.

    NARCIS (Netherlands)

    Kraan, TC van der Pouw; Gaalen, van FA; Huizinga, T.W.; Pieterman, E; Breedveld, F.C.; Verweij, C.L.

    2003-01-01

    Rheumatoid arthritis (RA) is a heterogeneous disease. We used cDNA microarray technology to subclassify RA patients and disclose disease pathways in rheumatoid synovium. Hierarchical clustering of gene expression data identified two main groups of tissues (RA-I and RA-II). A total of 121 genes were

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

    Science.gov (United States)

    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. Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray

    Directory of Open Access Journals (Sweden)

    Nobumasa Hitoshi

    2007-04-01

    Full Text Available Abstract Background Mycotoxins are fungal secondary metabolites commonly present in feed and food, and are widely regarded as hazardous contaminants. Citrinin, one of the very well known mycotoxins that was first isolated from Penicillium citrinum, is produced by more than 10 kinds of fungi, and is possibly spread all over the world. However, the information on the action mechanism of the toxin is limited. Thus, we investigated the citrinin-induced genomic response for evaluating its toxicity. Results Citrinin inhibited growth of yeast cells at a concentration higher than 100 ppm. We monitored the citrinin-induced mRNA expression profiles in yeast using the ORF DNA microarray and Oligo DNA microarray, and the expression profiles were compared with those of the other stress-inducing agents. Results obtained from both microarray experiments clustered together, but were different from those of the mycotoxin patulin. The oxidative stress response genes – AADs, FLR1, OYE3, GRE2, and MET17 – were significantly induced. In the functional category, expression of genes involved in "metabolism", "cell rescue, defense and virulence", and "energy" were significantly activated. In the category of "metabolism", genes involved in the glutathione synthesis pathway were activated, and in the category of "cell rescue, defense and virulence", the ABC transporter genes were induced. To alleviate the induced stress, these cells might pump out the citrinin after modification with glutathione. While, the citrinin treatment did not induce the genes involved in the DNA repair. Conclusion Results from both microarray studies suggest that citrinin treatment induced oxidative stress in yeast cells. The genotoxicity was less severe than the patulin, suggesting that citrinin is less toxic than patulin. The reproducibility of the expression profiles was much better with the Oligo DNA microarray. However, the Oligo DNA microarray did not completely overcome cross

  14. Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution

    DEFF Research Database (Denmark)

    Novak, Jaroslav P; Kim, Seon-Young; Xu, Jun

    2006-01-01

    BACKGROUND: DNA microarrays are a powerful technology that can provide a wealth of gene expression data for disease studies, drug development, and a wide scope of other investigations. Because of the large volume and inherent variability of DNA microarray data, many new statistical methods have...

  15. Comparing transformation methods for DNA microarray data

    Directory of Open Access Journals (Sweden)

    Zwinderman Aeilko H

    2004-06-01

    Full Text Available Abstract Background When DNA microarray data are used for gene clustering, genotype/phenotype correlation studies, or tissue classification the signal intensities are usually transformed and normalized in several steps in order to improve comparability and signal/noise ratio. These steps may include subtraction of an estimated background signal, subtracting the reference signal, smoothing (to account for nonlinear measurement effects, and more. Different authors use different approaches, and it is generally not clear to users which method they should prefer. Results We used the ratio between biological variance and measurement variance (which is an F-like statistic as a quality measure for transformation methods, and we demonstrate a method for maximizing that variance ratio on real data. We explore a number of transformations issues, including Box-Cox transformation, baseline shift, partial subtraction of the log-reference signal and smoothing. It appears that the optimal choice of parameters for the transformation methods depends on the data. Further, the behavior of the variance ratio, under the null hypothesis of zero biological variance, appears to depend on the choice of parameters. Conclusions The use of replicates in microarray experiments is important. Adjustment for the null-hypothesis behavior of the variance ratio is critical to the selection of transformation method.

  16. Uses of Dendrimers for DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Majoral

    2006-08-01

    Full Text Available Biosensors such as DNA microarrays and microchips are gaining an increasingimportance in medicinal, forensic, and environmental analyses. Such devices are based onthe detection of supramolecular interactions called hybridizations that occur betweencomplementary oligonucleotides, one linked to a solid surface (the probe, and the other oneto be analyzed (the target. This paper focuses on the improvements that hyperbranched andperfectly defined nanomolecules called dendrimers can provide to this methodology. Twomain uses of dendrimers for such purpose have been described up to now; either thedendrimer is used as linker between the solid surface and the probe oligonucleotide, or thedendrimer is used as a multilabeled entity linked to the target oligonucleotide. In the firstcase 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 smallquantities of biological entities.

  17. The application of DNA microarrays in gene expression analysis

    NARCIS (Netherlands)

    Hal, van N.L.W.; Vorst, O.; Houwelingen, van A.M.M.L.; Kok, E.J.; Peijnenburg, A.A.C.M.; Aharoni, A.; Tunen, van A.J.; Keijer, J.

    2000-01-01

    DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed.

  18. Microarray of DNA probes on carboxylate functional beads surface

    Institute of Scientific and Technical Information of China (English)

    黄承志; 李原芳; 黄新华; 范美坤

    2000-01-01

    The microarray of DNA probes with 5’ -NH2 and 5’ -Tex/3’ -NH2 modified terminus on 10 um carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) is characterized in the preseni paper. it was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentra-tion of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

  19. Microarray of DNA probes on carboxylate functional beads surface

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The microarray of DNA probes with 5′-NH2 and 5′-Tex/3′-NH2 modified terminus on 10 m m carboxylate functional beads surface in the presence of 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC) is characterized in the present paper. It was found that the microarray capacity of DNA probes on the beads surface depends on the pH of the aqueous solution, the concentration of DNA probe and the total surface area of the beads. On optimal conditions, the minimum distance of 20 mer single-stranded DNA probe microarrayed on beads surface is about 14 nm, while that of 20 mer double-stranded DNA probes is about 27 nm. If the probe length increases from 20 mer to 35 mer, its microarray density decreases correspondingly. Mechanism study shows that the binding mode of DNA probes on the beads surface is nearly parallel to the beads surface.

  20. The application of DNA microarrays in gene expression analysis.

    Science.gov (United States)

    van Hal, N L; Vorst, O; van Houwelingen, A M; Kok, E J; Peijnenburg, A; Aharoni, A; van Tunen, A J; Keijer, J

    2000-03-31

    DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

  1. cDNA microarray screening in food safety

    International Nuclear Information System (INIS)

    Roy, Sashwati; Sen, Chandan K.

    2006-01-01

    The cDNA microarray technology and related bioinformatics tools presents a wide range of novel application opportunities. The technology may be productively applied to address food safety. In this mini-review article, we present an update highlighting the late breaking discoveries that demonstrate the vitality of cDNA microarray technology as a tool to analyze food safety with reference to microbial pathogens and genetically modified foods. In order to bring the microarray technology to mainstream food safety, it is important to develop robust user-friendly tools that may be applied in a field setting. In addition, there needs to be a standardized process for regulatory agencies to interpret and act upon microarray-based data. The cDNA microarray approach is an emergent technology in diagnostics. Its values lie in being able to provide complimentary molecular insight when employed in addition to traditional tests for food safety, as part of a more comprehensive battery of tests

  2. Chromosomal Localization of DNA Amplifications in Neuroblastoma Tumors Using cDNA Microarray Comparative Genomic Hybridization

    Directory of Open Access Journals (Sweden)

    Ben Beheshti

    2003-01-01

    Full Text Available 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.

  3. Metric learning for DNA microarray data analysis

    International Nuclear Information System (INIS)

    Takeuchi, Ichiro; Nakagawa, Masao; Seto, Masao

    2009-01-01

    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.

  4. Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

    International Nuclear Information System (INIS)

    Railo, Antti; Pajunen, Antti; Itaeranta, Petri; Naillat, Florence; Vuoristo, Jussi; Kilpelaeinen, Pekka; Vainio, Seppo

    2009-01-01

    Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-β-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of β-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays of gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.

  5. Mining meiosis and gametogenesis with DNA microarrays.

    Science.gov (United States)

    Schlecht, Ulrich; Primig, Michael

    2003-04-01

    Gametogenesis is a key developmental process that involves complex transcriptional regulation of numerous genes including many that are conserved between unicellular eukaryotes and mammals. Recent expression-profiling experiments using microarrays have provided insight into the co-ordinated transcription of several hundred genes during mitotic growth and meiotic development in budding and fission yeast. Furthermore, microarray-based studies have identified numerous loci that are regulated during the cell cycle or expressed in a germ-cell specific manner in eukaryotic model systems like Caenorhabditis elegans, Mus musculus as well as Homo sapiens. The unprecedented amount of information produced by post-genome biology has spawned novel approaches to organizing biological knowledge using currently available information technology. This review outlines experiments that contribute to an emerging comprehensive picture of the molecular machinery governing sexual reproduction in eukaryotes.

  6. Probe Selection for DNA Microarrays using OligoWiz

    DEFF Research Database (Denmark)

    Wernersson, Rasmus; Juncker, Agnieszka; Nielsen, Henrik Bjørn

    2007-01-01

    Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client-server appl......Nucleotide abundance measurements using DNA microarray technology are possible only if appropriate probes complementary to the target nucleotides can be identified. Here we present a protocol for selecting DNA probes for microarrays using the OligoWiz application. OligoWiz is a client......-server application that offers a detailed graphical interface and real-time user interaction on the client side, and massive computer power and a large collection of species databases (400, summer 2007) on the server side. Probes are selected according to five weighted scores: cross-hybridization, deltaT(m), folding...... computer skills and can be executed from any Internet-connected computer. The probe selection procedure for a standard microarray design targeting all yeast transcripts can be completed in 1 h....

  7. DNA microarray technique for detecting food-borne pathogens

    Directory of Open Access Journals (Sweden)

    Xing GAO

    2012-08-01

    Full Text Available Objective To study the application of DNA microarray technique for screening and identifying multiple food-borne pathogens. Methods The oligonucleotide probes were designed by Clustal X and Oligo 6.0 at the conserved regions of specific genes of multiple food-borne pathogens, and then were validated by bioinformatic analyses. The 5' end of each probe was modified by amino-group and 10 Poly-T, and the optimized probes were synthesized and spotted on aldehyde-coated slides. The bacteria DNA template incubated with Klenow enzyme was amplified by arbitrarily primed PCR, and PCR products incorporated into Aminoallyl-dUTP were coupled with fluorescent dye. After hybridization of the purified PCR products with DNA microarray, the hybridization image and fluorescence intensity analysis was acquired by ScanArray and GenePix Pro 5.1 software. A series of detection conditions such as arbitrarily primed PCR and microarray hybridization were optimized. The specificity of this approach was evaluated by 16 different bacteria DNA, and the sensitivity and reproducibility were verified by 4 food-borne pathogens DNA. The samples of multiple bacteria DNA and simulated water samples of Shigella dysenteriae were detected. Results Nine different food-borne bacteria were successfully discriminated under the same condition. The sensitivity of genomic DNA was 102 -103pg/ μl, and the coefficient of variation (CV of the reproducibility of assay was less than 15%. The corresponding specific hybridization maps of the multiple bacteria DNA samples were obtained, and the detection limit of simulated water sample of Shigella dysenteriae was 3.54×105cfu/ml. Conclusions The DNA microarray detection system based on arbitrarily primed PCR can be employed for effective detection of multiple food-borne pathogens, and this assay may offer a new method for high-throughput platform for detecting bacteria.

  8. DNA microarray data and contextual analysis of correlation graphs

    Directory of Open Access Journals (Sweden)

    Hingamp Pascal

    2003-04-01

    Full Text Available Abstract Background DNA microarrays are used to produce large sets of expression measurements from which specific biological information is sought. Their analysis requires efficient and reliable algorithms for dimensional reduction, classification and annotation. Results We study networks of co-expressed genes obtained from DNA microarray experiments. The mathematical concept of curvature on graphs is used to group genes or samples into clusters to which relevant gene or sample annotations are automatically assigned. Application to publicly available yeast and human lymphoma data demonstrates the reliability of the method in spite of its simplicity, especially with respect to the small number of parameters involved. Conclusions We provide a method for automatically determining relevant gene clusters among the many genes monitored with microarrays. The automatic annotations and the graphical interface improve the readability of the data. A C++ implementation, called Trixy, is available from http://tagc.univ-mrs.fr/bioinformatics/trixy.html.

  9. GenePublisher: automated analysis of DNA microarray data

    DEFF Research Database (Denmark)

    Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, T.

    2003-01-01

    GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with aspecification of the data. The server performs normalization...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. A comparative analysis of DNA barcode microarray feature size

    Directory of Open Access Journals (Sweden)

    Smith Andrew M

    2009-10-01

    Full Text Available Abstract 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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Alvaro Díaz-Badillo

    2014-04-01

    Full Text Available 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.

  14. Spot detection and image segmentation in DNA microarray data.

    Science.gov (United States)

    Qin, Li; Rueda, Luis; Ali, Adnan; Ngom, Alioune

    2005-01-01

    Following the invention of microarrays in 1994, the development and applications of this technology have grown exponentially. The numerous applications of microarray technology include clinical diagnosis and treatment, drug design and discovery, tumour detection, and environmental health research. One of the key issues in the experimental approaches utilising microarrays is to extract quantitative information from the spots, which represent genes in a given experiment. For this process, the initial stages are important and they influence future steps in the analysis. Identifying the spots and separating the background from the foreground is a fundamental problem in DNA microarray data analysis. In this review, we present an overview of state-of-the-art methods for microarray image segmentation. We discuss the foundations of the circle-shaped approach, adaptive shape segmentation, histogram-based methods and the recently introduced clustering-based techniques. We analytically show that clustering-based techniques are equivalent to the one-dimensional, standard k-means clustering algorithm that utilises the Euclidean distance.

  15. DNA microarray technology in nutraceutical and food safety.

    Science.gov (United States)

    Liu-Stratton, Yiwen; Roy, Sashwati; Sen, Chandan K

    2004-04-15

    The quality and quantity of diet is a key determinant of health and disease. Molecular diagnostics may play a key role in food safety related to genetically modified foods, food-borne pathogens and novel nutraceuticals. Functional outcomes in biology are determined, for the most part, by net balance between sets of genes related to the specific outcome in question. The DNA microarray technology offers a new dimension of strength in molecular diagnostics by permitting the simultaneous analysis of large sets of genes. Automation of assay and novel bioinformatics tools make DNA microarrays a robust technology for diagnostics. Since its development a few years ago, this technology has been used for the applications of toxicogenomics, pharmacogenomics, cell biology, and clinical investigations addressing the prevention and intervention of diseases. Optimization of this technology to specifically address food safety is a vast resource that remains to be mined. Efforts to develop diagnostic custom arrays and simplified bioinformatics tools for field use are warranted.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  18. DNA microarray analysis of fim mutations in Escherichia coli

    DEFF Research Database (Denmark)

    Schembri, Mark; Ussery, David; Workman, Christopher

    2002-01-01

    Bacterial adhesion is often mediated by complex polymeric surface structures referred to as fimbriae. Type I fimbriae of Escherichia coli represent the archetypical and best characterised fimbrial system. These adhesive organelles mediate binding to D-mannose and are directly associated...... we have used DNA microarray analysis to examine the molecular events involved in response to fimbrial gene expression in E. coli K-12. Observed differential expression levels of the fim genes were in good agreement with our current knowledge of the stoichiometry of type I fimbriae. Changes in fim...

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

    International Nuclear Information System (INIS)

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

    2011-01-01

    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 µm 2 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

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

    Directory of Open Access Journals (Sweden)

    Tewfik Ahmed H

    2006-01-01

    Full Text Available 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.

  1. The effects of radiation on p53-mutated glioma cells using cDNA microarray technique

    International Nuclear Information System (INIS)

    Ngo, F.Q.H.; Hsiao, Y.-Y.H.

    2003-01-01

    Full text: In this study, we investigated the effects of 10-Gy irradiation on cell-cycle arrest, apoptosis and clonogenic death in the p53-mutated human U138MG (malignant glioblastoma) cell line. In order to evaluate time-dependent events in cellular responses to radiation, we did a time course study by incubating cells ranging from 0.5 to 48 hours after irradiation. Cell-cycle distribution and apoptosis were evaluated by flow cytometry using propidium iodide (PI) and annexin-V plus PI staining. Cell viability and proliferative capacity were studied by colony formation assay. Dual fluorescence cDNA microarray technique was used to examine the differential expression patterns of the irradiated cells. The cDNA microarray chips used contained DNA sequences corresponding to 12,814 human genes. From the flow cytometry data, it can be observed that radiation induced G2/M phase arrest and that late apoptosis was more evident following G2/M arrest. After 36 hours, some cells underwent senescence and the remains continued on with the cell cycle. Microarray analyses revealed changes in the expression of a small number of cell-cycle-related genes (p21, cyclin B1, etc.) and cell-death genes (tumor necrosis factors, DDB2, etc.) suggesting their involvement in radiation-induced cell-cycle arrest and apoptosis. In silico interpretations of the molecular mechanisms responsible for these radiation effects are in progress

  2. Sensitivity and fidelity of DNA microarray improved with integration of Amplified Differential Gene Expression (ADGE

    Directory of Open Access Journals (Sweden)

    Ile Kristina E

    2003-07-01

    Full Text Available Abstract Background The ADGE technique is a method designed to magnify the ratios of gene expression before detection. It improves the detection sensitivity to small change of gene expression and requires small amount of starting material. However, the throughput of ADGE is low. We integrated ADGE with DNA microarray (ADGE microarray and compared it with regular microarray. Results When ADGE was integrated with DNA microarray, a quantitative relationship of a power function between detected and input ratios was found. Because of ratio magnification, ADGE microarray was better able to detect small changes in gene expression in a drug resistant model cell line system. The PCR amplification of templates and efficient labeling reduced the requirement of starting material to as little as 125 ng of total RNA for one slide hybridization and enhanced the signal intensity. Integration of ratio magnification, template amplification and efficient labeling in ADGE microarray reduced artifacts in microarray data and improved detection fidelity. The results of ADGE microarray were less variable and more reproducible than those of regular microarray. A gene expression profile generated with ADGE microarray characterized the drug resistant phenotype, particularly with reference to glutathione, proliferation and kinase pathways. Conclusion ADGE microarray magnified the ratios of differential gene expression in a power function, improved the detection sensitivity and fidelity and reduced the requirement for starting material while maintaining high throughput. ADGE microarray generated a more informative expression pattern than regular microarray.

  3. Design issues in toxicogenomics using DNA microarray experiment

    International Nuclear Information System (INIS)

    Lee, Kyoung-Mu; Kim, Ju-Han; Kang, Daehee

    2005-01-01

    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

  4. Elucidation of the antibacterial mechanism of the Curvularia haloperoxidase system by DNA microarray profiling

    DEFF Research Database (Denmark)

    Hansen, E.H.; Schembri, Mark; Klemm, Per

    2004-01-01

    was the wild type. Our results demonstrate that DNA microarray technology cannot be used as the only technique to investigate the mechanisms of action of new antimicrobial compounds. However, by combining DNA microarray analysis with the subsequent creation of knockout mutants, we were able to pinpoint one...

  5. Increasing the specificity and function of DNA microarrays by processing arrays at different stringencies

    DEFF Research Database (Denmark)

    Dufva, Martin; Petersen, Jesper; Poulsen, Lena

    2009-01-01

    DNA microarrays have for a decade been the only platform for genome-wide analysis and have provided a wealth of information about living organisms. DNA microarrays are processed today under one condition only, which puts large demands on assay development because all probes on the array need to f...

  6. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  7. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    International Nuclear Information System (INIS)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon

    2003-01-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

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

    Science.gov (United States)

    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.

  9. An Overview of DNA Microarray Grid Alignment and Foreground Separation Approaches

    Directory of Open Access Journals (Sweden)

    Bajcsy Peter

    2006-01-01

    Full Text Available This paper overviews DNA microarray grid alignment and foreground separation approaches. Microarray grid alignment and foreground separation are the basic processing steps of DNA microarray images that affect the quality of gene expression information, and hence impact our confidence in any data-derived biological conclusions. Thus, understanding microarray data processing steps becomes critical for performing optimal microarray data analysis. In the past, the grid alignment and foreground separation steps have not been covered extensively in the survey literature. We present several classifications of existing algorithms, and describe the fundamental principles of these algorithms. Challenges related to automation and reliability of processed image data are outlined at the end of this overview paper.

  10. Construction of a cDNA microarray derived from the ascidian Ciona intestinalis.

    Science.gov (United States)

    Azumi, Kaoru; Takahashi, Hiroki; Miki, Yasufumi; Fujie, Manabu; Usami, Takeshi; Ishikawa, Hisayoshi; Kitayama, Atsusi; Satou, Yutaka; Ueno, Naoto; Satoh, Nori

    2003-10-01

    A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

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

    Science.gov (United States)

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Xylella fastidiosa gene expression analysis by DNA microarrays

    OpenAIRE

    Travensolo,Regiane F.; Carareto-Alves,Lucia M.; Costa,Maria V.C.G.; Lopes,Tiago J.S.; Carrilho,Emanuel; Lemos,Eliana G.M.

    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 transcrip...

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

    Directory of Open Access Journals (Sweden)

    Jee SH

    2015-10-01

    Full Text Available Seung Hyun Jee,1 Jong Won Kim,2 Ji Hyeong Lee,2 Young Soo Yoon11Department of Chemical and Biological Engineering, Gachon University, Seongnam, Gyeonggi, Republic of Korea; 2Genomics Clinical Research Institute, LabGenomics Co., Ltd., Bundang-gu, Seongnam-si, Gyeonggi-do, Republic of KoreaAbstract: 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. Keywords: DNA microarray, glass platform, bubbling method, self-assambled monolayer

  16. An efficient algorithm for the stochastic simulation of the hybridization of DNA to microarrays

    Directory of Open Access Journals (Sweden)

    Laurenzi Ian J

    2009-12-01

    Full Text Available Abstract Background Although oligonucleotide microarray technology is ubiquitous in genomic research, reproducibility and standardization of expression measurements still concern many researchers. Cross-hybridization between microarray probes and non-target ssDNA has been implicated as a primary factor in sensitivity and selectivity loss. Since hybridization is a chemical process, it may be modeled at a population-level using a combination of material balance equations and thermodynamics. However, the hybridization reaction network may be exceptionally large for commercial arrays, which often possess at least one reporter per transcript. Quantification of the kinetics and equilibrium of exceptionally large chemical systems of this type is numerically infeasible with customary approaches. Results In this paper, we present a robust and computationally efficient algorithm for the simulation of hybridization processes underlying microarray assays. Our method may be utilized to identify the extent to which nucleic acid targets (e.g. cDNA will cross-hybridize with probes, and by extension, characterize probe robustnessusing the information specified by MAGE-TAB. Using this algorithm, we characterize cross-hybridization in a modified commercial microarray assay. Conclusions By integrating stochastic simulation with thermodynamic prediction tools for DNA hybridization, one may robustly and rapidly characterize of the selectivity of a proposed microarray design at the probe and "system" levels. Our code is available at http://www.laurenzi.net.

  17. Microarrays in ecological research: A case study of a cDNA microarray for plant-herbivore interactions

    Directory of Open Access Journals (Sweden)

    Gase Klaus

    2004-09-01

    Full Text Available Abstract Background Microarray technology allows researchers to simultaneously monitor changes in the expression ratios (ERs of hundreds of genes and has thereby revolutionized most of biology. Although this technique has the potential of elucidating early stages in an organism's phenotypic response to complex ecological interactions, to date, it has not been fully incorporated into ecological research. This is partially due to a lack of simple procedures of handling and analyzing the expression ratio (ER data produced from microarrays. Results We describe an analysis of the sources of variation in ERs from 73 hybridized cDNA microarrays, each with 234 herbivory-elicited genes from the model ecological expression system, Nicotiana attenuata, using procedures that are commonly used in ecologic research. Each gene is represented by two independently labeled PCR products and each product was arrayed in quadruplicate. We present a robust method of normalizing and analyzing ERs based on arbitrary thresholds and statistical criteria, and characterize a "norm of reaction" of ERs for 6 genes (4 of known function, 2 of unknown with different ERs as determined across all analyzed arrays to provide a biologically-informed alternative to the use of arbitrary expression ratios in determining significance of expression. These gene-specific ERs and their variance (gene CV were used to calculate array-based variances (array CV, which, in turn, were used to study the effects of array age, probe cDNA quantity and quality, and quality of spotted PCR products as estimates of technical variation. Cluster analysis and a Principal Component Analysis (PCA were used to reveal associations among the transcriptional "imprints" of arrays hybridized with cDNA probes derived from mRNA from N. attenuata plants variously elicited and attacked by different herbivore species and from three congeners: N. quadrivalis, N. longiflora and N. clevelandii. Additionally, the PCA

  18. Microfluidic DNA microarrays in PMMA chips: streamlined fabrication via simultaneous DNA immobilization and bonding activation by brief UV exposure

    DEFF Research Database (Denmark)

    Sabourin, David; Petersen, J; Snakenborg, Detlef

    2010-01-01

    This report presents and describes a simple and scalable method for producing functional DNA microarrays within enclosed polymeric, PMMA, microfluidic devices. Brief (30 s) exposure to UV simultaneously immobilized poly(T)poly(C)-tagged DNA probes to the surface of unmodified PMMA and activated...... the surface for bonding below the glass transition temperature of the bulk PMMA. Functionality and validation of the enclosed PMMA microarrays was demonstrated as 18 patients were correctly genotyped for all eight mutation sites in the HBB gene interrogated. The fabrication process therefore produced probes...... with desired hybridization properties and sufficient bonding between PMMA layers to allow construction of microfluidic devices. The streamlined fabrication method is suited to the production of low-cost microfluidic microarray-based diagnostic devices and, as such, is equally applicable to the development...

  19. Observation of intermittency in gene expression on cDNA microarrays

    CERN Document Server

    Peterson, L E

    2002-01-01

    We used scaled factorial moments to search for intermittency in the log expression ratios (LERs) for thousands of genes spotted on cDNA microarrays (gene chips). Results indicate varying levels of intermittency in gene expression. The observation of intermittency in the data analyzed provides a complimentary handle on moderately expressed genes, generally not tackled by conventional techniques.

  20. Use of the cDNA microarray technology in thesafety assessment of GM food plants

    DEFF Research Database (Denmark)

    Pedersen, Jan W.; Knudsen, Ib; Eriksen, Folmer Damsted

    This report focuses on new analytical approaches that might give more insight into possible changes in a genetically modified plant. Primarily the focus is on the new DNA microarray technique but also proteomics and metabolomics are discussed.The report describes the new techniques and evaluates ...

  1. Use of the cDNA microarray technology in the safety assessment of GM food plants

    NARCIS (Netherlands)

    Kok, E.J.; Kleter, G.A.; Dijk, van J.P.

    2003-01-01

    This report focuses on new analytical approaches that might give more insight into possible changes in a genetically modified plant. Primarily the focus is on the new DNA microarray technique but also proteomics and metabolomics are discussed.The report describes the new techniques and evaluates the

  2. Transcription analysis of apple fruit development using cDNA microarrays

    NARCIS (Netherlands)

    Soglio, V.; Costa, F.; Molthoff, J.W.; Weemen-Hendriks, M.; Schouten, H.J.; Gianfranceschi, L.

    2009-01-01

    The knowledge of the molecular mechanisms underlying fruit quality traits is fundamental to devise efficient marker-assisted selection strategies and to improve apple breeding. In this study, cDNA microarray technology was used to identify genes whose expression changes during fruit development and

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

  5. On the statistical assessment of classifiers using DNA microarray data

    Directory of Open Access Journals (Sweden)

    Carella M

    2006-08-01

    Full Text Available Abstract Background In this paper we present a method for the statistical assessment of cancer predictors which make use of gene expression profiles. The methodology is applied to a new data set of microarray gene expression data collected in Casa Sollievo della Sofferenza Hospital, Foggia – Italy. The data set is made up of normal (22 and tumor (25 specimens extracted from 25 patients affected by colon cancer. We propose to give answers to some questions which are relevant for the automatic diagnosis of cancer such as: Is the size of the available data set sufficient to build accurate classifiers? What is the statistical significance of the associated error rates? In what ways can accuracy be considered dependant on the adopted classification scheme? How many genes are correlated with the pathology and how many are sufficient for an accurate colon cancer classification? The method we propose answers these questions whilst avoiding the potential pitfalls hidden in the analysis and interpretation of microarray data. Results We estimate the generalization error, evaluated through the Leave-K-Out Cross Validation error, for three different classification schemes by varying the number of training examples and the number of the genes used. The statistical significance of the error rate is measured by using a permutation test. We provide a statistical analysis in terms of the frequencies of the genes involved in the classification. Using the whole set of genes, we found that the Weighted Voting Algorithm (WVA classifier learns the distinction between normal and tumor specimens with 25 training examples, providing e = 21% (p = 0.045 as an error rate. This remains constant even when the number of examples increases. Moreover, Regularized Least Squares (RLS and Support Vector Machines (SVM classifiers can learn with only 15 training examples, with an error rate of e = 19% (p = 0.035 and e = 18% (p = 0.037 respectively. Moreover, the error rate

  6. An automated microfluidic DNA microarray platform for genetic variant detection in inherited arrhythmic diseases.

    Science.gov (United States)

    Huang, Shu-Hong; Chang, Yu-Shin; Juang, Jyh-Ming Jimmy; Chang, Kai-Wei; Tsai, Mong-Hsun; Lu, Tzu-Pin; Lai, Liang-Chuan; Chuang, Eric Y; Huang, Nien-Tsu

    2018-03-12

    In this study, we developed an automated microfluidic DNA microarray (AMDM) platform for point mutation detection of genetic variants in inherited arrhythmic diseases. The platform allows for automated and programmable reagent sequencing under precise conditions of hybridization flow and temperature control. It is composed of a commercial microfluidic control system, a microfluidic microarray device, and a temperature control unit. The automated and rapid hybridization process can be performed in the AMDM platform using Cy3 labeled oligonucleotide exons of SCN5A genetic DNA, which produces proteins associated with sodium channels abundant in the heart (cardiac) muscle cells. We then introduce a graphene oxide (GO)-assisted DNA microarray hybridization protocol to enable point mutation detection. In this protocol, a GO solution is added after the staining step to quench dyes bound to single-stranded DNA or non-perfectly matched DNA, which can improve point mutation specificity. As proof-of-concept we extracted the wild-type and mutant of exon 12 and exon 17 of SCN5A genetic DNA from patients with long QT syndrome or Brugada syndrome by touchdown PCR and performed a successful point mutation discrimination in the AMDM platform. Overall, the AMDM platform can greatly reduce laborious and time-consuming hybridization steps and prevent potential contamination. Furthermore, by introducing the reciprocating flow into the microchannel during the hybridization process, the total assay time can be reduced to 3 hours, which is 6 times faster than the conventional DNA microarray. Given the automatic assay operation, shorter assay time, and high point mutation discrimination, we believe that the AMDM platform has potential for low-cost, rapid and sensitive genetic testing in a simple and user-friendly manner, which may benefit gene screening in medical practice.

  7. [Preparation of the cDNA microarray on the differential expressed cDNA of senescence-accelerated mouse's hippocampus].

    Science.gov (United States)

    Cheng, Xiao-Rui; Zhou, Wen-Xia; Zhang, Yong-Xiang

    2006-05-01

    Alzheimer' s disease (AD) is the most common form of dementia in the elderly. AD is an invariably fatal neurodegenerative disorder with no effective treatment. Senescence-accelerated mouse prone 8 (SAMP8) is a model for studying age-related cognitive impairments and also is a good model to study brain aging and one of mouse model of AD. The technique of cDNA microarray can monitor the expression levels of thousands of genes simultaneously and can be used to study AD with the character of multi-mechanism, multi-targets and multi-pathway. In order to disclose the mechanism of AD and find the drug targets of AD, cDNA microarray containing 3136 cDNAs amplified from the suppression subtracted cDNA library of hippocampus of SAMP8 and SAMR1 was prepared with 16 blocks and 14 x 14 pins, the housekeeping gene beta-actin and G3PDH as inner conference. The background of this microarray was low and unanimous, and dots divided evenly. The conditions of hybridization and washing were optimized during the hybridization of probe and target molecule. After the data of hybridization analysis, the differential expressed cDNAs were sequenced and analyzed by the bioinformatics, and some of genes were quantified by the real time RT-PCR and the reliability of this cDNA microarray were validated. This cDNA microarray may be the good means to select the differential expressed genes and disclose the molecular mechanism of SAMP8's brain aging and AD.

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

    Science.gov (United States)

    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.

  9. Xylella fastidiosa gene expression analysis by DNA microarrays

    Directory of Open Access Journals (Sweden)

    Regiane F. Travensolo

    2009-01-01

    Full Text Available 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.

  10. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.

    Science.gov (United States)

    Alba, Rob; Fei, Zhangjun; Payton, Paxton; Liu, Yang; Moore, Shanna L; Debbie, Paul; Cohn, Jonathan; D'Ascenzo, Mark; Gordon, Jeffrey S; Rose, Jocelyn K C; Martin, Gregory; Tanksley, Steven D; Bouzayen, Mondher; Jahn, Molly M; Giovannoni, Jim

    2004-09-01

    Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

  11. A statistical model for investigating binding probabilities of DNA nucleotide sequences using microarrays.

    Science.gov (United States)

    Lee, Mei-Ling Ting; Bulyk, Martha L; Whitmore, G A; Church, George M

    2002-12-01

    There is considerable scientific interest in knowing the probability that a site-specific transcription factor will bind to a given DNA sequence. Microarray methods provide an effective means for assessing the binding affinities of a large number of DNA sequences as demonstrated by Bulyk et al. (2001, Proceedings of the National Academy of Sciences, USA 98, 7158-7163) in their study of the DNA-binding specificities of Zif268 zinc fingers using microarray technology. In a follow-up investigation, Bulyk, Johnson, and Church (2002, Nucleic Acid Research 30, 1255-1261) studied the interdependence of nucleotides on the binding affinities of transcription proteins. Our article is motivated by this pair of studies. We present a general statistical methodology for analyzing microarray intensity measurements reflecting DNA-protein interactions. The log probability of a protein binding to a DNA sequence on an array is modeled using a linear ANOVA model. This model is convenient because it employs familiar statistical concepts and procedures and also because it is effective for investigating the probability structure of the binding mechanism.

  12. Visual Analysis of DNA Microarray Data for Accurate Molecular Identification of Non-albicans Candida Isolates from Patients with Candidemia Episodes

    OpenAIRE

    De Luca Ferrari, Michela; Ribeiro Resende, Mariângela; Sakai, Kanae; Muraosa, Yasunori; Lyra, Luzia; Gonoi, Tohru; Mikami, Yuzuru; Tominaga, Kenichiro; Kamei, Katsuhiko; Zaninelli Schreiber, Angelica; Trabasso, Plinio; Moretti, Maria Luiza

    2013-01-01

    The performance of a visual slide-based DNA microarray for the identification of non-albicans Candida spp. was evaluated. Among 167 isolates that had previously been identified by Vitek 2, the agreement between DNA microarray and sequencing results was 97.6%. This DNA microarray platform showed excellent performance.

  13. Development and assessment of microarray-based DNA fingerprinting in Eucalyptus grandis.

    Science.gov (United States)

    Lezar, Sabine; Myburg, A A; Berger, D K; Wingfield, M J; Wingfield, B D

    2004-11-01

    Development of improved Eucalyptus genotypes involves the routine identification of breeding stock and superior clones. Currently, microsatellites and random amplified polymorphic DNA markers are the most widely used DNA-based techniques for fingerprinting of these trees. While these techniques have provided rapid and powerful fingerprinting assays, they are constrained by their reliance on gel or capillary electrophoresis, and therefore, relatively low throughput of fragment analysis. In contrast, recently developed microarray technology holds the promise of parallel analysis of thousands of markers in plant genomes. The aim of this study was to develop a DNA fingerprinting chip for Eucalyptus grandis and to investigate its usefulness for fingerprinting of eucalypt trees. A prototype chip was prepared using a partial genomic library from total genomic DNA of 23 E. grandis trees, of which 22 were full siblings. A total of 384 cloned genomic fragments were individually amplified and arrayed onto glass slides. DNA fingerprints were obtained for 17 individuals by hybridizing labeled genome representations of the individual trees to the 384-element chip. Polymorphic DNA fragments were identified by evaluating the binary distribution of their background-corrected signal intensities across full-sib individuals. Among 384 DNA fragments on the chip, 104 (27%) were found to be polymorphic. Hybridization of these polymorphic fragments was highly repeatable (R2>0.91) within the E. grandis individuals, and they allowed us to identify all 17 full-sib individuals. Our results suggest that DNA microarrays can be used to effectively fingerprint large numbers of closely related Eucalyptus trees.

  14. Study of hepatitis B virus gene mutations with enzymatic colorimetry-based DNA microarray.

    Science.gov (United States)

    Mao, Hailei; Wang, Huimin; Zhang, Donglei; Mao, Hongju; Zhao, Jianlong; Shi, Jian; Cui, Zhichu

    2006-01-01

    To establish a modified microarray method for detecting HBV gene mutations in the clinic. Site-specific oligonucleotide probes were immobilized to microarray slides and hybridized to biotin-labeled HBV gene fragments amplified from two-step PCR. Hybridized targets were transferred to nitrocellulose membranes, followed by intensity measurement using BCIP/NBT colorimetry. HBV genes from 99 Hepatitis B patients and 40 healthy blood donors were analyzed. Mutation frequencies of HBV pre-core/core and basic core promoter (BCP) regions were found to be significantly higher in the patient group (42%, 40% versus 2.5%, 5%, P colorimetry method exhibited the same level of sensitivity and reproducibility. An enzymatic colorimetry-based DNA microarray assay was successfully established to monitor HBV mutations. Pre-core/core and BCP mutations of HBV genes could be major causes of HBV infection in HBeAg-negative patients and could also be relevant to chronicity and aggravation of hepatitis B.

  15. Autoregressive-model-based missing value estimation for DNA microarray time series data.

    Science.gov (United States)

    Choong, Miew Keen; Charbit, Maurice; Yan, Hong

    2009-01-01

    Missing value estimation is important in DNA microarray data analysis. A number of algorithms have been developed to solve this problem, but they have several limitations. Most existing algorithms are not able to deal with the situation where a particular time point (column) of the data is missing entirely. In this paper, we present an autoregressive-model-based missing value estimation method (ARLSimpute) that takes into account the dynamic property of microarray temporal data and the local similarity structures in the data. ARLSimpute is especially effective for the situation where a particular time point contains many missing values or where the entire time point is missing. Experiment results suggest that our proposed algorithm is an accurate missing value estimator in comparison with other imputation methods on simulated as well as real microarray time series datasets.

  16. Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens.

    Science.gov (United States)

    Li, Yongjin

    2016-01-01

    The accurate detection and identification of food-borne pathogenic microorganisms is critical for food safety nowadays. In the present work, a visual DNA microarray was established and applied to detect pathogens commonly found in food, including Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in food samples. Multiplex PCR (mPCR) was employed to simultaneously amplify specific gene fragments, fimY for Salmonella, ipaH for Shigella, iap for L. monocytogenes and ECs2841 for E. coli O157:H7, respectively. Biotinylated PCR amplicons annealed to the microarray probes were then reacted with a streptavidin-alkaline phosphatase conjugate and nitro blue tetrazolium/5-bromo-4-chloro-3'-indolylphosphate, p-toluidine salt (NBT/BCIP); the positive results were easily visualized as blue dots formatted on the microarray surface. The performance of a DNA microarray was tested against 14 representative collection strains and mock-contamination food samples. The combination of mPCR and a visual micro-plate chip specifically and sensitively detected Salmonella enterica, Shigella flexneri, E. coli O157:H7 and Listeria monocytogenes in standard strains and food matrices with a sensitivity of ∼10(2) CFU/mL of bacterial culture. Thus, the developed method is advantageous because of its high throughput, cost-effectiveness and ease of use.

  17. Position dependent mismatch discrimination on DNA microarrays – experiments and model

    Directory of Open Access Journals (Sweden)

    Michel Wolfgang

    2008-12-01

    Full Text Available Abstract Background The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study. Results We observe that mismatch discrimination is mostly determined by the defect position (relative to the duplex ends as well as by the sequence context. We investigate the thermodynamics of the oligonucleotide duplexes on the basis of double-ended molecular zipper. Theoretical predictions of defect positional influence as well as long range sequence influence agree well with the experimental results. Conclusion Molecular zipping at thermodynamic equilibrium explains the binding affinity of mismatched DNA duplexes on microarrays well. The position dependent nearest neighbor model (PDNN can be inferred from it. Quantitative understanding of microarray experiments from first principles is in reach.

  18. DNA microarrays of baculovirus genomes: differential expression of viral genes in two susceptible insect cell lines.

    Science.gov (United States)

    Yamagishi, J; Isobe, R; Takebuchi, T; Bando, H

    2003-03-01

    We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef- 6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.

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

    Directory of Open Access Journals (Sweden)

    Vikalo Haris

    2010-01-01

    Full Text Available 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.

  20. Production of DNA microarray and expression analysis of genes from Xylella fastidiosa in different culture media

    Directory of Open Access Journals (Sweden)

    Regiane de Fátima Travensolo

    2009-06-01

    Full Text Available DNA Microarray was developed to monitor the expression of many genes from Xylella fastidiosa, allowing the side by-side comparison of two situations in a single experiment. The experiments were performed using X. fastidiosa cells grown in two culture media: BCYE and XDM2. The primers were synthesized, spotted onto glass slides and the array was hybridized against fluorescently labeled cDNAs. The emitted signals were quantified, normalized and the data were statistically analyzed to verify the differentially expressed genes. According to the data, 104 genes were differentially expressed in XDM2 and 30 genes in BCYE media. The present study showed that DNA microarray technique efficiently differentiate the expressed genes under different conditions.DNA Microarray foi desenvolvida para monitorar a expressão de muitos genes de Xylella fastidiosa, permitindo a comparação de duas situações distintas em um único experimento. Os experimentos foram feitos utilizando células de X. fastidiosa cultivada em dois meios de cultura: BCYE e XDM2. Pares de oligonucleotídeos iniciadores foram sintetizados, depositados em lâminas de vidro e o arranjo foi hibridizado contra cDNAs marcados fluorescentemente. Os sinais emitidos foram quantificados, normalizados e os dados foram estatisticamente analisados para verificar os genes diferencialmente expressos. De acordo com nossos dados, 104 genes foram diferencialmente expressos para o meio de cultura XDM2 e 30 genes para o BCYE. No presente estudo, nós demonstramos que a técnica de DNA microarrays eficientemente diferencia genes expressos sob diferentes condições de cultivo.

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

    Science.gov (United States)

    2012-01-01

    Background 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. Results 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. Conclusion 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

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

    Science.gov (United States)

    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.

  3. Clustering approaches to identifying gene expression patterns from DNA microarray data.

    Science.gov (United States)

    Do, Jin Hwan; Choi, Dong-Kug

    2008-04-30

    The analysis of microarray data is essential for large amounts of gene expression data. In this review we focus on clustering techniques. The biological rationale for this approach is the fact that many co-expressed genes are co-regulated, and identifying co-expressed genes could aid in functional annotation of novel genes, de novo identification of transcription factor binding sites and elucidation of complex biological pathways. Co-expressed genes are usually identified in microarray experiments by clustering techniques. There are many such methods, and the results obtained even for the same datasets may vary considerably depending on the algorithms and metrics for dissimilarity measures used, as well as on user-selectable parameters such as desired number of clusters and initial values. Therefore, biologists who want to interpret microarray data should be aware of the weakness and strengths of the clustering methods used. In this review, we survey the basic principles of clustering of DNA microarray data from crisp clustering algorithms such as hierarchical clustering, K-means and self-organizing maps, to complex clustering algorithms like fuzzy clustering.

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

    Directory of Open Access Journals (Sweden)

    Bordoni Roberta

    2007-11-01

    Full Text Available Abstract 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

  5. Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

    Directory of Open Access Journals (Sweden)

    Kreil David P

    2008-08-01

    Full Text Available Abstract Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer. To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes

  6. SiPM as miniaturised optical biosensor for DNA-microarray applications

    Directory of Open Access Journals (Sweden)

    M.F. Santangelo

    2015-12-01

    Full Text Available A miniaturized optical biosensor for low-level fluorescence emitted by DNA strands labelled with CY5 is showed. Aim of this work is to demonstrate that a Si-based photodetector, having a low noise and a high sensitivity, can replace traditional detection systems in DNA-microarray applications. The photodetector used is a photomultiplier (SiPM, with 25 pixels. It exhibits a higher sensitivity than commercial optical readers and we experimentally found a detection limit for spotted dried samples of ∼1 nM. We measured the fluorescence signal in different operating conditions (angle of analysis, fluorophores concentrations, solution volumes and support. Once fixed the angle of analysis, for samples spotted on Al-TEOS slide dried, the system is proportional to the concentration of the analyte in the sample and is linear in the range 1 nM–1 μM. For solutions, the range of linearity ranges from 100 fM to 10 nM. The system potentialities and the device low costs suggest it as basic component for the design and fabrication of a cheap, easy and portable optical system. Keywords: Optical Biosensor, SiPM, DNA microarray, Fluorophore detection

  7. Rational design of DNA sequences for nanotechnology, microarrays and molecular computers using Eulerian graphs.

    Science.gov (United States)

    Pancoska, Petr; Moravek, Zdenek; Moll, Ute M

    2004-01-01

    Nucleic acids are molecules of choice for both established and emerging nanoscale technologies. These technologies benefit from large functional densities of 'DNA processing elements' that can be readily manufactured. To achieve the desired functionality, polynucleotide sequences are currently designed by a process that involves tedious and laborious filtering of potential candidates against a series of requirements and parameters. Here, we present a complete novel methodology for the rapid rational design of large sets of DNA sequences. This method allows for the direct implementation of very complex and detailed requirements for the generated sequences, thus avoiding 'brute force' filtering. At the same time, these sequences have narrow distributions of melting temperatures. The molecular part of the design process can be done without computer assistance, using an efficient 'human engineering' approach by drawing a single blueprint graph that represents all generated sequences. Moreover, the method eliminates the necessity for extensive thermodynamic calculations. Melting temperature can be calculated only once (or not at all). In addition, the isostability of the sequences is independent of the selection of a particular set of thermodynamic parameters. Applications are presented for DNA sequence designs for microarrays, universal microarray zip sequences and electron transfer experiments.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  10. Development of a porcine skeletal muscle cDNA microarray: analysis of differential transcript expression in phenotypically distinct muscles

    Directory of Open Access Journals (Sweden)

    Stear Michael

    2003-03-01

    Full Text Available Abstract Background Microarray profiling has the potential to illuminate the molecular processes that govern the phenotypic characteristics of porcine skeletal muscles, such as hypertrophy or atrophy, and the expression of specific fibre types. This information is not only important for understanding basic muscle biology but also provides underpinning knowledge for enhancing the efficiency of livestock production. Results We report on the de novo development of a composite skeletal muscle cDNA microarray, comprising 5500 clones from two developmentally distinct cDNA libraries (longissimus dorsi of a 50-day porcine foetus and the gastrocnemius of a 3-day-old pig. Clones selected for the microarray assembly were of low to moderate abundance, as indicated by colony hybridisation. We profiled the differential expression of genes between the psoas (red muscle and the longissimus dorsi (white muscle, by co-hybridisation of Cy3 and Cy5 labelled cDNA derived from these two muscles. Results from seven microarray slides (replicates correctly identified genes that were expected to be differentially expressed, as well as a number of novel candidate regulatory genes. Quantitative real-time RT-PCR on selected genes was used to confirm the results from the microarray. Conclusion We have developed a porcine skeletal muscle cDNA microarray and have identified a number of candidate genes that could be involved in muscle phenotype determination, including several members of the casein kinase 2 signalling pathway.

  11. Robust multi-scale clustering of large DNA microarray datasets with the consensus algorithm

    DEFF Research Database (Denmark)

    Grotkjær, Thomas; Winther, Ole; Regenberg, Birgitte

    2006-01-01

    Motivation: Hierarchical and relocation clustering (e.g. K-means and self-organizing maps) have been successful tools in the display and analysis of whole genome DNA microarray expression data. However, the results of hierarchical clustering are sensitive to outliers, and most relocation methods...... analysis by collecting re-occurring clustering patterns in a co-occurrence matrix. The results show that consensus clustering obtained from clustering multiple times with Variational Bayes Mixtures of Gaussians or K-means significantly reduces the classification error rate for a simulated dataset...

  12. Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA.

    Science.gov (United States)

    Gibbons, Brian; Datta, Parikkhit; Wu, Ying; Chan, Alan; Al Armour, John

    2006-06-30

    Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH) we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A). Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

  13. Methods for interpreting lists of affected genes obtained in a DNA microarray experiment

    Directory of Open Access Journals (Sweden)

    Hedegaard Jakob

    2009-07-01

    Full Text Available Abstract Background The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. Results Several conceptually different analytical approaches, using both commercial and public available software, were applied by the participating groups. The following tools were used: Ingenuity Pathway Analysis, MAPPFinder, LIMMA, GOstats, GOEAST, GOTM, Globaltest, TopGO, ArrayUnlock, Pathway Studio, GIST and AnnotationDbi. The main focus of the approaches was to utilise the relation between probes/genes and their gene ontology and pathways to interpret the affected probes/genes. The lack of a well-annotated chicken genome did though limit the possibilities to fully explore the tools. The main results from these analyses showed that the biological interpretation is highly dependent on the statistical method used but that some common biological conclusions could be reached. Conclusion It is highly recommended to test different analytical methods on the same data set and compare the results to obtain a reliable biological interpretation of the affected genes in a DNA microarray experiment.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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 cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

  16. High-throughput screening of suppression subtractive hybridization cDNA libraries using DNA microarray analysis

    CSIR Research Space (South Africa)

    Van den Berg, N

    2004-11-01

    Full Text Available Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression...

  17. A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses.

    Science.gov (United States)

    Yamamoto, F; Yamamoto, M

    2004-07-01

    We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme NotI in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15 x 2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Deising Holger B

    2011-01-01

    Full Text Available Abstract 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

  1. Examination of gene expression in mice exposed to low dose radiation using affymetrix cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Morris, D.; Knox, D.; Lavoie, J.; Lemon, J.; Boreham, D. [McMaster Univ., Hamilton, Ontario (Canada)

    2005-07-01

    'Full text:' Gamma radiation acts via the indirect effect to damage cells by producing reactive oxygen species (ROS). These ROS are capable damaging macromolecules and, altering signal pathways and gene transcription. Cells have evolved enzymes and mechanisms to scavenge ROS and repair oxidative damage. Microarrays allow the survey of the gene transcription activity of thousands of genes simultaneously. Messenger RNA is extracted from cells, hybridized with the complementary DNA (cDNA) of a microarray chip, and examined with a chip reader. Affymetrix microarray chips have been produced by the CSCHAH in Winnipeg containing 26000 murine genes. Groups of female mice have been exposed to low dose whole body chronic gamma radiation exposures of 0,50,100, and 120 mGy, corresponding to 15,30,60, and 75 weeks, respectively. MRNA from mice brain tissue has been extracted, isolated, converted to cDNA and labeled. Gene expression in each irradiated mouse was compared to the pooled expression of the control mice. Analysis of gene expression levels are performed with microarray analytical software, Array Pro by Media Cybernetics, and powerful statistical software, BRB microarray tools. Differences in gene expressions, focusing on genes for cytokines, DNA repair mechanisms, immuno-modulators, apoptosis pathways, and enzymatic anti-oxidant systems, are being examined and will be reported. (author)

  2. Microarray and cDNA sequence analysis of transcription during nerve-dependent limb regeneration

    Directory of Open Access Journals (Sweden)

    Bryant Susan V

    2009-01-01

    Full Text Available Abstract Background Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR and denervated (DL forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Results Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa. Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST contigs from the Ambystoma EST database more than doubled (3935 to 9411 the number of non-redundant human-A. mexicanum orthologous sequences. Conclusion Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

  3. Dissection of the inflammatory bowel disease transcriptome using genome-wide cDNA microarrays.

    Directory of Open Access Journals (Sweden)

    Christine M Costello

    2005-08-01

    Full Text Available BACKGROUND: The differential pathophysiologic mechanisms that trigger and maintain the two forms of inflammatory bowel disease (IBD, Crohn disease (CD, and ulcerative colitis (UC are only partially understood. cDNA microarrays can be used to decipher gene regulation events at a genome-wide level and to identify novel unknown genes that might be involved in perpetuating inflammatory disease progression. METHODS AND FINDINGS: High-density cDNA microarrays representing 33,792 UniGene clusters were prepared. Biopsies were taken from the sigmoid colon of normal controls (n = 11, CD patients (n = 10 and UC patients (n = 10. 33P-radiolabeled cDNA from purified poly(A+ RNA extracted from biopsies (unpooled was hybridized to the arrays. We identified 500 and 272 transcripts differentially regulated in CD and UC, respectively. Interesting hits were independently verified by real-time PCR in a second sample of 100 individuals, and immunohistochemistry was used for exemplary localization. The main findings point to novel molecules important in abnormal immune regulation and the highly disturbed cell biology of colonic epithelial cells in IBD pathogenesis, e.g., CYLD (cylindromatosis, turban tumor syndrome and CDH11 (cadherin 11, type 2. By the nature of the array setup, many of the genes identified were to our knowledge previously uncharacterized, and prediction of the putative function of a subsection of these genes indicate that some could be involved in early events in disease pathophysiology. CONCLUSION: A comprehensive set of candidate genes not previously associated with IBD was revealed, which underlines the polygenic and complex nature of the disease. It points out substantial differences in pathophysiology between CD and UC. The multiple unknown genes identified may stimulate new research in the fields of barrier mechanisms and cell signalling in the context of IBD, and ultimately new therapeutic approaches.

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

    KAUST Repository

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

    2015-01-01

    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=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build 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 using 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.

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

    KAUST Repository

    Wong, Ka-Chun

    2015-06-11

    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=810). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build 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 using 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. Systematic spatial bias in DNA microarray hybridization is caused by probe spot position-dependent variability in lateral diffusion.

    Science.gov (United States)

    Steger, Doris; Berry, David; Haider, Susanne; Horn, Matthias; Wagner, Michael; Stocker, Roman; Loy, Alexander

    2011-01-01

    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. 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. 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.

  7. Normal uniform mixture differential gene expression detection for cDNA microarrays

    Directory of Open Access Journals (Sweden)

    Raftery Adrian E

    2005-07-01

    Full Text Available Abstract Background One of the primary tasks in analysing gene expression data is finding genes that are differentially expressed in different samples. Multiple testing issues due to the thousands of tests run make some of the more popular methods for doing this problematic. Results We propose a simple method, Normal Uniform Differential Gene Expression (NUDGE detection for finding differentially expressed genes in cDNA microarrays. The method uses a simple univariate normal-uniform mixture model, in combination with new normalization methods for spread as well as mean that extend the lowess normalization of Dudoit, Yang, Callow and Speed (2002 1. It takes account of multiple testing, and gives probabilities of differential expression as part of its output. It can be applied to either single-slide or replicated experiments, and it is very fast. Three datasets are analyzed using NUDGE, and the results are compared to those given by other popular methods: unadjusted and Bonferroni-adjusted t tests, Significance Analysis of Microarrays (SAM, and Empirical Bayes for microarrays (EBarrays with both Gamma-Gamma and Lognormal-Normal models. Conclusion The method gives a high probability of differential expression to genes known/suspected a priori to be differentially expressed and a low probability to the others. In terms of known false positives and false negatives, the method outperforms all multiple-replicate methods except for the Gamma-Gamma EBarrays method to which it offers comparable results with the added advantages of greater simplicity, speed, fewer assumptions and applicability to the single replicate case. An R package called nudge to implement the methods in this paper will be made available soon at http://www.bioconductor.org.

  8. Microarray MAPH: accurate array-based detection of relative copy number in genomic DNA

    Directory of Open Access Journals (Sweden)

    Chan Alan

    2006-06-01

    Full Text Available Abstract Background Current methods for measurement of copy number do not combine all the desirable qualities of convenience, throughput, economy, accuracy and resolution. In this study, to improve the throughput associated with Multiplex Amplifiable Probe Hybridisation (MAPH we aimed to develop a modification based on the 3-Dimensional, Flow-Through Microarray Platform from PamGene International. In this new method, electrophoretic analysis of amplified products is replaced with photometric analysis of a probed oligonucleotide array. Copy number analysis of hybridised probes is based on a dual-label approach by comparing the intensity of Cy3-labelled MAPH probes amplified from test samples co-hybridised with similarly amplified Cy5-labelled reference MAPH probes. The key feature of using a hybridisation-based end point with MAPH is that discrimination of amplified probes is based on sequence and not fragment length. Results In this study we showed that microarray MAPH measurement of PMP22 gene dosage correlates well with PMP22 gene dosage determined by capillary MAPH and that copy number was accurately reported in analyses of DNA from 38 individuals, 12 of which were known to have Charcot-Marie-Tooth disease type 1A (CMT1A. Conclusion Measurement of microarray-based endpoints for MAPH appears to be of comparable accuracy to electrophoretic methods, and holds the prospect of fully exploiting the potential multiplicity of MAPH. The technology has the potential to simplify copy number assays for genes with a large number of exons, or of expanded sets of probes from dispersed genomic locations.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

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

    Directory of Open Access Journals (Sweden)

    Sunwoo Chun

    Full Text Available 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

  11. Incorporation of gene-specific variability improves expression analysis using high-density DNA microarrays

    Directory of Open Access Journals (Sweden)

    Spitznagel Edward

    2003-11-01

    Full Text Available Abstract Background The assessment of data reproducibility is essential for application of microarray technology to exploration of biological pathways and disease states. Technical variability in data analysis largely depends on signal intensity. Within that context, the reproducibility of individual probe sets has not been hitherto addressed. Results We used an extraordinarily large replicate data set derived from human placental trophoblast to analyze probe-specific contribution to variability of gene expression. We found that signal variability, in addition to being signal-intensity dependant, is probe set-specific. Importantly, we developed a novel method to quantify the contribution of this probe set-specific variability. Furthermore, we devised a formula that incorporates a priori-computed, replicate-based information on probe set- and intensity-specific variability in determination of expression changes even without technical replicates. Conclusion The strategy of incorporating probe set-specific variability is superior to analysis based on arbitrary fold-change thresholds. We recommend its incorporation to any computation of gene expression changes using high-density DNA microarrays. A Java application implementing our T-score is available at http://www.sadovsky.wustl.edu/tscore.html.

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

    Science.gov (United States)

    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

  13. Detection of perturbation phases and developmental stages in organisms from DNA microarray time series data.

    Directory of Open Access Journals (Sweden)

    Marianne Rooman

    Full Text Available Available DNA microarray time series that record gene expression along the developmental stages of multicellular eukaryotes, or in unicellular organisms subject to external perturbations such as stress and diauxie, are analyzed. By pairwise comparison of the gene expression profiles on the basis of a translation-invariant and scale-invariant distance measure corresponding to least-rectangle regression, it is shown that peaks in the average distance values are noticeable and are localized around specific time points. These points systematically coincide with the transition points between developmental phases or just follow the external perturbations. This approach can thus be used to identify automatically, from microarray time series alone, the presence of external perturbations or the succession of developmental stages in arbitrary cell systems. Moreover, our results show that there is a striking similarity between the gene expression responses to these a priori very different phenomena. In contrast, the cell cycle does not involve a perturbation-like phase, but rather continuous gene expression remodeling. Similar analyses were conducted using three other standard distance measures, showing that the one we introduced was superior. Based on these findings, we set up an adapted clustering method that uses this distance measure and classifies the genes on the basis of their expression profiles within each developmental stage or between perturbation phases.

  14. A Bayesian decision procedure for testing multiple hypotheses in DNA microarray experiments.

    Science.gov (United States)

    Gómez-Villegas, Miguel A; Salazar, Isabel; Sanz, Luis

    2014-02-01

    DNA microarray experiments require the use of multiple hypothesis testing procedures because thousands of hypotheses are simultaneously tested. We deal with this problem from a Bayesian decision theory perspective. We propose a decision criterion based on an estimation of the number of false null hypotheses (FNH), taking as an error measure the proportion of the posterior expected number of false positives with respect to the estimated number of true null hypotheses. The methodology is applied to a Gaussian model when testing bilateral hypotheses. The procedure is illustrated with both simulated and real data examples and the results are compared to those obtained by the Bayes rule when an additive loss function is considered for each joint action and the generalized loss 0-1 function for each individual action. Our procedure significantly reduced the percentage of false negatives whereas the percentage of false positives remains at an acceptable level.

  15. Microarray-based analysis of plasma cirDNA epigenetic modification profiling in xenografted mice exposed to intermittent hypoxia

    Directory of Open Access Journals (Sweden)

    Rene Cortese

    2015-09-01

    Full Text Available Intermittent hypoxia (IH during sleep is one of the major abnormalities occurring in patients suffering from obstructive sleep apnea (OSA, a highly prevalent disorder affecting 6–15% of the general population, particularly among obese people. IH has been proposed as a major determinant of oncogenetically-related processes such as tumor growth, invasion and metastasis. During the growth and expansion of tumors, fragmented DNA is released into the bloodstream and enters the circulation. Circulating tumor DNA (cirDNA conserves the genetic and epigenetic profiles from the tumor of origin and can be isolated from the plasma fraction. Here we report a microarray-based epigenetic profiling of cirDNA isolated from blood samples of mice engrafted with TC1 epithelial lung cancer cells and controls, which were exposed to IH during sleep (XenoIH group, n = 3 or control conditions, (i.e., room air (RA; XenoRA group, n = 3 conditions. To prepare the targets for microarray hybridization, we applied a previously developed method that enriches the modified fraction of the cirDNA without amplification of genomic DNA. Regions of differential cirDNA modification between the two groups were identified by hybridizing the enriched fractions for each sample to Affymetrix GeneChip Human Promoter Arrays 1.0R. Microarray raw and processed data were deposited in NCBI's Gene Expression Omnibus (GEO database (accession number: GSE61070.

  16. FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota

    Directory of Open Access Journals (Sweden)

    Sophie Comtet-Marre

    2018-02-01

    Full Text Available Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6 were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium Fibrobacter succinogenes S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the F. succinogenes genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen.

  17. Early Gene Expression in Wounded Human Keratinocytes Revealed by DNA Microarray Analysis

    Directory of Open Access Journals (Sweden)

    Pascal Barbry

    2006-04-01

    Full Text Available Wound healing involves several steps: spreading of the cells, migration and proliferation. We have profiled gene expression during the early events of wound healing in normal human keratinocytes with a home-made DNA microarray containing about 1000 relevant human probes. An original wounding machine was used, that allows the wounding of up to 40% of the surface of a confluent monolayer of cultured cells grown on a Petri dish (compared with 5% with a classical ‘scratch’ method. The two aims of the present study were: (a to validate a limited number of genes by comparing the expression levels obtained with this technique with those found in the literature; (b to combine the use of the wounding machine with DNA microarray analysis for large-scale detection of the molecular events triggered during the early stages of the wound-healing process. The time-courses of RNA expression observed at 0.5, 1.5, 3, 6 and 15 h after wounding for genes such as c-Fos, c-Jun, Egr1, the plasminogen activator PLAU (uPA and the signal transducer and transcription activator STAT3, were consistent with previously published data. This suggests that our methodologies are able to perform quantitative measurement of gene expression. Transcripts encoding two zinc finger proteins, ZFP36 and ZNF161, and the tumour necrosis factor α-induced protein TNFAIP3, were also overexpressed after wounding. The role of the p38 mitogen-activated protein kinase (p38MAPK in wound healing was shown after the inhibition of p38 by SB203580, but our results also suggest the existence of surrogate activating pathways.

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

    Science.gov (United States)

    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

  19. FibroChip, a Functional DNA Microarray to Monitor Cellulolytic and Hemicellulolytic Activities of Rumen Microbiota.

    Science.gov (United States)

    Comtet-Marre, Sophie; Chaucheyras-Durand, Frédérique; Bouzid, Ourdia; Mosoni, Pascale; Bayat, Ali R; Peyret, Pierre; Forano, Evelyne

    2018-01-01

    Ruminants fulfill their energy needs for growth primarily through microbial breakdown of plant biomass in the rumen. Several biotic and abiotic factors influence the efficiency of fiber degradation, which can ultimately impact animal productivity and health. To provide more insight into mechanisms involved in the modulation of fibrolytic activity, a functional DNA microarray targeting genes encoding key enzymes involved in cellulose and hemicellulose degradation by rumen microbiota was designed. Eight carbohydrate-active enzyme (CAZyme) families (GH5, GH9, GH10, GH11, GH43, GH48, CE1, and CE6) were selected which represented 392 genes from bacteria, protozoa, and fungi. The DNA microarray, designated as FibroChip, was validated using targets of increasing complexity and demonstrated sensitivity and specificity. In addition, FibroChip was evaluated for its explorative and semi-quantitative potential. Differential expression of CAZyme genes was evidenced in the rumen bacterium Fibrobacter succinogenes S85 grown on wheat straw or cellobiose. FibroChip was used to identify the expressed CAZyme genes from the targeted families in the rumen of a cow fed a mixed diet based on grass silage. Among expressed genes, those encoding GH43, GH5, and GH10 families were the most represented. Most of the F. succinogenes genes detected by the FibroChip were also detected following RNA-seq analysis of RNA transcripts obtained from the rumen fluid sample. Use of the FibroChip also indicated that transcripts of fiber degrading enzymes derived from eukaryotes (protozoa and anaerobic fungi) represented a significant proportion of the total microbial mRNA pool. FibroChip represents a reliable and high-throughput tool that enables researchers to monitor active members of fiber degradation in the rumen.

  20. Isolation of Microarray-Grade Total RNA, MicroRNA, and DNA from a Single PAXgene Blood RNA Tube

    DEFF Research Database (Denmark)

    Kruhøffer, Mogens; Andersen, Lars Dyrskjøt; Voss, Thorsten

    2007-01-01

    We have developed a procedure for isolation of microRNA and genomic DNA in addition to total RNA from whole blood stabilized in PAXgene Blood RNA tubes. The procedure is based on automatic extraction on a BioRobot MDx and includes isolation of DNA from a fraction of the stabilized blood...... and recovery of small RNA species that are otherwise lost. The procedure presented here is suitable for large-scale experiments and is amenable to further automation. Procured total RNA and DNA was tested using Affymetrix Expression and single-nucleotide polymorphism GeneChips, respectively, and isolated micro......RNA was tested using spotted locked nucleic acid-based microarrays. We conclude that the yield and quality of total RNA, microRNA, and DNA from a single PAXgene blood RNA tube is sufficient for downstream microarray analysis....

  1. The intraclass correlation coefficient applied for evaluation of data correction, labeling methods and rectal biopsy sampling in DNA microarray experiments

    NARCIS (Netherlands)

    Pellis, E.P.M.; Franssen-Hal, van N.L.W.; Burema, J.; Keijer, J.

    2003-01-01

    We show that the intraclass correlation coefficient (ICC) can be used as a relatively simple statistical measure to assess methodological and biological variation in DNA microarray analysis. The ICC is a measure that determines the reproducibility of a variable, which can easily be calculated from

  2. Use of a multi-thermal washer for DNA microarrays simplifies probe design and gives robust genotyping assays

    DEFF Research Database (Denmark)

    Petersen, J.; Poulsen, Lena; Petronis, S.

    2008-01-01

    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......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...

  3. The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

    Science.gov (United States)

    Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

    2009-01-01

    We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

  4. The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy

    Directory of Open Access Journals (Sweden)

    Pesta David

    2003-06-01

    Full Text Available Abstract Background DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein. We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions The technique detailed

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

    Directory of Open Access Journals (Sweden)

    Gravelat Fabrice

    2010-09-01

    Full Text Available Abstract 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lu [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Xianwei [School of Life Sciences, Shandong University, Jinan 250100 (China); Zhang, Xiaoli [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China); Wang, Jinxing [School of Life Sciences, Shandong University, Jinan 250100 (China); Jin, Wenrui, E-mail: jwr@sdu.edu.cn [School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100 (China)

    2015-01-07

    Highlights: • A single-molecule-detection (SMD) microarray for 10 samples is fabricated. • The based-SMD microarray assay (SMA) can determine 8 DNAs for each sample. • The limit of detection of SMA is as low as 1.3 × 10{sup −16} mol L{sup −1}. • The SMA can be applied in single-cell multiple gene expression analysis. - Abstract: 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{sup −16} mol L{sup −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

  7. Integration of Multiplexed Microfluidic Electrokinetic Concentrators with a Morpholino Microarray via Reversible Surface Bonding for Enhanced DNA Hybridization.

    Science.gov (United States)

    Martins, Diogo; Wei, Xi; Levicky, Rastislav; Song, Yong-Ak

    2016-04-05

    We describe a microfluidic concentration device to accelerate the surface hybridization reaction between DNA and morpholinos (MOs) for enhanced detection. The microfluidic concentrator comprises a single polydimethylsiloxane (PDMS) microchannel onto which an ion-selective layer of conductive polymer poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) ( PSS) was directly printed and then reversibly surface bonded onto a morpholino microarray for hybridization. Using this electrokinetic trapping concentrator, we could achieve a maximum concentration factor of ∼800 for DNA and a limit of detection of 10 nM within 15 min. In terms of the detection speed, it enabled faster hybridization by around 10-fold when compared to conventional diffusion-based hybridization. A significant advantage of our approach is that the fabrication of the microfluidic concentrator is completely decoupled from the microarray; by eliminating the need to deposit an ion-selective layer on the microarray surface prior to device integration, interfacing between both modules, the PDMS chip for electrokinetic concentration and the substrate for DNA sensing are easier and applicable to any microarray platform. Furthermore, this fabrication strategy facilitates a multiplexing of concentrators. We have demonstrated the proof-of-concept for multiplexing by building a device with 5 parallel concentrators connected to a single inlet/outlet and applying it to parallel concentration and hybridization. Such device yielded similar concentration and hybridization efficiency compared to that of a single-channel device without adding any complexity to the fabrication and setup. These results demonstrate that our concentrator concept can be applied to the development of a highly multiplexed concentrator-enhanced microarray detection system for either genetic analysis or other diagnostic assays.

  8. A simple gold nanoparticle-mediated immobilization method to fabricate highly homogeneous DNA microarrays having higher capacities than those prepared by using conventional techniques

    International Nuclear Information System (INIS)

    Jung, Cheulhee; Mun, Hyo Young; Li, Taihua; Park, Hyun Gyu

    2009-01-01

    A simple, highly efficient immobilization method to fabricate DNA microarrays, that utilizes gold nanoparticles as the mediator, has been developed. The fabrication method begins with electrostatic attachment of amine-modified DNA to gold nanoparticles. The resulting gold-DNA complexes are immobilized on conventional amine or aldehyde functionalized glass slides. By employing gold nanoparticles as the immobilization mediator, implementation of this procedure yields highly homogeneous microarrays that have higher binding capacities than those produced by conventional methods. This outcome is due to the increased three-dimensional immobilization surface provided by the gold nanoparticles as well as the intrinsic effects of gold on emission properties. This novel immobilization strategy gives microarrays that produce more intense hybridization signals for the complementary DNA. Furthermore, the silver enhancement technique, made possible only in the case of immobilized gold nanoparticles on the microarrays, enables simple monitoring of the integrity of the immobilized DNA probe.

  9. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with {sup 33}P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells.

  10. Verification of animal species in ham and salami by dna microarray and real time pcr methods

    Directory of Open Access Journals (Sweden)

    Zuzana Drdolová

    2017-01-01

    Full Text Available Consumer protection and detecting of adulteration is very important and has a wide societal impact in the economic sphere. Detection of animal species in meat products and the use of combining different methods is one of the means to achieve relevant product status. The aim of this study was to reveal whether or not the products label clearly meets the content declared by producer. In our study, 29 samples of meat products such as salami and ham obtained from stores and supermarkets in Slovakia were analyzed to detect the existing animal species according to the product label the use of Chipron LCD Array Analysis System, Meat 5.0. Products in which the presence of non-declared animal species has been detected were subjected to testing by the innuDETECT PCR Real-Time Kit, repeatedly. The results showed that 20 (68.96% samples were improperly labeled. From in total 14 tested ham samples 11 (78.57% products exhibited non-conformity with declared composition. Tested salami samples (15 revealed 9 (60% incorrectly labelled products. The results obtained by DNA Microarray and Real Time PCR methods were identical, and both methods should be extensively promoted for the detection of animal species in the meat and meat products. Normal 0 21 false false false EN-GB X-NONE X-NONE

  11. Gene expression of panaxydol-treated human melanoma cells using radioactive cDNA microarrays

    International Nuclear Information System (INIS)

    Cho, Joong Youn; Yu, Su Jin; Soh, Jeong Won; Kim, Meyoung Kon

    2001-01-01

    Polyacetylenic alcohols derived from Panax ginseng have been studied to be an anticancer reagent previously. One of the Panax ginseng polyacetylenic alcohols, i.e., panaxydol, has been studied to possess an antiproliferative effect on human melanoma cell line (SK-MEL-1). In ths study, radioactive cDNA microarrays enabled an efficient approach to analyze the pattern of gene expression (3.194 genes in a total) simultaneously. The bioinformatics selection of human cDNAs, which is specifically designed for immunology, apoptosis and signal transduction, were arrayed on nylon membranes. Using with 33 P labeled probes, this method provided highly sensitive gene expression profiles of our interest including apoptosis, cell proliferation, cell cycle, and signal transduction. Gene expression profiles were also classified into several categories in accordance with the duration of panaxydol treatment. Consequently, the gene profiles of our interest were significantly up (199 genes, > 2.0 of Z-ratio) or down-(196 genes, < 2.0 of Z-ratio) regulated in panaxydol-treated human melanoma cells

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

  14. Characterization of methicillin-resistant Staphylococcus aureus isolated from healthy turkeys and broilers using DNA microarrays

    Directory of Open Access Journals (Sweden)

    Hosny El-Adawy

    2016-12-01

    Full Text Available Methicillin-resistant Staphylococcus aureus (MRSA is a major human health problem and recently, domestic animals, in particular pigs and poultry are discussed as carriers and possible reservoirs of MRSA. 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

  15. Global pathway analysis using DNA microarrays in skeletal muscle of women with polycystic ovary syndrome

    DEFF Research Database (Denmark)

    Skov, Vibe

    2007-01-01

    (study 1), to investigate whether pioglitazone therapy could reverse abnormalities in the transcriptional profile of muscle associated with insulin resistance in skeletal muscle of obese PCOS patients (study 2), and to develop a microarray platform for global gene expression profiling (study 3). In study...... comparable to other commercial and custom made microarrays and is a cost-effective alternative especially in larger epidemiological studies....

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  18. Ancient bacteria show evidence of DNA repair

    DEFF Research Database (Denmark)

    Johnson, Sarah Stewart; Hebsgaard, Martin B; Christensen, Torben R

    2007-01-01

    -term survival of bacteria sealed in frozen conditions for up to one million years. Our results show evidence of bacterial survival in samples up to half a million years in age, making this the oldest independently authenticated DNA to date obtained from viable cells. Additionally, we find strong evidence...... geological timescales. There has been no direct evidence in ancient microbes for the most likely mechanism, active DNA repair, or for the metabolic activity necessary to sustain it. In this paper, we couple PCR and enzymatic treatment of DNA with direct respiration measurements to investigate long...... that this long-term survival is closely tied to cellular metabolic activity and DNA repair that over time proves to be superior to dormancy as a mechanism in sustaining bacteria viability....

  19. Construction and application of a bovine immune-endocrine cDNA microarray.

    Science.gov (United States)

    Tao, Wenjing; Mallard, Bonnie; Karrow, Niel; Bridle, Byram

    2004-09-01

    A variety of commercial DNA arrays specific for humans and rodents are widely available; however, microarrays containing well-characterized genes to study pathway-specific gene expression are not as accessible for domestic animals, such as cattle, sheep and pigs. Therefore, a small-scale application-targeted bovine immune-endocrine cDNA array was developed to evaluate genetic pathways involved in the immune-endocrine axis of cattle during periods of altered homeostasis provoked by physiological or environmental stressors, such as infection, vaccination or disease. For this purpose, 167 cDNA sequences corresponding to immune, endocrine and inflammatory response genes were collected and categorized. Positive controls included 5 housekeeping genes (glyceraldehydes-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase, ribosomal protein L19, beta-actin, beta2-microglobulin) and bovine genomic DNA. Negative controls were a bacterial gene (Rhodococcus equi 17-kDa virulence-associated protein) and a partial sequence of the plasmid pACYC177. In addition, RNA extracted from un-stimulated, as well as superantigen (Staphylococcus aureus enterotoxin-A, S. aureus Cowan Pansorbin Cells) and mitogen-stimulated (LPS, ConA) bovine blood leukocytes was mixed, reverse transcribed and PCR amplified using gene-specific primers. The endocrine-associated genes were amplified from cDNA derived from un-stimulated bovine hypothalamus, pituitary, adrenal and thyroid gland tissues. The array was constructed in 4 repeating grids of 180 duplicated spots by coupling the PCR amplified 213-630 bp gene fragments onto poly-l-lysine coated glass slides. The bovine immune-endocrine arrays were standardized and preliminary gene expression profiles generated using Cy3 and Cy5 labelled cDNA from un-stimulated and ConA (5 microg/ml) stimulated PBMC of 4 healthy Holstein cows (2-4 replicate arrays/cow) in a time course study. Mononuclear cell-derived cytokine and chemokine (IL-2, IL-1alpha

  20. DNA microarray global gene expression analysis of influenza virus-infected chicken and duck cells

    Directory of Open Access Journals (Sweden)

    Suresh V. Kuchipudi

    2015-06-01

    Full Text Available The data described in this article pertain to the article by Kuchipudi et al. (2014 titled “Highly Pathogenic Avian Influenza Virus Infection in Chickens But Not Ducks Is Associated with Elevated Host Immune and Pro-inflammatory Responses” [1]. While infection of chickens with highly pathogenic avian influenza (HPAI H5N1 virus subtypes often leads to 100% mortality within 1 to 2 days, infection of ducks in contrast causes mild or no clinical signs. The rapid onset of fatal disease in chickens, but with no evidence of severe clinical symptoms in ducks, suggests underlying differences in their innate immune mechanisms. We used Chicken Genechip microarrays (Affymetrix to analyse the gene expression profiles of primary chicken and duck lung cells infected with a low pathogenic avian influenza (LPAI H2N3 virus and two HPAI H5N1 virus subtypes to understand the molecular basis of host susceptibility and resistance in chickens and ducks. Here, we described the experimental design, quality control and analysis that were performed on the data set. The data are publicly available through the Gene Expression Omnibus (GEOdatabase with accession number GSE33389, and the analysis and interpretation of these data are included in Kuchipudi et al. (2014 [1].

  1. DNA-microarrays identification of Streptococcus mutans genes associated with biofilm thickness

    Directory of Open Access Journals (Sweden)

    Feldman Mark

    2008-12-01

    Full Text Available Abstract Background A biofilm is a complex community of microorganisms that develop on surfaces in diverse environments. The thickness of the biofilm plays a crucial role in the physiology of the immobilized bacteria. The most cariogenic bacteria, mutans streptococci, are common inhabitants of a dental biofilm community. In this study, DNA-microarray analysis was used to identify differentially expressed genes associated with the thickness of S. mutans biofilms. Results Comparative transcriptome analyses indicated that expression of 29 genes was differentially altered in 400- vs. 100-microns depth and 39 genes in 200- vs. 100-microns biofilms. Only 10 S. mutans genes showed differential expression in both 400- vs. 100-microns and 200- vs. 100-microns biofilms. All of these genes were upregulated. As sucrose is a predominant factor in oral biofilm development, its influence was evaluated on selected genes expression in the various depths of biofilms. The presence of sucrose did not noticeably change the regulation of these genes in 400- vs. 100-microns and/or 200- vs. 100-microns biofilms tested by real-time RT-PCR. Furthermore, we analyzed the expression profile of selected biofilm thickness associated genes in the luxS- mutant strain. The expression of those genes was not radically changed in the mutant strain compared to wild-type bacteria in planktonic condition. Only slight downregulation was recorded in SMU.2146c, SMU.574, SMU.609, and SMU.987 genes expression in luxS- bacteria in biofilm vs. planktonic environments. Conclusion These findings reveal genes associated with the thickness of biofilms of S. mutans. Expression of these genes is apparently not regulated directly by luxS and is not necessarily influenced by the presence of sucrose in the growth media.

  2. DNA Microarrays: a Powerful Genomic Tool for Biomedical and Clinical Research

    OpenAIRE

    Trevino, Victor; Falciani, Francesco; Barrera-Saldaña, Hugo A

    2007-01-01

    Among the many benefits of the Human Genome Project are new and powerful tools such as the genome-wide hybridization devices referred to as microarrays. Initially designed to measure gene transcriptional levels, microarray technologies are now used for comparing other genome features among individuals and their tissues and cells. Results provide valuable information on disease subcategories, disease prognosis, and treatment outcome. Likewise, they reveal differences in genetic makeup, regulat...

  3. An information gap in DNA evidence interpretation.

    Directory of Open Access Journals (Sweden)

    Mark W Perlin

    Full Text Available Forensic DNA evidence often contains mixtures of multiple contributors, or is present in low template amounts. The resulting data signals may appear to be relatively uninformative when interpreted using qualitative inclusion-based methods. However, these same data can yield greater identification information when interpreted by computer using quantitative data-modeling methods. This study applies both qualitative and quantitative interpretation methods to a well-characterized DNA mixture and dilution data set, and compares the inferred match information. The results show that qualitative interpretation loses identification power at low culprit DNA quantities (below 100 pg, but that quantitative methods produce useful information down into the 10 pg range. Thus there is a ten-fold information gap that separates the qualitative and quantitative DNA mixture interpretation approaches. With low quantities of culprit DNA (10 pg to 100 pg, computer-based quantitative interpretation provides greater match sensitivity.

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

    Science.gov (United States)

    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.

  5. cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

    Science.gov (United States)

    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

    2018-01-01

    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.

  6. Effects of ingested turmeric oleoresin on glucose and lipid metabolisms in obese diabetic mice: a DNA microarray study.

    Science.gov (United States)

    Honda, Shinichi; Aoki, Fumiki; Tanaka, Hozumi; Kishida, Hideyuki; Nishiyama, Tozo; Okada, Shinji; Matsumoto, Ichiro; Abe, Keiko; Mae, Tatsumasa

    2006-11-29

    Turmeric, the rhizome of Curcuma longa L., has a wide range of effects on human health. Turmeric oleoresin, an extract of turmeric, is often used for flavoring and coloring. Curcuminoids and turmeric essential oil are both contained in turmeric oleoresin, and both of these fractions have hypoglycemic effects. In the present study, we comprehensively assessed the effect of turmeric oleoresin on hepatic gene expression in obese diabetic KK-Ay mice using DNA microarray analysis and quantitative real-time polymerase chain reaction (PCR). Female KK-Ay mice aged 6 weeks (n = 6/group) were fed a high-fat diet containing turmeric oleoresin, curcuminoids, and essential oil for 5 weeks. The same diet without any of these fractions was used as a control diet. Ingestion of turmeric oleoresin and essential oil inhibited the development of increased blood glucose and abdominal fat mass, while curcuminoids only inhibited the increase in blood glucose. DNA microarray analysis indicated that turmeric oleoresin ingestion up-regulated the expression of genes related to glycolysis, beta-oxidation, and cholesterol metabolism in the liver of KK-Ay mice, while expression of gluconeogenesis-related genes was down-regulated. Real-time PCR analysis was conducted to assess the contribution of the curcuminoids and essential oil in turmeric oleoresin to the changes in expression of representative genes selected by DNA microarray analysis. This analysis suggested that curcuminoids regulated turmeric oleoresin ingestion-induced expression of glycolysis-related genes and also that curcuminoids and turmeric essential oil acted synergistically to regulate the peroxisomal beta-oxidation-related gene expression induced by turmeric oleoresin ingestion. These changes in gene expression were considered to be the mechanism by which the turmeric oleoresin affected the control of both blood glucose levels and abdominal adipose tissue masses. All of these results suggest that the use of whole turmeric

  7. cDNA microarrays as a tool for identification of biomineralization proteins in the coccolithophorid Emiliania huxleyi (Haptophyta).

    Science.gov (United States)

    Quinn, Patrick; Bowers, Robert M; Zhang, Xiaoyu; Wahlund, Thomas M; Fanelli, Michael A; Olszova, Daniela; Read, Betsy A

    2006-08-01

    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.

  8. Methods for interpreting lists of affected genes obtained in a DNA microarray experiment

    DEFF Research Database (Denmark)

    Hedegaard, Jakob; Arce, Christina; Bicciato, Silvio

    2009-01-01

    The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microa...... a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria...

  9. PNA-PEG modified silicon platforms as functional bio-interfaces for applications in DNA microarrays and biosensors.

    Science.gov (United States)

    Cattani-Scholz, Anna; Pedone, Daniel; Blobner, Florian; Abstreiter, Gerhard; Schwartz, Jeffrey; Tornow, Marc; Andruzzi, Luisa

    2009-03-09

    promising substrates for DNA microarray applications.

  10. Identifying molecular subtypes in human colon cancer using gene expression and DNA methylation microarray data.

    Science.gov (United States)

    Ren, Zhonglu; Wang, Wenhui; Li, Jinming

    2016-02-01

    Identifying colon cancer subtypes based on molecular signatures may allow for a more rational, patient-specific approach to therapy in the future. Classifications using gene expression data have been attempted before with little concordance between the different studies carried out. In this study we aimed to uncover subtypes of colon cancer that have distinct biological characteristics and identify a set of novel biomarkers which could best reflect the clinical and/or biological characteristics of each subtype. Clustering analysis and discriminant analysis were utilized to discover the subtypes in two different molecular levels on 153 colon cancer samples from The Cancer Genome Atlas (TCGA) Data Portal. At gene expression level, we identified two major subtypes, ECL1 (expression cluster 1) and ECL2 (expression cluster 2) and a list of signature genes. Due to the heterogeneity of colon cancer, the subtype ECL1 can be further subdivided into three nested subclasses, and HOTAIR were found upregulated in subclass 2. At DNA methylation level, we uncovered three major subtypes, MCL1 (methylation cluster 1), MCL2 (methylation cluster 2) and MCL3 (methylation cluster 3). We found only three subtypes of CpG island methylator phenotype (CIMP) in colon cancer instead of the four subtypes in the previous reports, and we found no sufficient evidence to subdivide MCL3 into two distinct subgroups.

  11. Aligning ontologies and integrating textual evidence for pathway analysis of microarray data

    Energy Technology Data Exchange (ETDEWEB)

    Gopalan, Banu; Posse, Christian; Sanfilippo, Antonio P.; Stenzel-Poore, Mary; Stevens, S.L.; Castano, Jose; Beagley, Nathaniel; Riensche, Roderick M.; Baddeley, Bob; Simon, R.P.; Pustejovsky, James

    2006-10-08

    Expression arrays are introducing a paradigmatic change in biology by shifting experimental approaches from single gene studies to genome-level analysis, monitoring the ex-pression levels of several thousands of genes in parallel. The massive amounts of data obtained from the microarray data needs to be integrated and interpreted to infer biological meaning within the context of information-rich pathways. In this paper, we present a methodology that integrates textual information with annotations from cross-referenced ontolo-gies to map genes to pathways in a semi-automated way. We illustrate this approach and compare it favorably to other tools by analyzing the gene expression changes underlying the biological phenomena related to stroke. Stroke is the third leading cause of death and a major disabler in the United States. Through years of study, researchers have amassed a significant knowledge base about stroke, and this knowledge, coupled with new technologies, is providing a wealth of new scientific opportunities. The potential for neu-roprotective stroke therapy is enormous. However, the roles of neurogenesis, angiogenesis, and other proliferative re-sponses in the recovery process following ischemia and the molecular mechanisms that lead to these processes still need to be uncovered. Improved annotation of genomic and pro-teomic data, including annotation of pathways in which genes and proteins are involved, is required to facilitate their interpretation and clinical application. While our approach is not aimed at replacing existing curated pathway databases, it reveals multiple hidden relationships that are not evident with the way these databases analyze functional groupings of genes from the Gene Ontology.

  12. Prevalence, identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia.

    Science.gov (United States)

    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.

  13. Development of a microarray-based assay for efficient testing of new HSP70/DnaK inhibitors.

    Science.gov (United States)

    Mohammadi-Ostad-Kalayeh, Sona; Hrupins, Vjaceslavs; Helmsen, Sabine; Ahlbrecht, Christin; Stahl, Frank; Scheper, Thomas; Preller, Matthias; Surup, Frank; Stadler, Marc; Kirschning, Andreas; Zeilinger, Carsten

    2017-12-15

    A facile method for testing ATP binding in a highly miniaturized microarray environment using human HSP70 and DnaK from Mycobacterium tuberculosis as biological targets is reported. Supported by molecular modelling studies we demonstrate that the position of the fluorescence label on ATP has a strong influence on the binding to human HSP70. Importantly, the label has to be positioned on the adenine ring and not to the terminal phosphate group. Unlabelled ATP displaced bound Cy5-ATP from HSP70 in the micromolar range. The affinity of a well-known HSP70 inhibitor VER155008 for the ATP binding site in HSP70 was determined, with a EC 50 in the micromolar range, whereas reblastin, a HSP90-inhibitor, did not compete for ATP in the presence of HSP70. The applicability of the method was demonstrated by screening a small compound library of natural products. This unraveled that terphenyls rickenyl A and D, recently isolated from cultures of the fungus Hypoxylon rickii, are inhibitors of HSP70. They compete with ATP for the chaperone in the range of 29 µM (Rickenyl D) and 49 µM (Rickenyl A). Furthermore, the microarray-based test system enabled protein-protein interaction analysis using full-length HSP70 and HSP90 proteins. The labelled full-length human HSP90 binds with a half-maximal affinity of 5.5 µg/ml (∼40 µM) to HSP70. The data also demonstrate that the microarray test has potency for many applications from inhibitor screening to target-oriented interaction studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens

    International Nuclear Information System (INIS)

    Sadi, Al Muktafi; Wang, Dong-Yu; Youngson, Bruce J; Miller, Naomi; Boerner, Scott; Done, Susan J; Leong, Wey L

    2011-01-01

    The ability of gene profiling to predict treatment response and prognosis in breast cancers has been demonstrated in many studies using DNA microarray analyses on RNA from fresh frozen tumor specimens. In certain clinical and research situations, performing such analyses on archival formalin fixed paraffin-embedded (FFPE) surgical specimens would be advantageous as large libraries of such specimens with long-term follow-up data are widely available. However, FFPE tissue processing can cause fragmentation and chemical modifications of the RNA. A number of recent technical advances have been reported to overcome these issues. Our current study evaluates whether or not the technology is ready for clinical applications. A modified RNA extraction method and a recent DNA microarray technique, cDNA-mediated annealing, selection, extension and ligation (DASL, Illumina Inc) were evaluated. The gene profiles generated from FFPE specimens were compared to those obtained from paired fresh fine needle aspiration biopsies (FNAB) of 25 breast cancers of different clinical subtypes (based on ER and Her2/neu status). Selected RNA levels were validated using RT-qPCR, and two public databases were used to demonstrate the prognostic significance of the gene profiles generated from FFPE specimens. Compared to FNAB, RNA isolated from FFPE samples was relatively more degraded, nonetheless, over 80% of the RNA samples were deemed suitable for subsequent DASL assay. Despite a higher noise level, a set of genes from FFPE specimens correlated very well with the gene profiles obtained from FNAB, and could differentiate breast cancer subtypes. Expression levels of these genes were validated using RT-qPCR. Finally, for the first time we correlated gene expression profiles from FFPE samples to survival using two independent microarray databases. Specifically, over-expression of ANLN and KIF2C, and under-expression of MAPT strongly correlated with poor outcomes in breast cancer patients. We

  15. Identification of Differentially Expressed IGFBP5-Related Genes in Breast Cancer Tumor Tissues Using cDNA Microarray Experiments.

    Science.gov (United States)

    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.

  16. Evaluation of DNA microarray results in the Toxicogenomics Project (TGP) consortium in Japan.

    Science.gov (United States)

    Noriyuki, Nakatsu; Igarashi, Yoshinobu; Ono, Atsushi; Yamada, Hiroshi; Ohno, Yasuo; Urushidani, Tetsuro

    2012-01-01

    An important technology used in toxicogenomic drug discovery research is the microarray, which enables researchers to simultaneously analyze the expression of a large number of genes. To build a database and data analysis system for use in assessing the safety of drugs and drug candidates, in 2002 we conducted a 5-year collaborative study in the Toxicogenomics Project (TGP1) in Japan. Experimental data generated by such studies must be validated by different laboratories for robust and accurate analysis. For this purpose, we conducted intra- and inter-laboratory validation studies with participating companies in the second collaborative study in the Toxicogenomics Project (TGP2). Gene expression in the liver of rats treated with acetaminophen (APAP) was independently examined by the participating companies using Affymetrix GeneChip microarrays. The intra- and inter-laboratory reproducibility of the data was evaluated using hierarchical clustering analysis. The toxicogenomics results were highly reproducible, indicating that the gene expression data generated in our TGP1 project is reliable and compatible with the data generated by the participating laboratories.

  17. cDNA Microarray Analysis of Serially Sampled Cervical Cancer Specimens From Patients Treated With Thermochemoradiotherapy

    International Nuclear Information System (INIS)

    Borkamo, Erling Dahl; Schem, Baard-Christian; Fluge, Oystein; Bruland, Ove; Dahl, Olav; Mella, Olav

    2009-01-01

    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 κB; 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.

  18. BioconductorBuntu: a Linux distribution that implements a web-based DNA microarray analysis server.

    Science.gov (United States)

    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).

  19. Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80. Analysis by cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Chan, John Y.H.; Chen, Lung-Kun; Chang, Jui-Feng [National Yang Ming Univ., Taipei, Taiwan (China). Inst. of Radiological Sciences] (and others)

    2001-12-01

    The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes. (author)

  20. Differential gene expression in a DNA double-strand-break repair mutant XRS-5 defective in Ku80. Analysis by cDNA microarray

    International Nuclear Information System (INIS)

    Chan, John Y.H.; Chen, Lung-Kun; Chang, Jui-Feng

    2001-01-01

    The ability of cells to rejoin DNA double-strand breaks (DSBs) usually correlates with their radiosensitivity. This correlation has been demonstrated in radiosensitive cells, including the Chinese hamster ovary mutant XRS-5. XRS-5 is defective in a DNA end-binding protein, Ku80, which is a component of a DNA-dependent protein kinase complex used for joining strand breaks. However, Ku80-deficient cells are known to be retarded in cell proliferation and growth as well as other yet to be identified defects. Using custom-made 600-gene cDNA microarray filters, we found differential gene expressions between the wild-type and XRS-5 cells. Defective Ku80 apparently affects the expression of several repair genes, including topoisomerase-I and -IIA, ERCC5, MLH1, and ATM. In contrast, other DNA repair-associated genes, such as GADD45A, EGR1 MDM2 and p53, were not affected. In addition, for large numbers of growth-associated genes, such as cyclins and clks, the growth factors and cytokines were also affected. Down-regulated expression was also found in several categories of seemingly unrelated genes, including apoptosis, angiogenesis, kinase and signaling, phosphatase, stress protein, proto-oncogenes and tumor suppressors, transcription and translation factors. A RT-PCR analysis confirmed that the XRS-5 cells used were defective in Ku80 expression. The diversified groups of genes being affected could mean that Ku80, a multi-functional DNA-binding protein, not only affects DNA repair, but is also involved in transcription regulation. Our data, taken together, indicate that there are specific genes being modulated in Ku80- deficient cells, and that some of the DNA repair pathways and other biological functions are apparently linked, suggesting that a defect in one gene could have global effects on many other processes. (author)

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

    Science.gov (United States)

    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.

  2. Discovery of possible gene relationships through the application of self-organizing maps to DNA microarray databases.

    Science.gov (United States)

    Chavez-Alvarez, Rocio; Chavoya, Arturo; Mendez-Vazquez, Andres

    2014-01-01

    DNA microarrays and cell cycle synchronization experiments have made possible the study of the mechanisms of cell cycle regulation of Saccharomyces cerevisiae by simultaneously monitoring the expression levels of thousands of genes at specific time points. On the other hand, pattern recognition techniques can contribute to the analysis of such massive measurements, providing a model of gene expression level evolution through the cell cycle process. In this paper, we propose the use of one of such techniques--an unsupervised artificial neural network called a Self-Organizing Map (SOM)-which has been successfully applied to processes involving very noisy signals, classifying and organizing them, and assisting in the discovery of behavior patterns without requiring prior knowledge about the process under analysis. As a test bed for the use of SOMs in finding possible relationships among genes and their possible contribution in some biological processes, we selected 282 S. cerevisiae genes that have been shown through biological experiments to have an activity during the cell cycle. The expression level of these genes was analyzed in five of the most cited time series DNA microarray databases used in the study of the cell cycle of this organism. With the use of SOM, it was possible to find clusters of genes with similar behavior in the five databases along two cell cycles. This result suggested that some of these genes might be biologically related or might have a regulatory relationship, as was corroborated by comparing some of the clusters obtained with SOMs against a previously reported regulatory network that was generated using biological knowledge, such as protein-protein interactions, gene expression levels, metabolism dynamics, promoter binding, and modification, regulation and transport of proteins. The methodology described in this paper could be applied to the study of gene relationships of other biological processes in different organisms.

  3. SVD identifies transcript length distribution functions from DNA microarray data and reveals evolutionary forces globally affecting GBM metabolism.

    Directory of Open Access Journals (Sweden)

    Nicolas M Bertagnolli

    Full Text Available 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.

  4. Development of a cDNA microarray for the measurement of gene expression in the sheep scab mite Psoroptes ovis

    Directory of Open Access Journals (Sweden)

    Burgess Stewart TG

    2012-02-01

    Full Text Available Abstract 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.

  5. Rapid extraction of genomic DNA from saliva for HLA typing on microarray based on magnetic nanobeads

    Energy Technology Data Exchange (ETDEWEB)

    Xie Xin; Zhang Xu E-mail: shinezhang@hotmail.com; 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. Optimization of the BLASTN substitution matrix for prediction of non-specific DNA microarray hybridization

    DEFF Research Database (Denmark)

    Eklund, Aron Charles; Friis, Pia; Wernersson, Rasmus

    2010-01-01

    BLASTN accuracy by modifying the substitution matrix and gap penalties. We generated gene expression microarray data for samples in which 1 or 10% of the target mass was an exogenous spike of known sequence. We found that the 10% spike induced 2-fold intensity changes in 3% of the probes, two......-third of which were decreases in intensity likely caused by bulk-hybridization. These changes were correlated with similarity between the spike and probe sequences. Interestingly, even very weak similarities tended to induce a change in probe intensity with the 10% spike. Using this data, we optimized the BLASTN...... substitution matrix to more accurately identify probes susceptible to non-specific hybridization with the spike. Relative to the default substitution matrix, the optimized matrix features a decreased score for A–T base pairs relative to G–C base pairs, resulting in a 5–15% increase in area under the ROC curve...

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

    Science.gov (United States)

    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.

  8. DNA microarray revealed and RNAi plants confirmed key genes conferring low Cd accumulation in barley grains

    DEFF Research Database (Denmark)

    Sun, Hongyan; Chen, Zhong-Hua; Chen, Fei

    2015-01-01

    Background Understanding the mechanism of low Cd accumulation in crops is crucial for sustainable safe food production in Cd-contaminated soils. Results Confocal microscopy, atomic absorption spectrometry, gas exchange and chlorophyll fluorescence analyses revealed a distinct difference in Cd...... with a substantial difference between the two genotypes. Cd stress led to higher expression of genes involved in transport, carbohydrate metabolism and signal transduction in the low-grain-Cd-accumulating genotype. Novel transporter genes such as zinc transporter genes were identified as being associated with low Cd...... accumulation. Quantitative RT-PCR confirmed our microarray data. Furthermore, suppression of the zinc transporter genes HvZIP3 and HvZIP8 by RNAi silencing showed increased Cd accumulation and reduced Zn and Mn concentrations in barley grains. Thus, HvZIP3 and HvZIP8 could be candidate genes related to low...

  9. Correlating gene expression to physiological parameters and environmental conditons during cold acclimation of Pinus sylvestris, identification of molecular markers using cDNA microarrays

    NARCIS (Netherlands)

    Joosen, R.V.L.; Lammers, M.; Balk, P.A.; Bronnum, P.; Konings, M.C.J.M.; Perks, M.; Stattin, E.; Wordragen, van M.F.; Geest, van der A.H.M.

    2006-01-01

    Scots pine (Pinus sylvestris L.) seedlings were grown under different conditions (three field locations, two seasons and two climate room regimes), and then analyzed for freezing tolerance of shoots and roots and for transcript abundance in apical buds based on a cDNA microarray containing about

  10. DNA microarray genotyping and virulence and antimicrobial resistance gene profiling of methicillin-resistant Staphylococcus aureus bloodstream isolates from renal patients.

    LENUS (Irish Health Repository)

    McNicholas, Sinead

    2012-02-01

    Thirty-six methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from renal patients were genetically characterized by DNA microarray analysis and spa typing. The isolates were highly clonal, belonging mainly to ST22-MRSA-IV. The immune evasion and enterotoxin gene clusters were found in 29\\/36 (80%) and 33\\/36 (92%) isolates, respectively.

  11. DNA microarray genotyping and virulence and antimicrobial resistance gene profiling of methicillin-resistant Staphylococcus aureus bloodstream isolates from renal patients.

    LENUS (Irish Health Repository)

    McNicholas, Sinead

    2011-12-01

    Thirty-six methicillin-resistant Staphylococcus aureus (MRSA) bloodstream isolates from renal patients were genetically characterized by DNA microarray analysis and spa typing. The isolates were highly clonal, belonging mainly to ST22-MRSA-IV. The immune evasion and enterotoxin gene clusters were found in 29\\/36 (80%) and 33\\/36 (92%) isolates, respectively.

  12. Monitoring expression profiles of rice (Oryza sativa L.) genes under abiotic stresses using cDNA Microarray Analysis (abstract)

    International Nuclear Information System (INIS)

    Rabbani, M.A.

    2005-01-01

    Transcript regulation in response to cold, drought, high salinity and ABA application was investigated in rice (Oryza sativa L., Nipponbare) with microarray analysis including approx. 1700 independent DNA elements derived from three cDNA libraries constructed from 15-day old rice seedlings stressed with drought, cold and high salinity. A total of 141 non-redundant genes were identified, whose expression ratios were more than three-fold compared with the control genes for at least one of stress treatments in microarray analysis. However, after RNA gel blot analysis, a total of 73 genes were identified, among them the transcripts of 36, 62, 57 and 43 genes were found increased after cold, drought, high salinity and ABA application, respectively. Sixteen of these identified genes have been reported previously to be stress inducible in rice, while 57 of which are novel that have not been reported earlier as stress responsive in rice. We observed a strong association in the expression patterns of stress responsive genes and found 15 stress inducible genes that responded to all four treatments. Based on Venn diagram analysis, 56 genes were induced by both drought and high salinity, whereas 22 genes were upregulated by both cold and high salinity stress. Similarly 43 genes were induced by both drought stress and ABA application, while only 17 genes were identified as cold and ABA inducible genes. These results indicated the existence of greater cross talk between drought, ABA and high salinity stress signaling processes than those between cold and ABA, and cold and high salinity stress signaling pathways. The cold, drought, high salinity and ABA inducible genes were classified into four gene groups from their expression profiles. Analysis of data enabled us to identify a number of promoters and possible cis-acting DNA elements of several genes induced by a variety of abiotic stresses by combining expression data with genomic sequence data of rice. Comparative analysis of

  13. Improving cluster-based missing value estimation of DNA microarray data.

    Science.gov (United States)

    Brás, Lígia P; Menezes, José C

    2007-06-01

    We present a modification of the weighted K-nearest neighbours imputation method (KNNimpute) for missing values (MVs) estimation in microarray data based on the reuse of estimated data. The method was called iterative KNN imputation (IKNNimpute) as the estimation is performed iteratively using the recently estimated values. The estimation efficiency of IKNNimpute was assessed under different conditions (data type, fraction and structure of missing data) by the normalized root mean squared error (NRMSE) and the correlation coefficients between estimated and true values, and compared with that of other cluster-based estimation methods (KNNimpute and sequential KNN). We further investigated the influence of imputation on the detection of differentially expressed genes using SAM by examining the differentially expressed genes that are lost after MV estimation. The performance measures give consistent results, indicating that the iterative procedure of IKNNimpute can enhance the prediction ability of cluster-based methods in the presence of high missing rates, in non-time series experiments and in data sets comprising both time series and non-time series data, because the information of the genes having MVs is used more efficiently and the iterative procedure allows refining the MV estimates. More importantly, IKNN has a smaller detrimental effect on the detection of differentially expressed genes.

  14. An Improved Fuzzy Based Missing Value Estimation in DNA Microarray Validated by Gene Ranking

    Directory of Open Access Journals (Sweden)

    Sujay Saha

    2016-01-01

    Full Text Available Most of the gene expression data analysis algorithms require the entire gene expression matrix without any missing values. Hence, it is necessary to devise methods which would impute missing data values accurately. There exist a number of imputation algorithms to estimate those missing values. This work starts with a microarray dataset containing multiple missing values. We first apply the modified version of the fuzzy theory based existing method LRFDVImpute to impute multiple missing values of time series gene expression data and then validate the result of imputation by genetic algorithm (GA based gene ranking methodology along with some regular statistical validation techniques, like RMSE method. Gene ranking, as far as our knowledge, has not been used yet to validate the result of missing value estimation. Firstly, the proposed method has been tested on the very popular Spellman dataset and results show that error margins have been drastically reduced compared to some previous works, which indirectly validates the statistical significance of the proposed method. Then it has been applied on four other 2-class benchmark datasets, like Colorectal Cancer tumours dataset (GDS4382, Breast Cancer dataset (GSE349-350, Prostate Cancer dataset, and DLBCL-FL (Leukaemia for both missing value estimation and ranking the genes, and the results show that the proposed method can reach 100% classification accuracy with very few dominant genes, which indirectly validates the biological significance of the proposed method.

  15. 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.

    Science.gov (United States)

    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

    2017-08-01

    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.

  16. Carbohydrate microarrays

    DEFF Research Database (Denmark)

    Park, Sungjin; Gildersleeve, Jeffrey C; Blixt, Klas Ola

    2012-01-01

    In the last decade, carbohydrate microarrays have been core technologies for analyzing carbohydrate-mediated recognition events in a high-throughput fashion. A number of methods have been exploited for immobilizing glycans on the solid surface in a microarray format. This microarray...... of substrate specificities of glycosyltransferases. This review covers the construction of carbohydrate microarrays, detection methods of carbohydrate microarrays and their applications in biological and biomedical research....

  17. Detection of mutations using microarrays of poly(C)10-poly(T)10 modified DNA probes immobilized on agarose films

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Petersen, Jesper; Stoltenborg, M.

    2006-01-01

    Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation to an ag......Allele-specific hybridization to a DNA microarray call be a useful method for genotyping patient DNA. In this article, we demonstrate that 13- to 17-base oligonucleotides tagged with a poly(T)10-poly(C)10 tail (TC tag), but otherwise unmodified, can be crosslinked by UV light irradiation...... to an agarose film grafted onto unmodified glass. Microarrays of TC-tagged probes immobilized on the agarose film can be used to diagnose Mutations in the human P-globin gene, which encodes the beta-chains in hemoglobin. Although the probes differed widely regarding inciting point temperature (similar to 20...... degrees C), a single stringency wash still gave sufficiently high discrimination signals between perfect match and mismatch probes to allow robust mutation detection. In all, 270 genotypings were performed on patient materials, and no genotype was incorrectly classified. Quality control experiments...

  18. A cDNA microarray, UniShrimpChip, for identification of genes relevant to testicular development in the black tiger shrimp (Penaeus monodon

    Directory of Open Access Journals (Sweden)

    Klinbunga Sirawut

    2011-04-01

    Full Text Available Abstract Background Poor reproductive maturation in captive male broodstock of the black tiger shrimp (Penaeus monodon is one of the serious problems to the farming industries. Without genome sequence, EST libraries of P. monodon were previously constructed to identify transcripts with important biological functions. In this study, a new version of cDNA microarray, UniShrimpChip, was constructed from the Peneaus monodon EST libraries of 12 tissues, containing 5,568 non-redundant cDNA clones from 10,536 unique cDNA in the P. monodon EST database. UniShrimpChip was used to study testicular development by comparing gene expression levels of wild brooders from the West and East coasts of Thailand and domesticated brooders with different ages (10-, 14-, 18-month-old. Results The overall gene expression patterns from the microarray experiments revealed distinct transcriptomic patterns between the wild and domesticated groups. Moreover, differentially expressed genes from the microarray comparisons were identified, and the expression patterns of eight selected transcripts were subsequently confirmed by reverse-transcriptase quantitative PCR (RT-qPCR. Among these, expression levels of six subunits (CSN2, 4, 5, 6, 7a, and 8 of the COP9 signalosome (CSN gene family in wild and different ages of domesticated brooders were examined by RT-qPCR. Among the six subunits, CSN5 and CSN6 were most highly expressed in wild brooders and least expressed in the 18-month-old domesticated group; therefore, their full-length cDNA sequences were characterized. Conclusions This study is the first report to employ cDNA microarray to study testicular development in the black tiger shrimp. We show that there are obvious differences between the wild and domesticated shrimp at the transcriptomic level. Furthermore, our study is the first to investigate the feasibility that the CSN gene family might have involved in reproduction and development of this economically important

  19. Evidence Suggesting Absence of Mitochondrial DNA Methylation

    DEFF Research Database (Denmark)

    Mechta, Mie; Ingerslev, Lars R; Fabre, Odile

    2017-01-01

    , 16S, ND5 and CYTB, suggesting that mtDNA supercoiled structure blocks the access to bisulfite conversion. Here, we identified an artifact of mtDNA bisulfite sequencing that can lead to an overestimation of mtDNA methylation levels. Our study supports that cytosine methylation is virtually absent...

  20. Elucidating the transcription cycle of the UV-inducible hyperthermophilic archaeal virus SSV1 by DNA microarrays

    International Nuclear Information System (INIS)

    Froels, Sabrina; Gordon, Paul M.K.; Panlilio, Mayi Arcellana; Schleper, Christa; Sensen, Christoph W.

    2007-01-01

    The spindle-shaped Sulfolobus virus SSV1 was the first of a series of unusual and uniquely shaped viruses isolated from hyperthermophilic Archaea. Using whole-genome microarrays we show here that the circular 15.5 kb DNA genome of SSV1 exhibits a chronological regulation of its transcription upon UV irradiation, reminiscent to the life cycles of bacteriophages and eukaryotic viruses. The transcriptional cycle starts with a small UV-specific transcript and continues with early transcripts on both its flanks. The late transcripts appear after the onset of viral replication and are extended to their full lengths towards the end of the approximately 8.5 h cycle. While we detected only small differences in genome-wide analysis of the host Sulfolobus solfataricus comparing infected versus uninfected strains, we found a marked difference with respect to the strength and speed of the general UV response of the host. Models for the regulation of the virus cycle, and putative functions of genes in SSV1 are presented

  1. Forensic DNA methylation profiling from evidence material for investigative leads

    Science.gov (United States)

    Lee, Hwan Young; Lee, Soong Deok; Shin, Kyoung-Jin

    2016-01-01

    DNA methylation is emerging as an attractive marker providing investigative leads to solve crimes in forensic genetics. The identification of body fluids that utilizes tissue-specific DNA methylation can contribute to solving crimes by predicting activity related to the evidence material. The age estimation based on DNA methylation is expected to reduce the number of potential suspects, when the DNA profile from the evidence does not match with any known person, including those stored in the forensic database. Moreover, the variation in DNA implicates environmental exposure, such as cigarette smoking and alcohol consumption, thereby suggesting the possibility to be used as a marker for predicting the lifestyle of potential suspect. In this review, we describe recent advances in our understanding of DNA methylation variations and the utility of DNA methylation as a forensic marker for advanced investigative leads from evidence materials. [BMB Reports 2016; 49(7): 359-369] PMID:27099236

  2. Temporal Gene Expression Profiling of the Wheat Leaf Rust Pathosystem Using cDNA Microarray Reveals Differences in Compatible and Incompatible Defence Pathways

    OpenAIRE

    Fofana, Bourlaye; Banks, Travis W.; McCallum, Brent; Strelkov, Stephen E.; Cloutier, Sylvie

    2007-01-01

    In this study, we detail the construction of a custom cDNA spotted microarray containing 7728 wheat ESTs and the use of the array to identify host genes that are differentially expressed upon challenges with leaf rust fungal pathogens. Wheat cultivar RL6003 (Thatcher Lr1) was inoculated with Puccinia triticina virulence phenotypes BBB (incompatible) or TJB (7-2) (compatible) and sampled at four different time points (3, 6, 12, and 24 hours) after inoculation. Transcript expression levels rela...

  3. DNA microarrays immobilized on unmodified plastics in a microfluidic biochip for rapid typing of Avian Influenza Virus

    DEFF Research Database (Denmark)

    Yi, Sun; Perch-Nielsen, Ivan R.; Dufva, Martin

    2011-01-01

    Polymers are widely used for microfluidic systems, but fabrication of microarrays on such materials often requires complicated chemical surface modifications, which hinders the integration of microarrays into microfluidic systems. In this paper, we demonstrate that UV irradiation can be used to d...

  4. DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7

    DEFF Research Database (Denmark)

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong

    2011-01-01

    of RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal....... In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification...

  5. Diagnostic and analytical applications of protein microarrays

    DEFF Research Database (Denmark)

    Dufva, Hans Martin; Christensen, C.B.V.

    2005-01-01

    DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the post...

  6. DNA microarray analysis of the cyanotroph Pseudomonas pseudoalcaligenes CECT5344 in response to nitrogen starvation, cyanide and a jewelry wastewater.

    Science.gov (United States)

    Luque-Almagro, V M; Escribano, M P; Manso, I; Sáez, L P; Cabello, P; Moreno-Vivián, C; Roldán, M D

    2015-11-20

    Pseudomonas pseudoalcaligenes CECT5344 is an alkaliphilic bacterium that can use cyanide as nitrogen source for growth, becoming a suitable candidate to be applied in biological treatment of cyanide-containing wastewaters. The assessment of the whole genome sequence of the strain CECT5344 has allowed the generation of DNA microarrays to analyze the response to different nitrogen sources. The mRNA of P. pseudoalcaligenes CECT5344 cells grown under nitrogen limiting conditions showed considerable changes when compared against the transcripts from cells grown with ammonium; up-regulated genes were, among others, the glnK gene encoding the nitrogen regulatory protein PII, the two-component ntrBC system involved in global nitrogen regulation, and the ammonium transporter-encoding amtB gene. The protein coding transcripts of P. pseudoalcaligenes CECT5344 cells grown with sodium cyanide or an industrial jewelry wastewater that contains high concentration of cyanide and metals like iron, copper and zinc, were also compared against the transcripts of cells grown with ammonium as nitrogen source. This analysis revealed the induction by cyanide and the cyanide-rich wastewater of four nitrilase-encoding genes, including the nitC gene that is essential for cyanide assimilation, the cyanase cynS gene involved in cyanate assimilation, the cioAB genes required for the cyanide-insensitive respiration, and the ahpC gene coding for an alkyl-hydroperoxide reductase that could be related with iron homeostasis and oxidative stress. The nitC and cynS genes were also induced in cells grown under nitrogen starvation conditions. In cells grown with the jewelry wastewater, a malate quinone:oxidoreductase mqoB gene and several genes coding for metal extrusion systems were specifically induced. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  7. The Evidentiary Value of DNA Fingerprint as Criminal Evidence

    Directory of Open Access Journals (Sweden)

    Mussa Masoud Irhouma

    2016-12-01

    Full Text Available The subject of criminal evidence is considered to be one of the greatest challenges that face authorities concerned with fighting crime at all levels. Due to this, authorities try to benefit as much as possible from scientific evidence due to the important role it plays in revealing the identity of criminals or victims in present or past criminal cases against unknown people through the physical traces that are found at the scene of an event, which include biological traces. DNA is one of these scientific evidences which can be benefited from in the field of crime investigation. Despite the importance of DNA technology in this area of work, there is still some debate surrounding its acceptance as criminal evidence. Some experts believe it to be of great importance whereas others cast doubt on its evidentiary value. They attribute this to a number of factors including the experts who are entrusted to examine DNA samples, the laboratories in which DNA analysis takes place, as well as the fact that resorting to DNA as a criminal evidence raises some legal complexities related to the permissibility of using it and the conditions and scope of its use. This paper sheds light on DNA and its evidentiary value among the judiciary in criminal cases by answering a number of questions such as the possibility of forcing a person to undergo DNA analysis or not to do so and to what extent it is to be relied upon as criminal evidence. This paper concluded the importance of DNA and its role in the field of criminal evidence. Despite this, even if the DNA evidence is sufficient in proving the innocence of the accused, it is only an indication that must not be solely relied upon and treated as a single conclusive evidence, particularly in cases that involve prescribed Islamic or retributive punishments.

  8. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparisons with Other Methods

    International Nuclear Information System (INIS)

    Wu, Liyou; Yi, T.Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-01-01

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site (Hanford Reach of the Columbia River (HRCR), 11 strains), Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  9. Phylogenetic Analysis of Shewanella Strains by DNA Relatedness Derived from Whole Genome Microarray DNA-DNA Hybridization and Comparison with Other Methods

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Liyou; Yi, T. Y.; Van Nostrand, Joy; Zhou, Jizhong

    2010-05-17

    Phylogenetic analyses were done for the Shewanella strains isolated from Baltic Sea (38 strains), US DOE Hanford Uranium bioremediation site [Hanford Reach of the Columbia River (HRCR), 11 strains], Pacific Ocean and Hawaiian sediments (8 strains), and strains from other resources (16 strains) with three out group strains, Rhodopseudomonas palustris, Clostridium cellulolyticum, and Thermoanaerobacter ethanolicus X514, using DNA relatedness derived from WCGA-based DNA-DNA hybridizations, sequence similarities of 16S rRNA gene and gyrB gene, and sequence similarities of 6 loci of Shewanella genome selected from a shared gene list of the Shewanella strains with whole genome sequenced based on the average nucleotide identity of them (ANI). The phylogenetic trees based on 16S rRNA and gyrB gene sequences, and DNA relatedness derived from WCGA hybridizations of the tested Shewanella strains share exactly the same sub-clusters with very few exceptions, in which the strains were basically grouped by species. However, the phylogenetic analysis based on DNA relatedness derived from WCGA hybridizations dramatically increased the differentiation resolution at species and strains level within Shewanella genus. When the tree based on DNA relatedness derived from WCGA hybridizations was compared to the tree based on the combined sequences of the selected functional genes (6 loci), we found that the resolutions of both methods are similar, but the clustering of the tree based on DNA relatedness derived from WMGA hybridizations was clearer. These results indicate that WCGA-based DNA-DNA hybridization is an idea alternative of conventional DNA-DNA hybridization methods and it is superior to the phylogenetics methods based on sequence similarities of single genes. Detailed analysis is being performed for the re-classification of the strains examined.

  10. Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis

    Directory of Open Access Journals (Sweden)

    Paniego Norma

    2008-01-01

    Full Text Available Abstract Background Considering that sunflower production is expanding to arid regions, tolerance to abiotic stresses as drought, low temperatures and salinity arises as one of the main constrains nowadays. Differential organ-specific sunflower ESTs (expressed sequence tags were previously generated by a subtractive hybridization method that included a considerable number of putative abiotic stress associated sequences. The objective of this work is to analyze concerted gene expression profiles of organ-specific ESTs by fluorescence microarray assay, in response to high sodium chloride concentration and chilling treatments with the aim to identify and follow up candidate genes for early responses to abiotic stress in sunflower. Results Abiotic-related expressed genes were the target of this characterization through a gene expression analysis using an organ-specific cDNA fluorescence microarray approach in response to high salinity and low temperatures. The experiment included three independent replicates from leaf samples. We analyzed 317 unigenes previously isolated from differential organ-specific cDNA libraries from leaf, stem and flower at R1 and R4 developmental stage. A statistical analysis based on mean comparison by ANOVA and ordination by Principal Component Analysis allowed the detection of 80 candidate genes for either salinity and/or chilling stresses. Out of them, 50 genes were up or down regulated under both stresses, supporting common regulatory mechanisms and general responses to chilling and salinity. Interestingly 15 and 12 sequences were up regulated or down regulated specifically in one stress but not in the other, respectively. These genes are potentially involved in different regulatory mechanisms including transcription/translation/protein degradation/protein folding/ROS production or ROS-scavenging. Differential gene expression patterns were confirmed by qRT-PCR for 12.5% of the microarray candidate sequences. Conclusion

  11. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

    Science.gov (United States)

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

  12. Molecular biological identification of Babesia, Theileria, and Anaplasma species in cattle in Egypt using PCR assays, gene sequence analysis and a novel DNA microarray.

    Science.gov (United States)

    El-Ashker, Maged; Hotzel, Helmut; Gwida, Mayada; El-Beskawy, Mohamed; Silaghi, Cornelia; Tomaso, Herbert

    2015-01-30

    In this preliminary study, a novel DNA microarray system was tested for the diagnosis of bovine piroplasmosis and anaplasmosis in comparison with microscopy and PCR assay results. In the Dakahlia Governorate, Egypt, 164 cattle were investigated for the presence of piroplasms and Anaplasma species. All investigated cattle were clinically examined. Blood samples were screened for the presence of blood parasites using microscopy and PCR assays. Seventy-one animals were acutely ill, whereas 93 were apparently healthy. In acutely ill cattle, Babesia/Theileria species (n=11) and Anaplasma marginale (n=10) were detected. Mixed infections with Babesia/Theileria spp. and A. marginale were present in two further cases. A. marginale infections were also detected in apparently healthy subjects (n=23). The results of PCR assays were confirmed by DNA sequencing. All samples that were positive by PCR for Babesia/Theileria spp. gave also positive results in the microarray analysis. The microarray chips identified Babesia bovis (n=12) and Babesia bigemina (n=2). Cattle with babesiosis were likely to have hemoglobinuria and nervous signs when compared to those with anaplasmosis that frequently had bloody feces. We conclude that clinical examination in combination with microscopy are still very useful in diagnosing acute cases of babesiosis and anaplasmosis, but a combination of molecular biological diagnostic assays will detect even asymptomatic carriers. In perspective, parallel detection of Babesia/Theileria spp. and A. marginale infections using a single microarray system will be a valuable improvement. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  13. DNA Microarray Workshop

    National Research Council Canada - National Science Library

    Lauro, Bernie

    1999-01-01

    .... Funding for the workshop was provided by the US Department of Defense (USAMRAA). The aim of the meeting was to discuss how to best use the data derived from the Malaria Genome Sequencing Project for the functional analysis of the genome...

  14. Interspecies hybridization on DNA resequencing microarrays: efficiency of sequence recovery and accuracy of SNP detection in human, ape, and codfish mitochondrial DNA genomes sequenced on a human-specific MitoChip

    Directory of Open Access Journals (Sweden)

    Carr Steven M

    2007-09-01

    Full Text Available Abstract Background Iterative DNA "resequencing" on oligonucleotide microarrays offers a high-throughput method to measure intraspecific biodiversity, one that is especially suited to SNP-dense gene regions such as vertebrate mitochondrial (mtDNA genomes. However, costs of single-species design and microarray fabrication are prohibitive. A cost-effective, multi-species strategy is to hybridize experimental DNAs from diverse species to a common microarray that is tiled with oligonucleotide sets from multiple, homologous reference genomes. Such a strategy requires that cross-hybridization between the experimental DNAs and reference oligos from the different species not interfere with the accurate recovery of species-specific data. To determine the pattern and limits of such interspecific hybridization, we compared the efficiency of sequence recovery and accuracy of SNP identification by a 15,452-base human-specific microarray challenged with human, chimpanzee, gorilla, and codfish mtDNA genomes. Results In the human genome, 99.67% of the sequence was recovered with 100.0% accuracy. Accuracy of SNP identification declines log-linearly with sequence divergence from the reference, from 0.067 to 0.247 errors per SNP in the chimpanzee and gorilla genomes, respectively. Efficiency of sequence recovery declines with the increase of the number of interspecific SNPs in the 25b interval tiled by the reference oligonucleotides. In the gorilla genome, which differs from the human reference by 10%, and in which 46% of these 25b regions contain 3 or more SNP differences from the reference, only 88% of the sequence is recoverable. In the codfish genome, which differs from the reference by > 30%, less than 4% of the sequence is recoverable, in short islands ≥ 12b that are conserved between primates and fish. Conclusion Experimental DNAs bind inefficiently to homologous reference oligonucleotide sets on a re-sequencing microarray when their sequences differ by

  15. Comparison of two DNA microarrays for detection of plasmid-mediated antimicrobial resistance and virulence factor genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae.

    LENUS (Irish Health Repository)

    Walsh, Fiona

    2010-06-01

    A DNA microarray was developed to detect plasmid-mediated antimicrobial resistance (AR) and virulence factor (VF) genes in clinical isolates of Enterobacteriaceae and non-Enterobacteriaceae. The array was validated with the following bacterial species: Escherichiacoli (n=17); Klebsiellapneumoniae (n=3); Enterobacter spp. (n=6); Acinetobacter genospecies 3 (n=1); Acinetobacterbaumannii (n=1); Pseudomonasaeruginosa (n=2); and Stenotrophomonasmaltophilia (n=2). The AR gene profiles of these isolates were identified by polymerase chain reaction (PCR). The DNA microarray consisted of 155 and 133 AR and VF gene probes, respectively. Results were compared with the commercially available Identibac AMR-ve Array Tube. Hybridisation results indicated that there was excellent correlation between PCR and array results for AR and VF genes. Genes conferring resistance to each antibiotic class were identified by the DNA array. Unusual resistance genes were also identified, such as bla(SHV-5) in a bla(OXA-23)-positive carbapenem-resistant A. baumannii. The phylogenetic group of each E. coli isolate was verified by the array. These data demonstrate that it is possible to screen simultaneously for all important classes of mobile AR and VF genes in Enterobacteriaceae and non-Enterobacteriaceae whilst also assigning a correct phylogenetic group to E. coli isolates. Therefore, it is feasible to test clinical Gram-negative bacteria for all known AR genes and to provide important information regarding pathogenicity simultaneously.

  16. Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs

    Directory of Open Access Journals (Sweden)

    Sebastian Boltaña

    2017-02-01

    Full Text Available This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS and peptidoglycan (PGN. Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs, carrier proteins/membrane transport (approximately 15%, effectors/modulators and cell communication (approximately 11%, nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5% and intracellular transducers/signal transduction (approximately 5%. Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ that provides a platform enriched for the study

  17. Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

    Science.gov (United States)

    Boltaña, Sebastian; Castellana, Barbara; Goetz, Giles; Tort, Lluis; Teles, Mariana; Mulero, Victor; Novoa, Beatriz; Figueras, Antonio; Goetz, Frederick W; Gallardo-Escarate, Cristian; Planas, Josep V; Mackenzie, Simon

    2017-02-03

    This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene

  18.  DNA microarray-based gene expression profiling in diagnosis, assessing prognosis and predicting response to therapy in colorectal cancer

    Directory of Open Access Journals (Sweden)

    Przemysław Kwiatkowski

    2012-06-01

    Full Text Available  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.

  19. Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: Implications for transcriptomics studies

    NARCIS (Netherlands)

    Ballerstedt, H.; Volkers, R.J.M.; Mars, A.E.; Hallsworth, J.E.; Santos, V.A.M.D.; Puchalka, J.; Duuren, J. van; Eggink, G.; Timmis, K.N.; Bont, J.A.M. de; Wery, J.

    2007-01-01

    Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for

  20. Forensic DNA evidence and the death penalty in the Philippines.

    Science.gov (United States)

    De Ungria, M C A; Sagum, M S; Calacal, G C; Delfin, F C; Tabbada, K A; Dalet, M R M; Te, T O; Diokno, J I; Diokno, M S I; Asplen, C A

    2008-09-01

    The death penalty remains a contentious issue even though it has been abolished in countries such as Australia, New Zealand, Canada, European Union member nations and some Asian countries such as Cambodia, East Timor and Nepal. Many argue that the irrevocability of the death penalty, in the face of potential erroneous convictions, can never justify its imposition. The Philippines, the first Asian country that abolished the death penalty in 1987, held the record for the most number of mandatory death offenses (30 offenses) and death eligible offenses (22 offenses) after it was re-imposed in 1994. Majority of death penalty convictions were decided based on testimonial evidence. While such cases undergo automatic review by the Supreme Court, the appellate process in the Philippines is not structured to accept post-conviction evidence, including DNA evidence. Because of the compelling nature of post-conviction DNA evidence in overturning death penalty convictions in the United States, different groups advocated its use in the Philippines. In one such case, People v Reynaldo de Villa, the defendant was charged with raping his 13-year-old niece that supposedly led to birth of a female child, a situation commonly known as 'criminal paternity'. This paper reports the results of the first post-conviction DNA test using 16 Short Tandem Repeat (STR) DNA markers in a criminal paternity case (People v Reynaldo de Villa) and discusses the implications of these results in the Philippine criminal justice system.

  1. Identification of late O{sub 3}-responsive genes in Arabidopsis thaliana by cDNA microarray analysis

    Energy Technology Data Exchange (ETDEWEB)

    D' Haese, D. [Univ. of Antwerp, Dept. of Biology, Antwerp (BE) and Univ. of Newcastle, School of Biology and Psychology, Div. of Biology, Newcastle-Upon-Tyne (United Kingdom); Horemans, N.; Coen, W. De; Guisez, Y. [Univ. of Antwerp, Dept. of Biology, Antwerp (Belgium)

    2006-09-15

    To better understand the response of a plant to 0{sub 3} stress, an integrated microarray analysis was performed on Arabidopsis plants exposed during 2 days to purified air or 150 nl l{sup -1} O{sub 3}, 8 h day-l. Agilent Arabidopsis 2 Oligo Microarrays were used of which the reliability was confirmed by quantitative real-time PCR of nine randomly selected genes. We confirmed the O{sub 3} responsiveness of heat shock proteins (HSPs), glutathione-S-tranferases and genes involved in cell wall stiffening and microbial defence. Whereas, a previous study revealed that during an early stage of the O{sub 3} stress response, gene expression was strongly dependent on jasmonic acid and ethylene, we report that at a later stage (48 h) synthesis of jasrnonic acid and ethylene was downregulated. In addition, we observed the simultaneous induction of salicylic acid synthesis and genes involved in programmed cell death and senescence. Also typically, the later stage of the response to O{sub 3} appeared to be the induction of the complete pathway leading to the biosynthesis of anthocyanin diglucosides and the induction of thioredoxin-based redox control. Surprisingly absent in the list of induced genes were genes involved in ASC-dependent antioxidation, few of which were found to be induced after 12 h of 0{sub 3} exposure in another study. We discuss these and other particular results of the microarray analysis and provide a map depicting significantly affected genes and their pathways highlighting their interrelationships and subcellular localization. (au)

  2. Hematopoietic Lineage Transcriptome Stability and Representation in PAXgeneTM Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray

    Directory of Open Access Journals (Sweden)

    Laura Kennedy

    2008-01-01

    Full Text Available Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgeneTM RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2TM enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgeneTM blood samples also advocate a short, fixed room temperature storage time for all PAXgeneTM blood samples collected for the purposes of global transcriptional profiling in clinical studies.

  3. Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray.

    Science.gov (United States)

    Kennedy, Laura; Vass, J Keith; Haggart, D Ross; Moore, Steve; Burczynski, Michael E; Crowther, Dan; Miele, Gino

    2008-08-25

    Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgene() RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2() enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgene() blood samples also advocate a short, fixed room temperature storage time for all PAXgene() blood samples collected for the purposes of global transcriptional profiling in clinical studies.

  4. Hematopoietic Lineage Transcriptome Stability and Representation in PAXgene™ Collected Peripheral Blood Utilising SPIA Single-Stranded cDNA Probes for Microarray

    Science.gov (United States)

    Kennedy, Laura; Vass, J. Keith; Haggart, D. Ross; Moore, Steve; Burczynski, Michael E.; Crowther, Dan; Miele, Gino

    2008-01-01

    Peripheral blood as a surrogate tissue for transcriptome profiling holds great promise for the discovery of diagnostic and prognostic disease biomarkers, particularly when target tissues of disease are not readily available. To maximize the reliability of gene expression data generated from clinical blood samples, both the sample collection and the microarray probe generation methods should be optimized to provide stabilized, reproducible and representative gene expression profiles faithfully representing the transcriptional profiles of the constituent blood cell types present in the circulation. Given the increasing innovation in this field in recent years, we investigated a combination of methodological advances in both RNA stabilisation and microarray probe generation with the goal of achieving robust, reliable and representative transcriptional profiles from whole blood. To assess the whole blood profiles, the transcriptomes of purified blood cell types were measured and compared with the global transcriptomes measured in whole blood. The results demonstrate that a combination of PAXgene™ RNA stabilising technology and single-stranded cDNA probe generation afforded by the NuGEN Ovation RNA amplification system V2™ enables an approach that yields faithful representation of specific hematopoietic cell lineage transcriptomes in whole blood without the necessity for prior sample fractionation, cell enrichment or globin reduction. Storage stability assessments of the PAXgene™ blood samples also advocate a short, fixed room temperature storage time for all PAXgene™ blood samples collected for the purposes of global transcriptional profiling in clinical studies. PMID:19578521

  5. Genome-Wide Screening of Genes Showing Altered Expression in Liver Metastases of Human Colorectal Cancers by cDNA Microarray

    Directory of Open Access Journals (Sweden)

    Rempei Yanagawa

    2001-01-01

    Full Text Available In spite of intensive and increasingly successful attempts to determine the multiple steps involved in colorectal carcinogenesis, the mechanisms responsible for metastasis of colorectal tumors to the liver remain to be clarified. To identify genes that are candidates for involvement in the metastatic process, we analyzed genome-wide expression profiles of 10 primary colorectal cancers and their corresponding metastatic lesions by means of a cDNA microarray consisting of 9121 human genes. This analysis identified 40 genes whose expression was commonly upregulated in metastatic lesions, and 7 that were commonly downregulated. The upregulated genes encoded proteins involved in cell adhesion, or remodeling of the actin cytoskeleton. Investigation of the functions of more of the altered genes should improve our understanding of metastasis and may identify diagnostic markers and/or novel molecular targets for prevention or therapy of metastatic lesions.

  6. Bifidobacterium breve B-3 exerts metabolic syndrome-suppressing effects in the liver of diet-induced obese mice: a DNA microarray analysis.

    Science.gov (United States)

    Kondo, S; Kamei, A; Xiao, J Z; Iwatsuki, K; Abe, K

    2013-09-01

    We previously reported that supplementation with Bifidobacterium breve B-3 reduced body weight gain and accumulation of visceral fat in a dose-dependent manner, and improved serum levels of total cholesterol, glucose and insulin in a mouse model of diet-induced obesity. In this study, we investigated the expression of genes in the liver using DNA microarray analysis and q-PCR to reveal the mechanism of these anti-obesity effects in this mouse model. Administration of B. breve B-3 led to regulated gene expression of pathways involved in lipid metabolism and response to stress. The results indicate that these regulations in the liver are related to the anti-metabolic syndrome effects of B. breve B-3.

  7. Advanced microarray technologies for clinical diagnostics

    NARCIS (Netherlands)

    Pierik, Anke

    2011-01-01

    DNA microarrays become increasingly important in the field of clinical diagnostics. These microarrays, also called DNA chips, are small solid substrates, typically having a maximum surface area of a few cm2, onto which many spots are arrayed in a pre-determined pattern. Each of these spots contains

  8. A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer.

    Science.gov (United States)

    Schwartzman, Jacob; Mongoue-Tchokote, Solange; Gibbs, Angela; Gao, Lina; Corless, Christopher L; Jin, Jennifer; Zarour, Luai; Higano, Celestia; True, Lawrence D; Vessella, Robert L; Wilmot, Beth; Bottomly, Daniel; McWeeney, Shannon K; Bova, G Steven; Partin, Alan W; Mori, Motomi; Alumkal, Joshi

    2011-10-01

    DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.

  9. Eureka-DMA: an easy-to-operate graphical user interface for fast comprehensive investigation and analysis of DNA microarray data.

    Science.gov (United States)

    Abelson, Sagi

    2014-02-24

    In the past decade, the field of molecular biology has become increasingly quantitative; rapid development of new technologies enables researchers to investigate and address fundamental issues quickly and in an efficient manner which were once impossible. Among these technologies, DNA microarray provides methodology for many applications such as gene discovery, diseases diagnosis, drug development and toxicological research and it has been used increasingly since it first emerged. Multiple tools have been developed to interpret the high-throughput data produced by microarrays. However, many times, less consideration has been given to the fact that an extensive and effective interpretation requires close interplay between the bioinformaticians who analyze the data and the biologists who generate it. To bridge this gap and to simplify the usability of such tools we developed Eureka-DMA - an easy-to-operate graphical user interface that allows bioinformaticians and bench-biologists alike to initiate analyses as well as to investigate the data produced by DNA microarrays. In this paper, we describe Eureka-DMA, a user-friendly software that comprises a set of methods for the interpretation of gene expression arrays. Eureka-DMA includes methods for the identification of genes with differential expression between conditions; it searches for enriched pathways and gene ontology terms and combines them with other relevant features. It thus enables the full understanding of the data for following testing as well as generating new hypotheses. Here we show two analyses, demonstrating examples of how Eureka-DMA can be used and its capability to produce relevant and reliable results. We have integrated several elementary expression analysis tools to provide a unified interface for their implementation. Eureka-DMA's simple graphical user interface provides effective and efficient framework in which the investigator has the full set of tools for the visualization and interpretation

  10. Molecular characterisation of the early response in pigs to experimental infection with Actinobacillus pleuropneumoniae using cDNA microarrays

    DEFF Research Database (Denmark)

    Hedegaard, Jakob; Skovgaard, Kerstin; Mortensen, Shila

    2007-01-01

    Background: The bacterium Actinobacillus pleuropneumoniae is responsible for porcine pleuropneumonia, a widespread, highly contagious and often fatal respiratory disease of pigs. The general porcine innate immune response after A. pleuropneumoniae infection is still not clarified. The objective...... lymph node tissue were hybridised to an expanded version of the porcine microarray with 26879 unique PCR products. Results: A total of 357 genes differed significantly in expression between infected and non-infected lung tissue, 713 genes differed in expression in liver tissue from infected versus non-infected...... animals and 130 genes differed in expression in tracheobronchial lymph node tissue from infected versus non-infected animals. Among these genes, several have previously been described to be part of a general host response to infections encoding immune response related proteins. In inflamed lung tissue...

  11. Gene organization in rice revealed by full-length cDNA mapping and gene expression analysis through microarray.

    Directory of Open Access Journals (Sweden)

    Kouji Satoh

    Full Text Available Rice (Oryza sativa L. is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE genes, 33K annotated non-expressed (ANE genes, and 5.5K non-annotated expressed (NAE genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria.

  12. Application of a cDNA microarray for profiling the gene expression of Echinococcus granulosus protoscoleces treated with albendazole and artemisinin.

    Science.gov (United States)

    Lü, Guodong; Zhang, Wenbao; Wang, Jianhua; Xiao, Yunfeng; Zhao, Jun; Zhao, Jianqin; Sun, Yimin; Zhang, Chuanshan; Wang, Junhua; Lin, Renyong; Liu, Hui; Zhang, Fuchun; Wen, Hao

    2014-12-01

    Cystic echinoccocosis (CE) is a neglected zoonosis that is caused by the dog-tapeworm Echinococcus granulosus. The disease is endemic worldwide. There is an urgent need for searching effective drug for the treatment of the disease. In this study, we sequenced a cDNA library constructed using RNA isolated from oncospheres, protoscoleces, cyst membrane and adult worms of E. granulosus. A total of 9065 non-redundant or unique sequences were obtained and spotted on chips as uniEST probes to profile the gene expression in protoscoleces of E. granulosus treated with the anthelmintic drugs albendazole and artemisinin, respectively. The results showed that 7 genes were up-regulated and 38 genes were down-regulated in the protoscoleces treated with albendazole. Gene analysis showed that these genes are responsible for energy metabolism, cell cycle and assembly of cell structure. We also identified 100 genes up-regulated and 6 genes down-regulated in the protoscoleces treated with artemisinin. These genes play roles in the transduction of environmental signals, and metabolism. Albendazole appeared its drug efficacy in damaging cell structure, while artemisinin was observed to increase the formation of the heterochromatin in protoscolex cells. Our results highlight the utility of using cDNA microarray methods to detect gene expression profiles of E. granulosus and, in particular, to understand the pharmacologic mechanism of anti-echinococcosis drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

  13. Analysis of the effects of sex hormone background on the rat choroid plexus transcriptome by cDNA microarrays.

    Directory of Open Access Journals (Sweden)

    Telma Quintela

    Full Text Available The choroid plexus (CP are highly vascularized branched structures that protrude into the ventricles of the brain, and form a unique interface between the blood and the cerebrospinal fluid (CSF, the blood-CSF barrier, that are the main site of production and secretion of CSF. Sex hormones are widely recognized as neuroprotective agents against several neurodegenerative diseases, and the presence of sex hormones cognate receptors suggest that it may be a target for these hormones. In an effort to provide further insight into the neuroprotective mechanisms triggered by sex hormones we analyzed gene expression differences in the CP of female and male rats subjected to gonadectomy, using microarray technology. In gonadectomized female and male animals, 3045 genes were differentially expressed by 1.5-fold change, compared to sham controls. Analysis of the CP transcriptome showed that the top-five pathways significantly regulated by the sex hormone background are olfactory transduction, taste transduction, metabolism, steroid hormone biosynthesis and circadian rhythm pathways. These results represent the first overview of global expression changes in CP of female and male rats induced by gonadectomy and suggest that sex hormones are implicated in pathways with central roles in CP functions and CSF homeostasis.

  14. The global response of Nostoc punctiforme ATCC 29133 to UVA stress, assessed in a temporal DNA microarray study.

    Science.gov (United States)

    Soule, Tanya; Gao, Qunjie; Stout, Valerie; Garcia-Pichel, Ferran

    2013-01-01

    Cyanobacteria in nature are exposed not only to the visible spectrum of sunlight but also to its harmful ultraviolet components (UVA and UVB). We used Nostoc punctiforme ATCC 29133 as a model to study the UVA response by analyzing global gene expression patterns using genomic microarrays. UVA exposure resulted in the statistically detectable differential expression of 573 genes of the 6903 that were probed, compared with that of the control cultures. Of those genes, 473 were up-regulated, while only 100 were down-regulated. Many of the down-regulated genes were involved in photosynthetic pigment biosynthesis, indicating a significant shift in this metabolism. As expected, we detected the up-regulation of genes encoding antioxidant enzymes and the sunscreen, scytonemin. However, a majority of the up-regulated genes, 47%, were unassignable bioinformatically to known functional categories, suggesting that the UVA stress response is not well understood. Interestingly, the most dramatic up-regulation involved several contiguous genes of unassigned metabolism on plasmid A. This is the first global UVA stress response analysis of any phototrophic microorganism and the differential expression of 8% of the genes of the Nostoc genome indicates that adaptation to UVA in Nostoc has been an evolutionary force of significance. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

  15. Microarray analysis of DNA damage repair gene expression profiles in cervical cancer cells radioresistant to 252Cf neutron and X-rays

    International Nuclear Information System (INIS)

    Qing, Yi; Wang, Ge; Wang, Dong; Yang, Xue-Qin; Zhong, Zhao-Yang; Lei, Xin; Xie, Jia-Yin; Li, Meng-Xia; Xiang, De-Bing; Li, Zeng-Peng; Yang, Zhen-Zhou

    2010-01-01

    The aim of the study was to obtain stable radioresistant sub-lines from the human cervical cancer cell line HeLa by prolonged exposure to 252 Cf neutron and X-rays. Radioresistance mechanisms were investigated in the resulting cells using microarray analysis of DNA damage repair genes. HeLa cells were treated with fractionated 252 Cf neutron and X-rays, with a cumulative dose of 75 Gy each, over 8 months, yielding the sub-lines HeLaNR and HeLaXR. Radioresistant characteristics were detected by clone formation assay, ultrastructural observations, cell doubling time, cell cycle distribution, and apoptosis assay. Gene expression patterns of the radioresistant sub-lines were studied through microarray analysis and verified by Western blotting and real-time PCR. The radioresistant sub-lines HeLaNR and HeLaXR were more radioresisitant to 252 Cf neutron and X-rays than parental HeLa cells by detecting their radioresistant characteristics, respectively. Compared to HeLa cells, the expression of 24 genes was significantly altered by at least 2-fold in HeLaNR cells. Of these, 19 genes were up-regulated and 5 down-regulated. In HeLaXR cells, 41 genes were significantly altered by at least 2-fold; 38 genes were up-regulated and 3 down-regulated. Chronic exposure of cells to ionizing radiation induces adaptive responses that enhance tolerance of ionizing radiation and allow investigations of cellular radioresistance mechanisms. The insights gained into the molecular mechanisms activated by these 'radioresistance' genes will lead to new therapeutic targets for cervical cancer

  16. Toxicity of Doxorubicin on Pig Liver After Chemoembolization with Doxorubicin-loaded Microspheres: A Pilot DNA-microarrays and Histology Study

    Energy Technology Data Exchange (ETDEWEB)

    Verret, Valentin, E-mail: valentin.verret@archimmed.com; Namur, Julien; Ghegediban, Saieda Homayra [ArchimMed (France); Wassef, Michel [University of Paris 7-Denis Diderot, Department of Pathology, Faculty of Medicine, AP-HP Hopital Lariboisiere (France); Moine, Laurence [Universite Paris Sud, Faculte de Pharmacie, UMR CNRS 8612, IFR 141-ITFM (France); Bonneau, Michel [AP-HP/INRA, Centre de Recherche En Imagerie Interventionnelle (France); Pelage, Jean-Pierre [AP-HP Hopital Ambroise Pare, Department of Interventional Radiology (France); Laurent, Alexandre [AP-HP/INRA, Centre de Recherche En Imagerie Interventionnelle (France)

    2013-02-15

    The potential mechanisms accounting for the hepatotoxicity of doxorubicin-loaded microspheres in chemoembolization were examined by combining histology and DNA-microarray techniques.The left hepatic arteries of two pigs were embolized with 1 mL of doxorubicin-loaded (25 mg; (DoxMS)) or non-loaded (BlandMS) microspheres. The histopathological effects of the embolization were analyzed at 1 week. RNAs extracted from both the embolized and control liver areas were hybridized onto Agilent porcine microarrays. Genes showing significantly different expression (p < 0.01; fold-change > 2) between two groups were classified by biological process. At 1 week after embolization, DoxMS caused arterial and parenchymal necrosis in 51 and 38 % of embolized vessels, respectively. By contrast, BlandMS did not cause any tissue damage. Up-regulated genes following embolization with DoxMS (vs. BlandMS, n = 353) were mainly involved in cell death, apoptosis, and metabolism of doxorubicin. Down-regulated genes (n = 120) were mainly related to hepatic functions, including enzymes of lipid and carbohydrate metabolisms. Up-regulated genes included genes related to cell proliferation (growth factors and transcription factors), tissue remodeling (MMPs and several collagen types), inflammatory reaction (interleukins and chemokines), and angiogenesis (angiogenic factors and HIF1a pathway), all of which play an important role in liver healing and regeneration. DoxMS caused lesions to the liver, provoked cell death, and disturbed liver metabolism. An inflammatory repair process with cell proliferation, tissue remodeling, and angiogenesis was rapidly initiated during the first week after chemoembolization. This pilot study provides a comprehensive method to compare different types of DoxMS in healthy animals or tumor models.

  17. A microarray analysis of sex- and gonad-biased gene expression in the zebrafish: Evidence for masculinization of the transcriptome

    Directory of Open Access Journals (Sweden)

    Mo Qianxing

    2009-12-01

    Full Text Available Abstract Background In many taxa, males and females are very distinct phenotypically, and these differences often reflect divergent selective pressures acting on the sexes. Phenotypic sexual dimorphism almost certainly reflects differing patterns of gene expression between the sexes, and microarray studies have documented widespread sexually dimorphic gene expression. Although the evolutionary significance of sexual dimorphism in gene expression remains unresolved, these studies have led to the formulation of a hypothesis that male-driven evolution has resulted in the masculinization of animal transcriptomes. Here we use a microarray assessment of sex- and gonad-biased gene expression to test this hypothesis in zebrafish. Results By using zebrafish Affymetrix microarrays to compare gene expression patterns in male and female somatic and gonadal tissues, we identified a large number of genes (5899 demonstrating differences in transcript abundance between male and female Danio rerio. Under conservative statistical significance criteria, all sex-biases in gene expression were due to differences between testes and ovaries. Male-enriched genes were more abundant than female-enriched genes, and expression bias for male-enriched genes was greater in magnitude than that for female-enriched genes. We also identified a large number of genes demonstrating elevated transcript abundance in testes and ovaries relative to male body and female body, respectively. Conclusion Overall our results support the hypothesis that male-biased evolutionary pressures have resulted in male-biased patterns of gene expression. Interestingly, our results seem to be at odds with a handful of other microarray-based studies of sex-specific gene expression patterns in zebrafish. However, ours was the only study designed to address this specific hypothesis, and major methodological differences among studies could explain the discrepancies. Regardless, all of these studies agree

  18. Direct calibration of PICKY-designed microarrays

    Directory of Open Access Journals (Sweden)

    Ronald Pamela C

    2009-10-01

    Full Text Available Abstract Background Few microarrays have been quantitatively calibrated to identify optimal hybridization conditions because it is difficult to precisely determine the hybridization characteristics of a microarray using biologically variable cDNA samples. Results Using synthesized samples with known concentrations of specific oligonucleotides, a series of microarray experiments was conducted to evaluate microarrays designed by PICKY, an oligo microarray design software tool, and to test a direct microarray calibration method based on the PICKY-predicted, thermodynamically closest nontarget information. The complete set of microarray experiment results is archived in the GEO database with series accession number GSE14717. Additional data files and Perl programs described in this paper can be obtained from the website http://www.complex.iastate.edu under the PICKY Download area. Conclusion PICKY-designed microarray probes are highly reliable over a wide range of hybridization temperatures and sample concentrations. The microarray calibration method reported here allows researchers to experimentally optimize their hybridization conditions. Because this method is straightforward, uses existing microarrays and relatively inexpensive synthesized samples, it can be used by any lab that uses microarrays designed by PICKY. In addition, other microarrays can be reanalyzed by PICKY to obtain the thermodynamically closest nontarget information for calibration.

  19. DNA microarray-based assessment of virulence potential of Shiga toxin gene-carrying Escherichia coli O104:H7 isolated from feedlot cattle feces.

    Directory of Open Access Journals (Sweden)

    Pragathi B Shridhar

    Full Text Available Escherichia coli O104:H4, a hybrid pathotype reported in a large 2011 foodborne outbreak in Germany, has not been detected in cattle feces. However, cattle harbor and shed in the feces other O104 serotypes, particularly O104:H7, which has been associated with sporadic cases of diarrhea in humans. The objective of our study was to assess the virulence potential of Shiga toxin-producing E. coli (STEC O104:H7 isolated from feces of feedlot cattle using DNA microarray. Six strains of STEC O104:H7 isolated from cattle feces were analyzed using FDA-E. coli Identification (ECID DNA microarray to determine their virulence profiles and compare them to the human strains (clinical of O104:H7, STEC O104:H4 (German outbreak strain, and O104:H21 (milk-associated Montana outbreak strain. Scatter plots were generated from the array data to visualize the gene-level differences between bovine and human O104 strains, and Pearson correlation coefficients (r were determined. Splits tree was generated to analyze relatedness between the strains. All O104:H7 strains, both bovine and human, similar to O104:H4 and O104:H21 outbreak strains were negative for intimin (eae. The bovine strains were positive for Shiga toxin 1 subtype c (stx1c, enterohemolysin (ehxA, tellurite resistance gene (terD, IrgA homolog protein (iha, type 1 fimbriae (fimH, and negative for genes that code for effector proteins of type III secretory system. The six cattle O104 strains were closely related (r = 0.86-0.98 to each other, except for a few differences in phage related and non-annotated genes. One of the human clinical O104:H7 strains (2011C-3665 was more closely related to the bovine O104:H7 strains (r = 0.81-0.85 than the other four human clinical O104:H7 strains (r = 0.75-0.79. Montana outbreak strain (O104:H21 was more closely related to four of the human clinical O104:H7 strains than the bovine O104:H7 strains. None of the bovine E. coli O104 strains carried genes characteristic of E

  20. Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    International Nuclear Information System (INIS)

    Guo Yingying; Breeden, Linda L.; Fan, Wenhong; Zhao Lueping; Eaton, David L.; Zarbl, Helmut

    2006-01-01

    Aflatoxin B1 (AFB 1 ) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB 1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N 7 -guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB 1 , a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB 1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB 1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB 1 -treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by

  1. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  2. USE OF cDNA MICROARRAY TECHNOLOGY FOR IDENTIFICATION OF NOVEL GENES RESPONDING TO ABSCISIC ACID PHYTOHORMONE

    OpenAIRE

    D. Cabezas; Sandra Pérez; R. Huelva; Dong Haitao; Li Debao

    2005-01-01

    Se utilizó el cDNA microarreglo con 4370 unigenes, provenientes de la biblioteca del endospermo del arroz y de los tejidos de las hojas, para detectar los niveles de expresión del mRNA de los tejidos del tallo del arroz tratados con agua y con la hormona ácido abscísico (ABA). Los resultados mostraron que los niveles de expresión de cinco genes fueron reprimidos por la fitohormona ABA. El Reverse Northern confirmó que uno de los cinco genes (H024g06) fue realmente reprimido por el ABA. Los an...

  3. DNA microarray-based genome comparison of a pathogenic and a nonpathogenic strain of Xylella fastidiosa delineates genes important for bacterial virulence.

    Science.gov (United States)

    Koide, Tie; Zaini, Paulo A; Moreira, Leandro M; Vêncio, Ricardo Z N; Matsukuma, Adriana Y; Durham, Alan M; Teixeira, Diva C; El-Dorry, Hamza; Monteiro, Patrícia B; da Silva, Ana Claudia R; Verjovski-Almeida, Sergio; da Silva, Aline M; Gomes, Suely L

    2004-08-01

    Xylella fastidiosa is a phytopathogenic bacterium that causes serious diseases in a wide range of economically important crops. Despite extensive comparative analyses of genome sequences of Xylella pathogenic strains from different plant hosts, nonpathogenic strains have not been studied. In this report, we show that X. fastidiosa strain J1a12, associated with citrus variegated chlorosis (CVC), is nonpathogenic when injected into citrus and tobacco plants. Furthermore, a DNA microarray-based comparison of J1a12 with 9a5c, a CVC strain that is highly pathogenic and had its genome completely sequenced, revealed that 14 coding sequences of strain 9a5c are absent or highly divergent in strain J1a12. Among them, we found an arginase and a fimbrial adhesin precursor of type III pilus, which were confirmed to be absent in the nonpathogenic strain by PCR and DNA sequencing. The absence of arginase can be correlated to the inability of J1a12 to multiply in host plants. This enzyme has been recently shown to act as a bacterial survival mechanism by down-regulating host nitric oxide production. The lack of the adhesin precursor gene is in accordance with the less aggregated phenotype observed for J1a12 cells growing in vitro. Thus, the absence of both genes can be associated with the failure of the J1a12 strain to establish and spread in citrus and tobacco plants. These results provide the first detailed comparison between a nonpathogenic strain and a pathogenic strain of X. fastidiosa, constituting an important step towards understanding the molecular basis of the disease.

  4. Genomic DNA Enrichment Using Sequence Capture Microarrays: a Novel Approach to Discover Sequence Nucleotide Polymorphisms (SNP) in Brassica napus L

    Science.gov (United States)

    Clarke, Wayne E.; Parkin, Isobel A.; Gajardo, Humberto A.; Gerhardt, Daniel J.; Higgins, Erin; Sidebottom, Christine; Sharpe, Andrew G.; Snowdon, Rod J.; Federico, Maria L.; Iniguez-Luy, Federico L.

    2013-01-01

    Targeted genomic selection methodologies, or sequence capture, allow for DNA enrichment and large-scale resequencing and characterization of natural genetic variation in species with complex genomes, such as rapeseed canola (Brassica napus L., AACC, 2n=38). The main goal of this project was to combine sequence capture with next generation sequencing (NGS) to discover single nucleotide polymorphisms (SNPs) in specific areas of the B. napus genome historically associated (via quantitative trait loci –QTL– analysis) to traits of agronomical and nutritional importance. A 2.1 million feature sequence capture platform was designed to interrogate DNA sequence variation across 47 specific genomic regions, representing 51.2 Mb of the Brassica A and C genomes, in ten diverse rapeseed genotypes. All ten genotypes were sequenced using the 454 Life Sciences chemistry and to assess the effect of increased sequence depth, two genotypes were also sequenced using Illumina HiSeq chemistry. As a result, 589,367 potentially useful SNPs were identified. Analysis of sequence coverage indicated a four-fold increased representation of target regions, with 57% of the filtered SNPs falling within these regions. Sixty percent of discovered SNPs corresponded to transitions while 40% were transversions. Interestingly, fifty eight percent of the SNPs were found in genic regions while 42% were found in intergenic regions. Further, a high percentage of genic SNPs was found in exons (65% and 64% for the A and C genomes, respectively). Two different genotyping assays were used to validate the discovered SNPs. Validation rates ranged from 61.5% to 84% of tested SNPs, underpinning the effectiveness of this SNP discovery approach. Most importantly, the discovered SNPs were associated with agronomically important regions of the B. napus genome generating a novel data resource for research and breeding this crop species. PMID:24312619

  5. A DNA Microarray Analysis of Chemokine and Receptor Genes in the Rat Dental Follicle – Role of Secreted Frizzled-Related Protein-1 in Osteoclastogenesis

    Science.gov (United States)

    Liu, Dawen; Wise, Gary E.

    2007-01-01

    The dental follicle, a loose connective tissue sac that surrounds the unerupted tooth, appears to regulate the osteoclastogenesis needed for eruption; i.e., bone resorption to form an eruption pathway. Thus, DNA microarray studies were conducted to determine which chemokines and their receptors were expressed chronologically in the dental follicle, chemokines that might attract osteoclast precursors. In the rat first mandibular molar, a major burst of osteoclastogenesis occurs at day 3 with a minor burst at day 10. The results of the microarray confirmed our previous studies showing the gene expression of molecules such as CSF-1 and MCP-1 in the dental follicle cells. Other new genes also were detected, including secreted frizzled-related protein-1 (SFRP-1), which was found to be down-regulated at days 3 and 9. Using rat bone marrow cultures to conduct in vitro osteoclastogenic assays, it was demonstrated that SFRP-1 inhibited osteoclast formation in a concentration-dependent fashion. However, with increasing concentrations of SFRP-1, the number of TRAP-positive mononuclear cells increased suggesting that SFRP-1 inhibits osteoclast formation by inhibiting the fusion of mononuclear cells (osteoclast precursors). Co-culturing bone marrow mononuclear cells and dental follicle cells demonstrated that the dental follicle cells were secreting a product(s) that inhibited osteoclastogenesis, as measured by counting of TRAP-positive osteoclasts. Adding an antibody either to SFRP-1 or OPG partially restored osteoclastogenesis. Adding both anti-SFRP-1 and anti-OPG fully negated the inhibitory effect of the follicle cells upon osteoclastogenesis. These results strongly suggest that SFRP-1 and OPG, both secreted by the dental follicle cells, use different pathways to exert their inhibitory effect on osteoclastogenesis. Based on these in vitro studies of osteoclastogenesis, it is likely that the down-regulation of SFRP-1 gene expression in the dental follicle at days 3 and 9 is

  6. Silanization of silica and glass slides for DNA microarrays by impregnation and gas phase protocols: A comparative study

    International Nuclear Information System (INIS)

    Phaner-Goutorbe, Magali; Dugas, Vincent; Chevolot, Yann; Souteyrand, Eliane

    2011-01-01

    Surface immobilization of oligonucleotide probes (oligoprobes) is a key issue in the development of DNA-chips. The immobilization protocol should guarantee good availability of the probes, low non-specific adsorption and reproducibility. We have previously reported a silanization protocol with tert-butyl-11-(dimethylamino)silylundecanoate performed by impregnation (Impregnation Protocol, IP) of silica substrates from dilute silane solutions, leading to surfaces bearing carboxylic groups. In this paper, the Impregnation protocol is compared with a Gas phase Protocol (GP) which is more suited to industrial requirements such as reliable and robust processing, cost efficiency, etc.... The morphology of the oligoprobe films at the nanoscale (characterized by Atomic Force Microscopy) and the reproducibility of subsequent oligoprobes immobilization steps have been investigated for the two protocols on thermal silica (Si/SiO 2 ) and glass slide substrates. IP leads to smooth surfaces whereas GP induces the formation of islands features suggesting a non-continuous silane layer. The reproducibility of the overall surface layer (18.75 mm 2 ) has been evaluated through the covalent immobilization of a fluorescent oligoprobes. Average fluorescent signals of 6 (a.u.) and 4 (a.u.) were observed for IP and GP, respectively, with a standard deviation of 1 for both protocols. Thus, despite a morphological difference of the silane layer at the nanometer scale, the density of the immobilized probes remained similar.

  7. Gene expression analysis of the rat testis after treatment with di(2-ethylhexyl) phthalate using cDNA microarray and real-time RT-PCR

    International Nuclear Information System (INIS)

    Kijima, Kazuyasu; Toyosawa, Kaoru; Yasuba, Masashi; Matsuoka, Nobuo; Adachi, Tetsuya; Komiyama, Masatoshi; Mori, Chisato

    2004-01-01

    To investigate the effects of di(2-ethylhexyl) phthalate (DEHP) on gene expression in rat testis, 6-week-old male Sprague-Dawley rats were given a single oral dose of 20 or 2000 mg/kg and euthanized 3, 6, 24, or 72 h thereafter. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells were significantly increased in the testis at 24 and 72 h after the exposure to 2000 mg/kg of DEHP. On cDNA microarray analysis, in addition to apoptosis-related genes, genes associated with atrophy, APEX nuclease, MutS homologue (E. coli), testosterone-repressed-prostatic-message-2 (TRPM-2), connective tissue growth factor, collagen alpha 2 type V, and cell adhesion kinase were differentially expressed. To investigate the relationship between histopathological alteration and gene expression, we selected genes associated with apoptosis and analyzed their expression by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR). With 20 mg/kg of DEHP treatment, bcl-2, key gene related to apoptosis, was increased. Up-regulation of bcl-2, inhibitor of Apaf-1/caspase-9/caspase-2 cascade of apoptosis, may be related to the fact that no morphological apoptotic change was induced after dosing of 20 mg/kg DEHP. With 2000 mg/kg of DEHP treatment, the apoptotic activator cascade, Fas/FasL, FADD/caspase-8/caspase-3 cascade, and Apaf-1/caspase-9/caspase-2 cascade were increased and bcl-2 was decreased. Thus, these gene regulations might lead the cells into apoptosis in the case of high exposure to DEHP. In contrast, FADD/caspase-10/caspase-6 cascade and caspase-11/caspase-3 cascade were not increased. These results indicate that the cascades of FADD/caspase-10/caspase-6 and caspase-11/caspase-3 are not related to apoptosis with DEHP treatment

  8. Searching for evidence of selection in avian DNA barcodes.

    Science.gov (United States)

    Kerr, Kevin C R

    2011-11-01

    The barcode of life project has assembled a tremendous number of mitochondrial cytochrome c oxidase I (COI) sequences. Although these sequences were gathered to develop a DNA-based system for species identification, it has been suggested that further biological inferences may also be derived from this wealth of data. Recurrent selective sweeps have been invoked as an evolutionary mechanism to explain limited intraspecific COI diversity, particularly in birds, but this hypothesis has not been formally tested. In this study, I collated COI sequences from previous barcoding studies on birds and tested them for evidence of selection. Using this expanded data set, I re-examined the relationships between intraspecific diversity and interspecific divergence and sampling effort, respectively. I employed the McDonald-Kreitman test to test for neutrality in sequence evolution between closely related pairs of species. Because amino acid sequences were generally constrained between closely related pairs, I also included broader intra-order comparisons to quantify patterns of protein variation in avian COI sequences. Lastly, using 22 published whole mitochondrial genomes, I compared the evolutionary rate of COI against the other 12 protein-coding mitochondrial genes to assess intragenomic variability. I found no conclusive evidence of selective sweeps. Most evidence pointed to an overall trend of strong purifying selection and functional constraint. The COI protein did vary across the class Aves, but to a very limited extent. COI was the least variable gene in the mitochondrial genome, suggesting that other genes might be more informative for probing factors constraining mitochondrial variation within species. © 2011 Blackwell Publishing Ltd.

  9. Evolution of finger millet: evidence from random amplified polymorphic DNA.

    Science.gov (United States)

    Hilu, K W

    1995-04-01

    Finger millet (Eleusine coracana ssp. coracana) is an annual tetraploid member of a predominantly African genus. The crop is believed to have been domesticated from the tetraploid E. coracana ssp. africana. Cytogenetic and isozyme data point to the allopolyploid nature of the species and molecular information has shown E. indica to be one of the genomic donors. A recent isozyme study questioned the proposed phylogenetic relationship between finger millet and its direct ancestor subspecies africana. An approach using random amplified polymorphic DNA (RAPD) was employed in this study to examine genetic diversity and to evaluate hypotheses concerning the evolution of domesticated and wild annual species of Eleusine. Unlike previous molecular approaches, the RAPD study revealed genetic diversity in the crop. The pattern of genetic variation was loosely correlated to geographic distribution. The allotetraploid nature of the crop was confirmed and molecular markers that can possibly identify the other genomic donor were proposed. Genotypes of subspecies africana did not group closely with those of the crop but showed higher affinities to E. indica, reflecting the pattern of similarity revealed by the isozyme study. The multiple origin of subspecies africana could explain the discrepancy between the isozyme-RAPD evidence and previous information. The RAPD study showed the close genetic affinity of E. tristachya to the E. coracana--E. indica group and understood the distinctness of E. multiflora.

  10. Touch DNA collection versus firearm fingerprinting: comparing evidence production and identification outcomes.

    Science.gov (United States)

    Nunn, Samuel

    2013-05-01

    A project by a metropolitan police agency in 2008-2009 had police use touch DNA kits to collect cell samples from seized firearms. To assess outcomes, results of touch DNA swabbing of firearms were compared to fingerprinting firearm evidence. The rationale was that fingerprinting, as the older technology, was the baseline against which to compare touch DNA. But little is known about ways to measure touch DNA productivity compared to fingerprinting. To examine differences between the two requires comparable measurements. Two measures were used: quantity of probative or investigative evidence produced and identification outcomes. When applied to firearms seized within an Indianapolis, IN police district, touch DNA produced a larger volume of evidence than fingerprinting, but identification outcomes for the two methods were equal. Because touch DNA was deployed by police patrol officers, there are implications for firearm forensics and the choice of forensic approaches used by police. © 2013 American Academy of Forensic Sciences.

  11. Forensic evidence collection and DNA identification in acute child sexual assault.

    Science.gov (United States)

    Thackeray, Jonathan D; Hornor, Gail; Benzinger, Elizabeth A; Scribano, Philip V

    2011-08-01

    To describe forensic evidence findings and reevaluate previous recommendations with respect to timing of evidence collection in acute child sexual assault and to identify factors associated with yield of DNA. This was a retrospective review of medical and legal records of patients aged 0 to 20 years who required forensic evidence collection. Ninety-seven of 388 (25%) processed evidence-collection kits were positive and 63 (65%) of them produced identifiable DNA. There were 20 positive samples obtained from children younger than 10 years; 17 of these samples were obtained from children seen within 24 hours of the assault. Three children had positive body samples beyond 24 hours after the assault, including 1 child positive for salivary amylase in the underwear and on the thighs 54 hours after the assault. DNA was found in 11 children aged younger than 10 years, including the child seen 54 hours after the assault. Collection of evidence within 24 hours of the assault was identified as an independent predictor of DNA detection. Identifiable DNA was collected from a child's body despite cases in which: evidence collection was performed >24 hours beyond the assault; the child had a normal/nonacute anogenital examination; there was no reported history of ejaculation; and the victim had bathed and/or changed clothes before evidence collection. Failure to conduct evidence collection on prepubertal children beyond 24 hours after the assault will result in rare missed opportunities to identify forensic evidence, including identification of DNA.

  12. Microarray BASICA: Background Adjustment, Segmentation, Image Compression and Analysis of Microarray Images

    Directory of Open Access Journals (Sweden)

    Jianping Hua

    2004-01-01

    Full Text Available This paper presents microarray BASICA: an integrated image processing tool for background adjustment, segmentation, image compression, and analysis of cDNA microarray images. BASICA uses a fast Mann-Whitney test-based algorithm to segment cDNA microarray images, and performs postprocessing to eliminate the segmentation irregularities. The segmentation results, along with the foreground and background intensities obtained with the background adjustment, are then used for independent compression of the foreground and background. We introduce a new distortion measurement for cDNA microarray image compression and devise a coding scheme by modifying the embedded block coding with optimized truncation (EBCOT algorithm (Taubman, 2000 to achieve optimal rate-distortion performance in lossy coding while still maintaining outstanding lossless compression performance. Experimental results show that the bit rate required to ensure sufficiently accurate gene expression measurement varies and depends on the quality of cDNA microarray images. For homogeneously hybridized cDNA microarray images, BASICA is able to provide from a bit rate as low as 5 bpp the gene expression data that are 99% in agreement with those of the original 32 bpp images.

  13. Environmental chemicals and DNA methylation in adults: a systematic review of the epidemiologic evidence

    Science.gov (United States)

    Current evidence supports the notion that environmental exposures are associated with DNA-methylation and expression changes that can impact human health. Our objective was to conduct a systematic review of epidemiologic studies evaluating the association between environmental chemicals with DNA met...

  14. Development and application of a microarray meter tool to optimize microarray experiments

    Directory of Open Access Journals (Sweden)

    Rouse Richard JD

    2008-07-01

    Full Text Available Abstract Background Successful microarray experimentation requires a complex interplay between the slide chemistry, the printing pins, the nucleic acid probes and targets, and the hybridization milieu. Optimization of these parameters and a careful evaluation of emerging slide chemistries are a prerequisite to any large scale array fabrication effort. We have developed a 'microarray meter' tool which assesses the inherent variations associated with microarray measurement prior to embarking on large scale projects. Findings The microarray meter consists of nucleic acid targets (reference and dynamic range control and probe components. Different plate designs containing identical probe material were formulated to accommodate different robotic and pin designs. We examined the variability in probe quality and quantity (as judged by the amount of DNA printed and remaining post-hybridization using three robots equipped with capillary printing pins. Discussion The generation of microarray data with minimal variation requires consistent quality control of the (DNA microarray manufacturing and experimental processes. Spot reproducibility is a measure primarily of the variations associated with printing. The microarray meter assesses array quality by measuring the DNA content for every feature. It provides a post-hybridization analysis of array quality by scoring probe performance using three metrics, a a measure of variability in the signal intensities, b a measure of the signal dynamic range and c a measure of variability of the spot morphologies.

  15. Science, truth, and forensic cultures: the exceptional legal status of DNA evidence.

    Science.gov (United States)

    Lynch, Michael

    2013-03-01

    Many epistemological terms, such as investigation, inquiry, argument, evidence, and fact were established in law well before being associated with science. However, while legal proof remained qualified by standards of 'moral certainty', scientific proof attained a reputation for objectivity. Although most forms of legal evidence (including expert evidence) continue to be treated as fallible 'opinions' rather than objective 'facts', forensic DNA evidence increasingly is being granted an exceptional factual status. It did not always enjoy such status. Two decades ago, the scientific status of forensic DNA evidence was challenged in the scientific literature and in courts of law, but by the late 1990s it was being granted exceptional legal status. This paper reviews the ascendancy of DNA profiling, and argues that its widely-heralded objective status is bound up with systems of administrative accountability. The 'administrative objectivity' of DNA evidence rests upon observable and reportable bureaucratic rules, records, recording devices, protocols, and architectural arrangements. By highlighting administrative sources of objectivity, this paper suggests that DNA evidence remains bound within the context of ordinary organisational and practical routines, and is not a transcendent source of 'truth' in the criminal justice system. Copyright © 2012. Published by Elsevier Ltd.

  16. The validation of forensic DNA extraction systems to utilize soil contaminated biological evidence.

    Science.gov (United States)

    Kasu, Mohaimin; Shires, Karen

    2015-07-01

    The production of full DNA profiles from biological evidence found in soil has a high failure rate due largely to the inhibitory substance humic acid (HA). Abundant in various natural soils, HA co-extracts with DNA during extraction and inhibits DNA profiling by binding to the molecular components of the genotyping assay. To successfully utilize traces of soil contaminated evidence, such as that found at many murder and rape crime scenes in South Africa, a reliable HA removal extraction system would often be selected based on previous validation studies. However, for many standard forensic DNA extraction systems, peer-reviewed publications detailing the efficacy on soil evidence is either lacking or is incomplete. Consequently, these sample types are often not collected or fail to yield suitable DNA material due to the use of unsuitable methodology. The aim of this study was to validate the common forensic DNA collection and extraction systems used in South Africa, namely DNA IQ, FTA elute and Nucleosave for processing blood and saliva contaminated with HA. A forensic appropriate volume of biological evidence was spiked with HA (0, 0.5, 1.5 and 2.5 mg/ml) and processed through each extraction protocol for the evaluation of HA removal using QPCR and STR-genotyping. The DNA IQ magnetic bead system effectively removed HA from highly contaminated blood and saliva, and generated consistently acceptable STR profiles from both artificially spiked samples and crude soil samples. This system is highly recommended for use on soil-contaminated evidence over the cellulose card-based systems currently being preferentially used for DNA sample collection. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae.

    Science.gov (United States)

    Garbacz, Marta A; Lujan, Scott A; Burkholder, Adam B; Cox, Phillip B; Wu, Qiuqin; Zhou, Zhi-Xiong; Haber, James E; Kunkel, Thomas A

    2018-02-27

    To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase ε (Pol ε). Although pol2-16 mutants survive, they present very tiny spore colonies, increased doubling time, larger than normal cells, aberrant nuclei, and rapid acquisition of suppressor mutations. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack only Pol ε proofreading (pol2-4), consistent with the idea that Pol ε is the major leading-strand polymerase used for unstressed DNA replication. Ribonucleotides are incorporated into the pol2-16 genome in patterns consistent with leading-strand replication by Pol δ when Pol ε is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.

  18. The Importance of Normalization on Large and Heterogeneous Microarray Datasets

    Science.gov (United States)

    DNA microarray technology is a powerful functional genomics tool increasingly used for investigating global gene expression in environmental studies. Microarrays can also be used in identifying biological networks, as they give insight on the complex gene-to-gene interactions, ne...

  19. Calculating the weight of evidence in low-template forensic DNA casework.

    Science.gov (United States)

    Lohmueller, Kirk E; Rudin, Norah

    2013-01-01

    Interpreting and assessing the weight of low-template DNA evidence presents a formidable challenge in forensic casework. This report describes a case in which a similar mixed DNA profile was obtained from four different bloodstains. The defense proposed that the low-level minor profile came from an alternate suspect, the defendant's mistress. The strength of the evidence was assessed using a probabilistic approach that employed likelihood ratios incorporating the probability of allelic drop-out. Logistic regression was used to model the probability of drop-out using empirical validation data from the government laboratory. The DNA profile obtained from the bloodstain described in this report is at least 47 billion times more likely if, in addition to the victim, the alternate suspect was the minor contributor, than if another unrelated individual was the minor contributor. This case illustrates the utility of the probabilistic approach for interpreting complex low-template DNA profiles. © 2012 American Academy of Forensic Sciences.

  20. Increased recovery of touch DNA evidence using FTA paper compared to conventional collection methods.

    Science.gov (United States)

    Kirgiz, Irina A; Calloway, Cassandra

    2017-04-01

    21 samples (69%) and a partial profile was observed for nine samples (25%); STR analysis failed for two samples collected using tape (6%). In conclusion, we show that the FTA paper scraping method has the potential to collect higher DNA yields from touch DNA evidence deposited on non-porous surfaces often encountered in criminal cases compared to conventional methods. Copyright © 2017 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.

  1. LNA-modified isothermal oligonucleotide microarray for ...

    Indian Academy of Sciences (India)

    2014-10-20

    Oct 20, 2014 ... the advent of DNA microarray techniques (Lee et al. 2007). ... atoms of ribose to form a bicyclic ribosyl structure. It is the .... 532 nm and emission at 570 nm. The signal ..... sis and validation using real-time PCR. Nucleic Acids ...

  2. Evaluating the efficacy of DNA differential extraction methods for sexual assault evidence.

    Science.gov (United States)

    Klein, Sonja B; Buoncristiani, Martin R

    2017-07-01

    Analysis of sexual assault evidence, often a mixture of spermatozoa and victim epithelial cells, represents a significant portion of a forensic DNA laboratory's case load. Successful genotyping of sperm DNA from these mixed cell samples, particularly with low amounts of sperm, depends on maximizing sperm DNA recovery and minimizing non-sperm DNA carryover. For evaluating the efficacy of the differential extraction, we present a method which uses a Separation Potential Ratio (SPRED) to consider both sperm DNA recovery and non-sperm DNA removal as variables for determining separation efficiency. In addition, we describe how the ratio of male-to-female DNA in the sperm fraction may be estimated by using the SPRED of the differential extraction method in conjunction with the estimated ratio of male-to-female DNA initially present on the mixed swab. This approach may be useful for evaluating or modifying differential extraction methods, as we demonstrate by comparing experimental results obtained from the traditional differential extraction and the Erase Sperm Isolation Kit (PTC © ) procedures. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Unraveling the rat blood genome-wide transcriptome after oral administration of lavender oil by a two-color dye-swap DNA microarray approach

    Directory of Open Access Journals (Sweden)

    Motohide Hori

    2016-06-01

    Full Text Available Lavender oil (LO is a commonly used essential oil in aromatherapy as non-traditional medicine. With an aim to demonstrate LO effects on the body, we have recently established an animal model investigating the influence of orally administered LO in rat tissues, genome-wide. In this brief, we investigate the effect of LO ingestion in the blood of rat. Rats were administered LO at usual therapeutic dose (5 mg/kg in humans, and following collection of the venous blood from the heart and extraction of total RNA, the differentially expressed genes were screened using a 4 × 44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA in conjunction with a two-color dye-swap approach. A total of 834 differentially expressed genes in the blood were identified: 362 up-regulated and 472 down-regulated. These genes were functionally categorized using bioinformatics tools. The gene expression inventory of rat blood transcriptome under LO, a first report, has been deposited into the Gene Expression Omnibus (GEO: GSE67499. The data will be a valuable resource in examining the effects of natural products, and which could also serve as a human model for further functional analysis and investigation.

  4. Prosecutor: parameter-free inference of gene function for prokaryotes using DNA microarray data, genomic context and multiple gene annotation sources

    Directory of Open Access Journals (Sweden)

    van Hijum Sacha AFT

    2008-10-01

    Full Text Available Abstract Background Despite a plethora of functional genomic efforts, the function of many genes in sequenced genomes remains unknown. The increasing amount of microarray data for many species allows employing the guilt-by-association principle to predict function on a large scale: genes exhibiting similar expression patterns are more likely to participate in shared biological processes. Results We developed Prosecutor, an application that enables researchers to rapidly infer gene function based on available gene expression data and functional annotations. Our parameter-free functional prediction method uses a sensitive algorithm to achieve a high association rate of linking genes with unknown function to annotated genes. Furthermore, Prosecutor utilizes additional biological information such as genomic context and known regulatory mechanisms that are specific for prokaryotes. We analyzed publicly available transcriptome data sets and used literature sources to validate putative functions suggested by Prosecutor. We supply the complete results of our analysis for 11 prokaryotic organisms on a dedicated website. Conclusion The Prosecutor software and supplementary datasets available at http://www.prosecutor.nl allow researchers working on any of the analyzed organisms to quickly identify the putative functions of their genes of interest. A de novo analysis allows new organisms to be studied.

  5. Fibre optic microarrays.

    Science.gov (United States)

    Walt, David R

    2010-01-01

    This tutorial review describes how fibre optic microarrays can be used to create a variety of sensing and measurement systems. This review covers the basics of optical fibres and arrays, the different microarray architectures, and describes a multitude of applications. Such arrays enable multiplexed sensing for a variety of analytes including nucleic acids, vapours, and biomolecules. Polymer-coated fibre arrays can be used for measuring microscopic chemical phenomena, such as corrosion and localized release of biochemicals from cells. In addition, these microarrays can serve as a substrate for fundamental studies of single molecules and single cells. The review covers topics of interest to chemists, biologists, materials scientists, and engineers.

  6. Cytological evidence for DNA chain elongation after UV irradiation in the S phase

    International Nuclear Information System (INIS)

    Minka, D.F.; Nath, J.

    1981-01-01

    Human cells irradiated with UV light synthesize lower molecular weight DNA than unirradiated cells. This reduction in molecular weight is greater in xeroderma pigmentosum (XP) cells than in normal cells. The molecular weight of DNA is further reduced by the addition of caffeine to XP cells. By several hours after irradiation, DNA fragments are barely detectable. Cells from excision-proficient and excision-deficient XP patients were studied autoradiographically to produce cytological evidence of DNA chain elongation. Replicate cultures with and without caffeine were synchronized and irradiated with UV light during the S phase. Caffeine was removed in G2, and the cells were labeled with 3 H-thymidine. Results showed significantly increased labeling during G2 of excision-deficient XP cells. Labeling was dependent on the time of irradiation and presence of caffeine. The XP variant cells had no increase in labeling for any irradiation time

  7. DNA microarray profiling of a diverse collection of nosocomial methicillin-resistant staphylococcus aureus isolates assigns the majority to the correct sequence type and staphylococcal cassette chromosome mec (SCCmec) type and results in the subsequent identification and characterization of novel SCCmec-SCCM1 composite islands.

    LENUS (Irish Health Repository)

    Shore, Anna C

    2012-10-01

    One hundred seventy-five isolates representative of methicillin-resistant Staphylococcus aureus (MRSA) clones that predominated in Irish hospitals between 1971 and 2004 and that previously underwent multilocus sequence typing (MLST) and staphylococcal cassette chromosome mec (SCCmec) typing were characterized by spa typing (175 isolates) and DNA microarray profiling (107 isolates). The isolates belonged to 26 sequence type (ST)-SCCmec types and subtypes and 35 spa types. The array assigned all isolates to the correct MLST clonal complex (CC), and 94% (100\\/107) were assigned an ST, with 98% (98\\/100) correlating with MLST. The array assigned all isolates to the correct SCCmec type, but subtyping of only some SCCmec elements was possible. Additional SCCmec\\/SCC genes or DNA sequence variation not detected by SCCmec typing was detected by array profiling, including the SCC-fusidic acid resistance determinant Q6GD50\\/fusC. Novel SCCmec\\/SCC composite islands (CIs) were detected among CC8 isolates and comprised SCCmec IIA-IIE, IVE, IVF, or IVg and a ccrAB4-SCC element with 99% DNA sequence identity to SCC(M1) from ST8\\/t024-MRSA, SCCmec VIII, and SCC-CI in Staphylococcus epidermidis. The array showed that the majority of isolates harbored one or more superantigen (94%; 100\\/107) and immune evasion cluster (91%; 97\\/107) genes. Apart from fusidic acid and trimethoprim resistance, the correlation between isolate antimicrobial resistance phenotype and the presence of specific resistance genes was ≥97%. Array profiling allowed high-throughput, accurate assignment of MRSA to CCs\\/STs and SCCmec types and provided further evidence of the diversity of SCCmec\\/SCC. In most cases, array profiling can accurately predict the resistance phenotype of an isolate.

  8. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R.

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells.

  9. A robotics platform for automated batch fabrication of high density, microfluidics-based DNA microarrays, with applications to single cell, multiplex assays of secreted proteins.

    Science.gov (United States)

    Ahmad, Habib; Sutherland, Alex; Shin, Young Shik; Hwang, Kiwook; Qin, Lidong; Krom, Russell-John; Heath, James R

    2011-09-01

    Microfluidics flow-patterning has been utilized for the construction of chip-scale miniaturized DNA and protein barcode arrays. Such arrays have been used for specific clinical and fundamental investigations in which many proteins are assayed from single cells or other small sample sizes. However, flow-patterned arrays are hand-prepared, and so are impractical for broad applications. We describe an integrated robotics/microfluidics platform for the automated preparation of such arrays, and we apply it to the batch fabrication of up to eighteen chips of flow-patterned DNA barcodes. The resulting substrates are comparable in quality with hand-made arrays and exhibit excellent substrate-to-substrate consistency. We demonstrate the utility and reproducibility of robotics-patterned barcodes by utilizing two flow-patterned chips for highly parallel assays of a panel of secreted proteins from single macrophage cells. © 2011 American Institute of Physics

  10. Biological Evidence Management for DNA Analysis in Cases of Sexual Assault

    Science.gov (United States)

    Magalhães, Teresa; Dinis-Oliveira, Ricardo Jorge; Silva, Benedita; Corte-Real, Francisco; Nuno Vieira, Duarte

    2015-01-01

    Biological evidence with forensic interest may be found in several cases of assault, being particularly relevant if sexually related. Sexual assault cases are characterized by low rates of disclosure, reporting, prosecution, and conviction. Biological evidence is sometimes the only way to prove the occurrence of sexual contact and to identify the perpetrator. The major focus of this review is to propose practical approaches and guidelines to help health, forensic, and law enforcement professionals to deal with biological evidence for DNA analysis. Attention should be devoted to avoiding contamination, degradation, and loss of biological evidence, as well as respecting specific measures to properly handle evidence (i.e., selection, collection, packing, sealing, labeling, storage, preservation, transport, and guarantee of the chain custody). Biological evidence must be carefully managed since the relevance of any finding in Forensic Genetics is determined, in the first instance, by the integrity and quantity of the samples submitted for analysis. PMID:26587562

  11. Biological Evidence Management for DNA Analysis in Cases of Sexual Assault

    Directory of Open Access Journals (Sweden)

    Teresa Magalhães

    2015-01-01

    Full Text Available Biological evidence with forensic interest may be found in several cases of assault, being particularly relevant if sexually related. Sexual assault cases are characterized by low rates of disclosure, reporting, prosecution, and conviction. Biological evidence is sometimes the only way to prove the occurrence of sexual contact and to identify the perpetrator. The major focus of this review is to propose practical approaches and guidelines to help health, forensic, and law enforcement professionals to deal with biological evidence for DNA analysis. Attention should be devoted to avoiding contamination, degradation, and loss of biological evidence, as well as respecting specific measures to properly handle evidence (i.e., selection, collection, packing, sealing, labeling, storage, preservation, transport, and guarantee of the chain custody. Biological evidence must be carefully managed since the relevance of any finding in Forensic Genetics is determined, in the first instance, by the integrity and quantity of the samples submitted for analysis.

  12. The use of microarrays in microbial ecology

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-09-15

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

  13. DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

    Directory of Open Access Journals (Sweden)

    Lenz Heinz-Josef

    2009-11-01

    Full Text Available Abstract Background Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes. Methods HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity® Pathway Analysis. Results Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed. Conclusion This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets

  14. Genetic Evidence for Elevated Pathogenicity of Mitochondrial DNA Heteroplasmy in Autism Spectrum Disorder.

    Directory of Open Access Journals (Sweden)

    Yiqin Wang

    2016-10-01

    Full Text Available Increasing clinical and biochemical evidence implicate mitochondrial dysfunction in the pathophysiology of Autism Spectrum Disorder (ASD, but little is known about the biological basis for this connection. A possible cause of ASD is the genetic variation in the mitochondrial DNA (mtDNA sequence, which has yet to be thoroughly investigated in large genomic studies of ASD. Here we evaluated mtDNA variation, including the mixture of different mtDNA molecules in the same individual (i.e., heteroplasmy, using whole-exome sequencing data from mother-proband-sibling trios from simplex families (n = 903 where only one child is affected by ASD. We found that heteroplasmic mutations in autistic probands were enriched at non-polymorphic mtDNA sites (P = 0.0015, which were more likely to confer deleterious effects than heteroplasmies at polymorphic mtDNA sites. Accordingly, we observed a ~1.5-fold enrichment of nonsynonymous mutations (P = 0.0028 as well as a ~2.2-fold enrichment of predicted pathogenic mutations (P = 0.0016 in autistic probands compared to their non-autistic siblings. Both nonsynonymous and predicted pathogenic mutations private to probands conferred increased risk of ASD (Odds Ratio, OR[95% CI] = 1.87[1.14-3.11] and 2.55[1.26-5.51], respectively, and their influence on ASD was most pronounced in families with probands showing diminished IQ and/or impaired social behavior compared to their non-autistic siblings. We also showed that the genetic transmission pattern of mtDNA heteroplasmies with high pathogenic potential differed between mother-autistic proband pairs and mother-sibling pairs, implicating developmental and possibly in utero contributions. Taken together, our genetic findings substantiate pathogenic mtDNA mutations as a potential cause for ASD and synergize with recent work calling attention to their unique metabolic phenotypes for diagnosis and treatment of children with ASD.

  15. Evaluating the weight of evidence by using quantitative short tandem repeat data in DNA mixtures

    DEFF Research Database (Denmark)

    Tvedebrink, Torben; Eriksen, Poul Svante; Mogensen, Helle Smidt

    2010-01-01

    he evaluation of results from mixtures of deoxyribonucleic acid (DNA) from two or more people in crime case investigations may be improved by taking not only the qualitative but also the quantitative part of the results into consideration. We present a statistical likelihood approach to assess...... the probability of observed peak heights and peak areas information for a pair of profiles matching the DNA mixture. Furthermore, we demonstrate how to incorporate this probability in the evaluation of the weight of the evidence by a likelihood ratio approach. Our model is based on a multivariate normal...... peak heights and areas. Complying with this latent structure, we used the EM algorithm to impute the missing variables on the basis of a compound symmetry model. The measurements were subject to intralocus and interlocus correlations not depending on the actual alleles of the DNA profiles. Owing...

  16. Autoradiographic evidence for reutilization of DNA catabolites by granulocytopoiesis in the rat

    International Nuclear Information System (INIS)

    Gerecke, D.; Gross, R.

    1976-01-01

    The proliferating granulocyte precursor pool of rat bone marrow was labelled during DNA synthesis by continuous infusion and by single injection of 3 H-thymidine ( 3 H-TdR), as well as by single injection of 125 I-iododeoxyuridine ( 125 I-UdR). The appearance of neutrophilic granulocytes in the blood stream after these various labelling procedures was studied by autoradiography. Labelling patterns of blood neutrophils were identical during continuous infusion and after single injection of 3 H-TdR, and 100 percent labelling of the blood compartment was achieved. This result indicated reutilization of DNA catabolites to occur in granulocytopoiesis leading to continuous availability of 3 H-labelled DNA precursors even after a single injection of 3 H-TdR. Attempts to suppress reutilization of label by infusion of cold thymidine 1 h after injection of 3 H-TdR were unsuccessful. However, a change in the labelling pattern of blood neutrophils was seen after single injection of 125 I-UdR, a DNA precursor poorly reutilized in comparison to 3 H-TdR. This result provided further evidence for reutilization of DNA catabolites by the cell system investigated. A comprehensive discussion of the results indicates that thymidinemonophosphate is the biochemical level of reutilization in granulocytopoiesis. (author)

  17. Autoradiographic evidence for reutilization of DNA catabolites by granulocytopoiesis in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Gerecke, D; Gross, R [Koeln Univ. (Germany, F.R.). Medizinische Klinik

    1976-01-01

    The proliferating granulocyte precursor pool of rat bone marrow was labelled during DNA synthesis by continuous infusion and by single injection of /sup 3/H-thymidine (/sup 3/H-TdR), as well as by single injection of /sup 125/I-iododeoxyuridine (/sup 125/I-UdR). The appearance of neutrophilic granulocytes in the blood stream after these various labelling procedures was studied by autoradiography. Labelling patterns of blood neutrophils were identical during continuous infusion and after single injection of /sup 3/H-TdR, and 100 percent labelling of the blood compartment was achieved. This result indicated reutilization of DNA catabolites to occur in granulocytopoiesis leading to continuous availability of /sup 3/H-labelled DNA precursors even after a single injection of /sup 3/H-TdR. Attempts to suppress reutilization of label by infusion of cold thymidine 1 h after injection of /sup 3/H-TdR were unsuccessful. However, a change in the labelling pattern of blood neutrophils was seen after single injection of /sup 125/I-UdR, a DNA precursor poorly reutilized in comparison to /sup 3/H-TdR. This result provided further evidence for reutilization of DNA catabolites by the cell system investigated. A comprehensive discussion of the results indicates that thymidinemonophosphate is the biochemical level of reutilization in granulocytopoiesis.

  18. Single cells for forensic DNA analysis--from evidence material to test tube.

    Science.gov (United States)

    Brück, Simon; Evers, Heidrun; Heidorn, Frank; Müller, Ute; Kilper, Roland; Verhoff, Marcel A

    2011-01-01

    The purpose of this project was to develop a method that, while providing morphological quality control, allows single cells to be obtained from the surfaces of various evidence materials and be made available for DNA analysis in cases where only small amounts of cell material are present or where only mixed traces are found. With the SteREO Lumar.V12 stereomicroscope and UV unit from Zeiss, it was possible to detect and assess single epithelial cells on the surfaces of various objects (e.g., glass, plastic, metal). A digitally operated micromanipulator developed by aura optik was used to lift a single cell from the surface of evidence material and to transfer it to a conventional PCR tube or to an AmpliGrid(®) from Advalytix. The actual lifting of the cells was performed with microglobes that acted as carriers. The microglobes were held with microtweezers and were transferred to the DNA analysis receptacles along with the adhering cells. In a next step, the PCR can be carried out in this receptacle without removing the microglobe. Our method allows a single cell to be isolated directly from evidence material and be made available for forensic DNA analysis. © 2010 American Academy of Forensic Sciences.

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

    Science.gov (United States)

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

    2014-01-01

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

  20. Vertebrate DNA in fecal samples from bonobos and gorillas: evidence for meat consumption or artefact?

    Directory of Open Access Journals (Sweden)

    Michael Hofreiter

    Full Text Available BACKGROUND: Deciphering the behavioral repertoire of great apes is a challenge for several reasons. First, due to their elusive behavior in dense forest environments, great ape populations are often difficult to observe. Second, members of the genus Pan are known to display a great variety in their behavioral repertoire; thus, observations from one population are not necessarily representative for other populations. For example, bonobos (Pan paniscus are generally believed to consume almost no vertebrate prey. However, recent observations show that at least some bonobo populations may consume vertebrate prey more commonly than previously believed. We investigated the extent of their meat consumption using PCR amplification of vertebrate mitochondrial DNA (mtDNA segments from DNA extracted from bonobo feces. As a control we also attempted PCR amplifications from gorilla feces, a species assumed to be strictly herbivorous. PRINCIPAL FINDINGS: We found evidence for consumption of a variety of mammalian species in about 16% of the samples investigated. Moreover, 40% of the positive DNA amplifications originated from arboreal monkeys. However, we also found duiker and monkey mtDNA in the gorilla feces, albeit in somewhat lower percentages. Notably, the DNA sequences isolated from the two ape species fit best to the species living in the respective regions. This result suggests that the sequences are of regional origin and do not represent laboratory contaminants. CONCLUSIONS: Our results allow at least three possible and mutually not exclusive conclusions. First, all results may represent contamination of the feces by vertebrate DNA from the local environment. Thus, studies investigating a species' diet from feces DNA may be unreliable due to the low copy number of DNA originating from diet items. Second, there is some inherent difference between the bonobo and gorilla feces, with only the later ones being contaminated. Third, similar to bonobos, for

  1. Vertebrate DNA in fecal samples from bonobos and gorillas: evidence for meat consumption or artefact?

    Science.gov (United States)

    Hofreiter, Michael; Kreuz, Eva; Eriksson, Jonas; Schubert, Grit; Hohmann, Gottfried

    2010-02-25

    Deciphering the behavioral repertoire of great apes is a challenge for several reasons. First, due to their elusive behavior in dense forest environments, great ape populations are often difficult to observe. Second, members of the genus Pan are known to display a great variety in their behavioral repertoire; thus, observations from one population are not necessarily representative for other populations. For example, bonobos (Pan paniscus) are generally believed to consume almost no vertebrate prey. However, recent observations show that at least some bonobo populations may consume vertebrate prey more commonly than previously believed. We investigated the extent of their meat consumption using PCR amplification of vertebrate mitochondrial DNA (mtDNA) segments from DNA extracted from bonobo feces. As a control we also attempted PCR amplifications from gorilla feces, a species assumed to be strictly herbivorous. We found evidence for consumption of a variety of mammalian species in about 16% of the samples investigated. Moreover, 40% of the positive DNA amplifications originated from arboreal monkeys. However, we also found duiker and monkey mtDNA in the gorilla feces, albeit in somewhat lower percentages. Notably, the DNA sequences isolated from the two ape species fit best to the species living in the respective regions. This result suggests that the sequences are of regional origin and do not represent laboratory contaminants. Our results allow at least three possible and mutually not exclusive conclusions. First, all results may represent contamination of the feces by vertebrate DNA from the local environment. Thus, studies investigating a species' diet from feces DNA may be unreliable due to the low copy number of DNA originating from diet items. Second, there is some inherent difference between the bonobo and gorilla feces, with only the later ones being contaminated. Third, similar to bonobos, for which the consumption of monkeys has only recently been

  2. Analysis of DNA evidence recovered from epithelial cells in penile swabs.

    Science.gov (United States)

    Drobnic, Katja

    2003-06-01

    In the rape case presented here, no semen, hair, or other biological evidence were left by the perpetuator at the crime scene or on the victim. The alleged assailant was arrested soon after the crime. A classical stain recovery technique using cotton swab moistened with sterile water was taken for recovering potential female epithelial cells and leukocytes deposited on the alleged assailant's penis during sexual assault. The organic method used for DNA extraction was quantified according to the slot-blot procedure and amplified at 9 and 15 polymorphic loci. Penile swab revealed a DNA profile of mixed origin. In addition to the suspect's DNA profile, DNA contribution from the victim was identified as a minor component in the mixture. Frequency of the profile resulted in a value of 5 x 10(-14) for the multiplex systems AmpFlSTR Plus and 2.5 x 10(-18) for the multiplex system PowerPlex 16, taking into account only non-overlapping alleles between the suspect and the victim from the minor component in the DNA mixture. Moreover, three additional alleles were observed at D21S11 locus by use of PowerPlex and STR SGM plus primers, which could not belong to the suspect. The victim's DNA profile showed the same three-banded genotype at this locus. The same pattern was detected when the victim's saliva or blood were used as reference samples. Our laboratory finding was consistent with the police report that the victim was a person with Down syndrome, a human genetic disease mainly resulting from trisomy (triplication) of the 21 chromosome.

  3. Noncovalent DNA Binding Drives DNA Alkylation by Leinamycin. Evidence That the Z,E-5-(Thiazol-4-yl)-penta-2,4-dienone Moiety of the Natural Product Serves As An Atypical DNA Intercalator

    Science.gov (United States)

    Fekry, Mostafa I.; Szekely, Jozsef; Dutta, Sanjay; Breydo, Leonid; Zang, Hong; Gates, Kent S.

    2012-01-01

    Molecular recognition and chemical modification of DNA are important in medicinal chemistry, toxicology, and biotechnology. Historically, natural products have revealed many interesting and unexpected mechanisms for noncovalent DNA binding and covalent DNA modification. The studies reported here characterize the molecular mechanisms underlying the efficient alkylation of duplex DNA by the Streptomyces-derived natural product leinamycin. Previous studies suggested that alkylation of duplex DNA by activated leinamycin (2) is driven by noncovalent association of the natural product with the double helix. This is striking because leinamycin does not contain a classical noncovalent DNA-binding motif such as an intercalating unit, a groove binder, or a polycation. The experiments described here provide evidence that leinamycin is an atypical DNA-intercalating agent. A competition binding assay involving daunomycin-mediated inhibition of DNA alkylation by leinamycin provided evidence that activated leinamycin binds to duplex DNA with an apparent binding constant of approximately 4.3 ± 0.4 × 103 M−1. Activated leinamycin caused duplex unwinding and hydrodynamic changes in DNA-containing solutions that are indicative of DNA intercalation. Characterization of the reaction of activated leinamycin with palindromic duplexes containing 5'-CG and 5'-GC target sites, bulge-containing duplexes, and 5-methylcytosine-containing duplexes provided evidence regarding the orientation of leinamycin with respect to target guanine residues. The data allows construction of a model for the leinamycin-DNA complex suggesting how a modest DNA-binding constant combines with proper positioning of the natural product to drive efficient alkylation of guanine residues in the major groove of duplex DNA. PMID:21954957

  4. "Harshlighting" small blemishes on microarrays

    Directory of Open Access Journals (Sweden)

    Wittkowski Knut M

    2005-03-01

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

  5. Systematic study of association of four GABAergic genes: glutamic acid decarboxylase 1 gene, glutamic acid decarboxylase 2 gene, GABA(B) receptor 1 gene and GABA(A) receptor subunit beta2 gene, with schizophrenia using a universal DNA microarray.

    Science.gov (United States)

    Zhao, Xu; Qin, Shengying; Shi, Yongyong; Zhang, Aiping; Zhang, Jing; Bian, Li; Wan, Chunling; Feng, Guoyin; Gu, Niufan; Zhang, Guangqi; He, Guang; He, Lin

    2007-07-01

    Several studies have suggested the dysfunction of the GABAergic system as a risk factor in the pathogenesis of schizophrenia. In the present study, case-control association analysis was conducted in four GABAergic genes: two glutamic acid decarboxylase genes (GAD1 and GAD2), a GABA(A) receptor subunit beta2 gene (GABRB2) and a GABA(B) receptor 1 gene (GABBR1). Using a universal DNA microarray procedure we genotyped a total of 20 SNPs on the above four genes in a study involving 292 patients and 286 controls of Chinese descent. Statistically significant differences were observed in the allelic frequencies of the rs187269C/T polymorphism in the GABRB2 gene (P=0.0450, chi(2)=12.40, OR=1.65) and the -292A/C polymorphism in the GAD1 gene (P=0.0450, chi(2)=14.64 OR=1.77). In addition, using an electrophoretic mobility shift assay (EMSA), we discovered differences in the U251 nuclear protein binding to oligonucleotides representing the -292 SNP on the GAD1 gene, which suggests that the -292C allele has reduced transcription factor binding efficiency compared with the 292A allele. Using the multifactor-dimensionality reduction method (MDR), we found that the interactions among the rs187269C/T polymorphism in the GABRB2 gene, the -243A/G polymorphism in the GAD2 gene and the 27379C/T and 661C/T polymorphisms in the GAD1 gene revealed a significant association with schizophrenia (Pschizophrenia in the Chinese population.

  6. Principles of gene microarray data analysis.

    Science.gov (United States)

    Mocellin, Simone; Rossi, Carlo Riccardo

    2007-01-01

    The development of several gene expression profiling methods, such as comparative genomic hybridization (CGH), differential display, serial analysis of gene expression (SAGE), and gene microarray, together with the sequencing of the human genome, has provided an opportunity to monitor and investigate the complex cascade of molecular events leading to tumor development and progression. The availability of such large amounts of information has shifted the attention of scientists towards a nonreductionist approach to biological phenomena. High throughput technologies can be used to follow changing patterns of gene expression over time. Among them, gene microarray has become prominent because it is easier to use, does not require large-scale DNA sequencing, and allows for the parallel quantification of thousands of genes from multiple samples. Gene microarray technology is rapidly spreading worldwide and has the potential to drastically change the therapeutic approach to patients affected with tumor. Therefore, it is of paramount importance for both researchers and clinicians to know the principles underlying the analysis of the huge amount of data generated with microarray technology.

  7. Trace DNA Sampling Success from Evidence Items Commonly Encountered in Forensic Casework.

    Science.gov (United States)

    Dziak, Renata; Peneder, Amy; Buetter, Alicia; Hageman, Cecilia

    2018-05-01

    Trace DNA analysis is a significant part of a forensic laboratory's workload. Knowing optimal sampling strategies and item success rates for particular item types can assist in evidence selection and examination processes and shorten turnaround times. In this study, forensic short tandem repeat (STR) casework results were reviewed to determine how often STR profiles suitable for comparison were obtained from "handler" and "wearer" areas of 764 items commonly submitted for examination. One hundred and fifty-five (155) items obtained from volunteers were also sampled. Items were analyzed for best sampling location and strategy. For casework items, headwear and gloves provided the highest success rates. Experimentally, eyeglasses and earphones, T-shirts, fabric gloves and watches provided the highest success rates. Eyeglasses and latex gloves provided optimal results if the entire surfaces were swabbed. In general, at least 10%, and up to 88% of all trace DNA analyses resulted in suitable STR profiles for comparison. © 2017 American Academy of Forensic Sciences.

  8. Forensic DNA identification of animal-derived trace evidence: tools for linking victims and suspects.

    Science.gov (United States)

    Halverson, Joy L; Basten, Christopher

    2005-08-01

    To evaluate the population substructure of purebred dogs and cats in order to estimate the true significance of a microsatellite-based DNA match for use as evidence in legal proceedings. The high frequency of animal hair as a forensic evidence submission necessitates the development of mitochondrial analysis tools as well. Random samples from a large convenience collection of veterinary diagnostic submissions from the western USA were used, as well as contributed samples of unrelated purebred cats and dogs. Dogs (n=558) were profiled with 17 microsatellites and the data evaluated for Hardy Weinberg and linkage equilibrium. The mitochondrial control region (D loop) of dogs (n=348) and cats (n=167) was sequenced to determine the haplotype distribution. Domestic dogs in the western United States showed significant population substructure with marked associations within loci but no disequilibrium between loci. A population substructure coefficient Theta=0.11 is recommended for calculating genotype frequencies. Mitochondrial haplotypes in cats and dogs show less variation than human haplotypes. Although population substructure occurs in domestic dogs (and can be inferred in cats), the discriminatory power of microsatellite analysis is dramatic with even partial DNA types, strongly supporting the prosecution of perpetrators in five discussed cases. Mitochondrial analysis, while less powerful, adds a layer of evidence in four discussed cases.

  9. [Screening of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) by cDNA microarray and influence of overexpression of PAG1 on biologic behavior of human metastatic prostatic cancer cell line in vitro].

    Science.gov (United States)

    Yu, Wen-juan; Wang, Yue-wei; Xie, Zhi-gang; You, Jiang-feng; Wang, Jie-liang; Cui, Xiang-lin; Pei, Fei; Zheng, Jie

    2010-02-01

    To screen for novel gene(s) associated with tumor metastasis, and to investigate the effect of overexpression of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behaviors of human prostatic cancer cell line PC-3M-1E8 in vitro. Four cDNA microarrays were constructed using cDNA library of prostatic cancer cells PC-3M-1E8 (high metastatic potential), PC-3M-2B4 (low metastatic potential), lung cancer cells PG-BE1 (high metastatic potential)and PG-LH7 (low metastatic potential)to screen genes which were differentially expressed according to their different metastatic properties. From a battery of differentially expressed genes, PAG1, which was markedly downregulated in both high metastatic sublines of PC-3M and PG was chosen for further investigation. Real-time PCR and Western blot were used to confirm the gene expression of PAG1 at mRNA and protein levels. Full-length coding sequence of human PAG1 was subcloned into plasmid pcDNA3.0 and the recombinant plasmids were stably transfected into PC-3M-1E8. The cell proliferation ability, anchorage-independent growth, cell cycle distribution, apoptosis rates and invasive ability were detected by MTT, and in addition, soft agar colony formation, flow cytometry analysis and matrigel invasion assay using Boyden chamber were also carried out respectively. All experiments contained pcDNA3.0-PAG1-transfected clones, vector transfected clones and non-transfected parental cells. A total of 327 differentially expressed genes were obtained between the high and low metastatic sublines of PC-3M cells, including 123 upregulated and 204 downregulated genes in PC-3M-1E8. A total of 281 genes, including 167 upregulated and 114 downregulated genes were obtained in PG-BE1 cells. Nine genes were simultaneously downregulated and 8 genes were upregulated in both high metastatic cell lines of PC-3M and PG. The expression of PAG1 at mRNA and protein level were decreased in the high metastatic subline PC-3M-1

  10. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

    International Nuclear Information System (INIS)

    Tao Weitao; Budd, Martin; Campbell, Judith L.

    2003-01-01

    We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1Δ double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1Δ alone. However, surprisingly, the dna2-2 sgs1Δ lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1Δ lethality is only partially suppressed by deletion of rad51Δ. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones

  11. Evidence that yeast SGS1, DNA2, SRS2, and FOB1 interact to maintain rDNA stability

    Energy Technology Data Exchange (ETDEWEB)

    Tao Weitao; Budd, Martin; Campbell, Judith L

    2003-11-27

    We and others have proposed that faulty processing of arrested replication forks leads to increases in recombination and chromosome instability in Saccharomyces cerevisiae. Now we use the ribosomal DNA locus, which is a good model for all stages of DNA replication, to test this hypothesis. We showed previously that DNA replication pausing at the ribosomal DNA replication fork barrier (RFB) is accompanied by the occurrence of double-strand breaks near the RFB. Both pausing and breakage are elevated in the hypomorphic dna2-2 helicase mutant. Deletion of FOB1 suppresses the elevated pausing and DSB formation. Our current work shows that mutation inactivating Sgs1, the yeast RecQ helicase ortholog, also causes accumulation of stalled replication forks and DSBs at the rDNA RFB. Either deletion of FOB1, which suppresses fork blocking and certain types of rDNA recombination, or an increase in SIR2 gene dosage, which suppresses rDNA recombination, reduces the number of forks persisting at the RFB. Although dna2-2 sgs1{delta} double mutants are conditionally lethal, they do not show enhanced rDNA defects compared to sgs1{delta} alone. However, surprisingly, the dna2-2 sgs1{delta} lethality is suppressed by deletion of FOB1. On the other hand, the dna2-2 sgs1{delta} lethality is only partially suppressed by deletion of rad51{delta}. We propose that the replication-associated defects that we document in the rDNA are characteristic of similar events occurring either stochastically throughout the genome or at other regions where replication forks move slowly or stall, such as telomeres, centromeres, or replication slow zones.

  12. Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

    Directory of Open Access Journals (Sweden)

    Almeida Igor C

    2007-04-01

    Full Text Available Abstract Background All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells. Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. Results The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. Conclusion Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein.

  13. DNA evidence of bowhead whale exploitation by Greenlandic Paleo-Inuit 4,000 years ago

    Science.gov (United States)

    Seersholm, Frederik Valeur; Pedersen, Mikkel Winther; Søe, Martin Jensen; Shokry, Hussein; Mak, Sarah Siu Tze; Ruter, Anthony; Raghavan, Maanasa; Fitzhugh, William; Kjær, Kurt H.; Willerslev, Eske; Meldgaard, Morten; Kapel, Christian M. O.; Hansen, Anders Johannes

    2016-11-01

    The demographic history of Greenland is characterized by recurrent migrations and extinctions since the first humans arrived 4,500 years ago. Our current understanding of these extinct cultures relies primarily on preserved fossils found in their archaeological deposits, which hold valuable information on past subsistence practices. However, some exploited taxa, though economically important, comprise only a small fraction of these sub-fossil assemblages. Here we reconstruct a comprehensive record of past subsistence economies in Greenland by sequencing ancient DNA from four well-described midden deposits. Our results confirm that the species found in the fossil record, like harp seal and ringed seal, were a vital part of Inuit subsistence, but also add a new dimension with evidence that caribou, walrus and whale species played a more prominent role for the survival of Paleo-Inuit cultures than previously reported. Most notably, we report evidence of bowhead whale exploitation by the Saqqaq culture 4,000 years ago.

  14. No evidence of chromosome damage in children and adolescents with differentiated thyroid carcinoma after receiving {sup 131}I radiometabolic therapy, as evaluated by micronucleus assay and microarray analysis

    Energy Technology Data Exchange (ETDEWEB)

    Federico, Giovanni; Fiore, Lisa; Massart, Francesco; Saggese, Giuseppe [Azienda Ospedaliero-Universitaria Pisana, Department of Pediatrics, Unit of Pediatric Endocrinology and Diabetes, Pisa (Italy); Boni, Giuseppe; Lazzeri, Patrizia; Mariani, Giuliano [Azienda Ospedaliero-Universitaria Pisana, Unit of Nuclear Medicine, Pisa (Italy); Fabiani, Barbara; Verola, Carmela; Scarpato, Roberto [University of Pisa, Department of Biology, Unit of Genetics, Mutagenesis and Environmental Epidemiology, Pisa (Italy); Traino, Claudio [Azienda Ospedaliero-Universitaria Pisana, Health Physics Service, Pisa (Italy)

    2008-11-15

    As {sup 131}I therapy, used to achieve ablation of thyroid gland remnant, can cause chromosome damage in cultured peripheral lymphocytes especially, we investigated whether administration of radioiodine may induce early genome damage in peripheral T lymphocytes of adolescents with differentiated thyroid carcinoma (DTC). We studied 11 patients, aged 14.8 {+-} 3.1 years, who assumed {sup 131}I (range: 1.11-4.44 GBq) to ablate thyroid remnant. A blood sample for micronucleus assay and for evaluating expression of some genes involved in the DNA repair or the apoptosis pathways was obtained from each patient 1 h before (T{sub 0}) and 24 (T{sub 1}) and 48 h (T{sub 2}) post-radioiodine administration. Compared to T{sub 0}, we did not find any difference in the number of micronucleated cells at both T{sub 1} and T{sub 2} in any subject. Nine out of 11 patients had altered expression levels in a majority of the DNA repair and apoptosis genes at T{sub 1}, which decreased at T{sub 2}. We demonstrated for the first time that peripheral cells of DTC children and adolescents who received {sup 131}I at a mean dosage of 3.50 {+-} 0.37 GBq did not show chromosome damage within 48 h from the end of radiometabolic therapy. This may be due to a prompt activation of the cell machinery that maintains the integrity of the genome to prevent harmful double-strand breaks from progressing to chromosome mutations, either by repairing the lesions or by eliminating the most seriously damaged cells via apoptosis. (orig.)

  15. DNA microarrays : a molecular cloning manual

    National Research Council Canada - National Science Library

    Sambrook, Joseph; Bowtell, David

    2002-01-01

    .... This manual, designed to extend and to complement the information in the best-selling Molecular Cloning, is a synthesis of the expertise and experience of more than 30 contributors all innovators in a fast moving field...

  16. Chemical elemental distribution and soil DNA fingerprints provide the critical evidence in murder case investigation.

    Directory of Open Access Journals (Sweden)

    Giuseppe Concheri

    Full Text Available BACKGROUND: The scientific contribution to the solution of crime cases, or throughout the consequent forensic trials, is a crucial aspect of the justice system. The possibility to extract meaningful information from trace amounts of samples, and to match and validate evidences with robust and unambiguous statistical tests, are the key points of such process. The present report is the authorized disclosure of an investigation, carried out by Attorney General appointment, on a murder case in northern Italy, which yielded the critical supporting evidence for the judicial trial. METHODOLOGY/PRINCIPAL FINDINGS: The proportional distribution of 54 chemical elements and the bacterial community DNA fingerprints were used as signature markers to prove the similarity of two soil samples. The first soil was collected on the crime scene, along a corn field, while the second was found in trace amounts on the carpet of a car impounded from the main suspect in a distant location. The matching similarity of the two soils was proven by crossing the results of two independent techniques: a elemental analysis via inductively coupled plasma mass spectrometry (ICP-MS and optical emission spectrometry (ICP-OES approaches, and b amplified ribosomal DNA restriction analysis by gel electrophoresis (ARDRA. CONCLUSIONS: Besides introducing the novel application of these methods to forensic disciplines, the highly accurate level of resolution observed, opens new possibilities also in the fields of soil typing and tracking, historical analyses, geochemical surveys and global land mapping.

  17. Further evidence for poly-ADP-ribosylated histones as DNA suppressors

    International Nuclear Information System (INIS)

    Yu, F.L.; Geronimo, I.H.; Bender, W.; Meginniss, K.E.

    1986-01-01

    For many years histones have been considered to be the gene suppressors in eukaryotic cells. Recently, the authors have found strong evidence indicating that poly-ADP-ribosylated histones, rather than histones, are the potent inhibitors of DNA-dependent RNA synthesis. They now report additional evidence for this concept: 1) using histone inhibitor isolated directly from nuclei, the authors are able to confirm their earlier findings that the inhibitor substances are sensitive to pronase, snake venom phosphodiesterase digestion and 0.1N KOH hydrolysis, and are resistant to DNase I and RNase A digestion, 2) the O.D. 260/O.D.280 ratio of the histone inhibitor is between pure protein and nuclei acid, suggesting the inhibitor substance is a nucleoprotein hybrid. This result directly supports the fact that the isolated histone inhibitor is radioactive poly (ADP-ribose) labeled, 3) commercial histones show big differences in inhibitor activity. The authors believe this reflects the variation in poly-ADP-ribosylation among commercial histones, and 4) 0.1N KOH hydrolysis eliminates the poly (ADP-ribose) radioactivity from the acceptor proteins as well as histone inhibitor activity. Yet, on gel, the inhibitor shows identical histone bands and stain intensity before and after hydrolysis, indicating the histones per se are qualitatively and quantitatively unaffected by alkaline treatment. This result strongly suggests that histones themselves are not capable of inhibiting DNA-dependent RNA synthesis

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

    Directory of Open Access Journals (Sweden)

    Manish Biyani

    2015-07-01

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

  19. Evidence of animal mtDNA recombination between divergent populations of the potato cyst nematode Globodera pallida.

    Science.gov (United States)

    Hoolahan, Angelique H; Blok, Vivian C; Gibson, Tracey; Dowton, Mark

    2012-03-01

    Recombination is typically assumed to be absent in animal mitochondrial genomes (mtDNA). However, the maternal mode of inheritance means that recombinant products are indistinguishable from their progenitor molecules. The majority of studies of mtDNA recombination assess past recombination events, where patterns of recombination are inferred by comparing the mtDNA of different individuals. Few studies assess contemporary mtDNA recombination, where recombinant molecules are observed as direct mosaics of known progenitor molecules. Here we use the potato cyst nematode, Globodera pallida, to investigate past and contemporary recombination. Past recombination was assessed within and between populations of G. pallida, and contemporary recombination was assessed in the progeny of experimental crosses of these populations. Breeding of genetically divergent organisms may cause paternal mtDNA leakage, resulting in heteroplasmy and facilitating the detection of recombination. To assess contemporary recombination we looked for evidence of recombination between the mtDNA of the parental populations within the mtDNA of progeny. Past recombination was detected between a South American population and several UK populations of G. pallida, as well as between two South American populations. This suggests that these populations may have interbred, paternal mtDNA leakage occurred, and the mtDNA of these populations subsequently recombined. This evidence challenges two dogmas of animal mtDNA evolution; no recombination and maternal inheritance. No contemporary recombination between the parental populations was detected in the progeny of the experimental crosses. This supports current arguments that mtDNA recombination events are rare. More sensitive detection methods may be required to adequately assess contemporary mtDNA recombination in animals.

  20. Journey of DNA Evidence in Legal Arena: An Insight on Its Legal Perspective Worldwide and Highlight on Admissibility in India

    Directory of Open Access Journals (Sweden)

    Ramakant Gupta

    2016-01-01

    Full Text Available DNA profiling is one of the powerful breakthroughs in forensics. This specialized technique has made the identification of an individual possible even by a tiny shred of tissue or drop of blood thus, has strongly revolutionized various criminal investigations. Rape, paternity, and murder cases are the type of criminal cases commonly solved by the use of this technique. It has been recently introduced to forensic odontology and is also used frequently. Although this is a powerful and reliable scientific technique but its forensic use is a major contribution to the debate on law reform. The application of DNA profiling in the criminal justice system, i.e., the admissibility of DNA evidence in court of law is an important issue which is being faced by the courts and forensic experts worldwide today. Thus, a proper legal outlook is required while dealing with this kind of scientific evidence. Therefore, this review intends to make forensic experts/odontologists aware about the admissibility of DNA evidence in court, with a highlight on the laws related to the admissibility of evidence worldwide, having a special focus on the laws related to admissibility of evidence in Indian judicial system. For this review, the literature was overviewed from articles on DNA evidence and admissibility retrieved by searches on electronic databases such as Google, PubMed, and EMBASE from 1975 through July 2015.

  1. Cell-Based Microarrays for In Vitro Toxicology

    Science.gov (United States)

    Wegener, Joachim

    2015-07-01

    DNA/RNA and protein microarrays have proven their outstanding bioanalytical performance throughout the past decades, given the unprecedented level of parallelization by which molecular recognition assays can be performed and analyzed. Cell microarrays (CMAs) make use of similar construction principles. They are applied to profile a given cell population with respect to the expression of specific molecular markers and also to measure functional cell responses to drugs and chemicals. This review focuses on the use of cell-based microarrays for assessing the cytotoxicity of drugs, toxins, or chemicals in general. It also summarizes CMA construction principles with respect to the cell types that are used for such microarrays, the readout parameters to assess toxicity, and the various formats that have been established and applied. The review ends with a critical comparison of CMAs and well-established microtiter plate (MTP) approaches.

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

    Science.gov (United States)

    Lodha, T D; Basak, J

    2012-01-01

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

  3. Statistical and population genetics issues of two Hungarian datasets from the aspect of DNA evidence interpretation.

    Science.gov (United States)

    Szabolcsi, Zoltán; Farkas, Zsuzsa; Borbély, Andrea; Bárány, Gusztáv; Varga, Dániel; Heinrich, Attila; Völgyi, Antónia; Pamjav, Horolma

    2015-11-01

    When the DNA profile from a crime-scene matches that of a suspect, the weight of DNA evidence depends on the unbiased estimation of the match probability of the profiles. For this reason, it is required to establish and expand the databases that reflect the actual allele frequencies in the population applied. 21,473 complete DNA profiles from Databank samples were used to establish the allele frequency database to represent the population of Hungarian suspects. We used fifteen STR loci (PowerPlex ESI16) including five, new ESS loci. The aim was to calculate the statistical, forensic efficiency parameters for the Databank samples and compare the newly detected data to the earlier report. The population substructure caused by relatedness may influence the frequency of profiles estimated. As our Databank profiles were considered non-random samples, possible relationships between the suspects can be assumed. Therefore, population inbreeding effect was estimated using the FIS calculation. The overall inbreeding parameter was found to be 0.0106. Furthermore, we tested the impact of the two allele frequency datasets on 101 randomly chosen STR profiles, including full and partial profiles. The 95% confidence interval estimates for the profile frequencies (pM) resulted in a tighter range when we used the new dataset compared to the previously published ones. We found that the FIS had less effect on frequency values in the 21,473 samples than the application of minimum allele frequency. No genetic substructure was detected by STRUCTURE analysis. Due to the low level of inbreeding effect and the high number of samples, the new dataset provides unbiased and precise estimates of LR for statistical interpretation of forensic casework and allows us to use lower allele frequencies. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Prehistoric introduction of domestic pigs onto the Okinawa Islands: ancient mitochondrial DNA evidence.

    Science.gov (United States)

    Watanobe, Takuma; Ishiguro, Naotaka; Nakano, Masuo; Takamiya, Hiroto; Matsui, Akira; Hongo, Hitomi

    2002-08-01

    Ancient DNAs of Sus scrofa specimens excavated from archaeological sites on the Okinawa islands were examined to clarify the genetic relationships among prehistoric Sus scrofa, modern wild boars and domestic pigs inhabiting the Ryukyu archipelago, the Japanese islands, and the Asian continent. We extracted remain DNA from 161 bone specimens excavated from 12 archaeological sites on the Okinawa islands and successfully amplified mitochondrial DNA control region fragments from 33 of 161 specimens. Pairwise difference between prehistoric and modern S. scrofa nucleotide sequences showed that haplotypes of the East Asian domestic pig lineage were found from archaeological specimens together with Ryukyu wild boars native to the Ryukyu archipelago. Phylogenetic analysis of 14 ancient sequences (11 haplotypes; 574 bp) indicated that S. scrofa specimens from two Yayoi-Heian sites (Kitahara and Ara shellmiddens) and two Recent Times sites (Wakuta Kiln and Kiyuna sites) are grouped with modern East Asian domestic pigs. Sus scrofa specimens from Shimizu shellmidden (Yayoi-Heian Period) were very closely related to modern Sus scrofa riukiuanus but had a unique nucleotide insertion, indicating that the population is genetically distinct from the lineage of modern Ryukyu wild boars. This genetic evidence suggests that domestic pigs from the Asian continent were introduced to the Okinawa islands in the early Yayoi-Heian period (1700-2000 BP), or earlier.

  5. Mitochondrial DNA variation of the common hippopotamus: evidence for a recent population expansion

    DEFF Research Database (Denmark)

    Okello, John Bosco A.; Nyakaana, Silvester; Masembe, C.

    2005-01-01

    Mitochondrial DNA control region sequence variation was obtained and the population history of the common hippopotamus was inferred from 109 individuals from 13 localities covering six populations in sub-Saharan Africa. In all, 100 haplotypes were defined, of which 98 were locality specific....... A relatively low overall nucleotide diversity was observed ( =1.9%), as compared to other large mammals so far studied from the same region. Within populations, nucleotide diversity varied from 1.52% in Zambia to 1.92% in Queen Elizabeth and Masai Mara. Overall, low but significant genetic differentiation...... was observed in the total data set (FST=0.138; P=0.001), and at the population level, patterns of differentiation support previously suggested hippopotamus subspecies designations (FCT=0.103; P=0.015). Evidence that the common hippopotamus recently expanded were revealed by: (i) lack of clear geographical...

  6. X linked neonatal centronuclear/myotubular myopathy: evidence for linkage to Xq28 DNA marker loci.

    Science.gov (United States)

    Thomas, N S; Williams, H; Cole, G; Roberts, K; Clarke, A; Liechti-Gallati, S; Braga, S; Gerber, A; Meier, C; Moser, H

    1990-05-01

    We have studied the inheritance of several polymorphic Xq27/28 DNA marker loci in two three generation families with the X linked neonatal lethal form of centronuclear/myotubular myopathy (XL MTM). We found complete linkage of XLMTM to all four informative Xq28 markers analysed, with GCP/RCP (Z = 3.876, theta = 0.00), with DXS15 (Z = 3.737, theta = 0.00), with DXS52 (Z = 2.709, theta = 0.00), and with F8C (Z = 1.020, theta = 0.00). In the absence of any observable recombination, we are unable to sublocalise the XLMTM locus further within the Xq28 region. This evidence for an Xq28 localisation may allow us to carry out useful genetic counselling within such families.

  7. Linking probe thermodynamics to microarray quantification

    International Nuclear Information System (INIS)

    Li, Shuzhao; Pozhitkov, Alexander; Brouwer, Marius

    2010-01-01

    Understanding the difference in probe properties holds the key to absolute quantification of DNA microarrays. So far, Langmuir-like models have failed to link sequence-specific properties to hybridization signals in the presence of a complex hybridization background. Data from washing experiments indicate that the post-hybridization washing has no major effect on the specifically bound targets, which give the final signals. Thus, the amount of specific targets bound to probes is likely determined before washing, by the competition against nonspecific binding. Our competitive hybridization model is a viable alternative to Langmuir-like models. (comment)

  8. Microarray analysis in the archaeon Halobacterium salinarum strain R1.

    Directory of Open Access Journals (Sweden)

    Jens Twellmeyer

    Full Text Available BACKGROUND: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. METHODOLOGY/PRINCIPAL FINDINGS: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. CONCLUSION/SIGNIFICANCE: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

  9. Lack of evidence from HPLC 32P-post-labelling for tamoxifen-DNA adducts in the human endometrium.

    Science.gov (United States)

    Carmichael, P L; Sardar, S; Crooks, N; Neven, P; Van Hoof, I; Ugwumadu, A; Bourne, T; Tomas, E; Hellberg, P; Hewer, A J; Phillips, D H

    1999-02-01

    Tamoxifen is associated with an increased incidence of endometrial cancer in women. It is also a potent carcinogen in rat liver and forms covalent DNA adducts in this tissue. A previous study exploring DNA adducts in human endometria, utilizing thin layer chromatography 32P-postlabelling, found no evidence for adducts in tamoxifen-treated women [Carmichael,P.L., Ugwumadu,A.H.N., Neven,P., Hewer,A.J., Poon,G.K. and Phillips,D.H. (1996) Cancer Res., 56, 1475-1479]. However, subsequent work utilizing HPLC 32P-post-labelling [Hemminki,K., Ranjaniemi,H., Lindahl,B. and Moberger,B. (1996) Cancer Res., 56, 4374-4377] suggested that very low levels could be detected. We have sought to investigate this question further by reproducing the HPLC methodology at two centres, and analysing endometrial DNA from 20 patients treated with 20 mg/day tamoxifen for between 22 and 65 months. Liver DNA isolated from tamoxifen-treated rats was used as a positive control. We found no convincing evidence for tamoxifen-derived DNA adducts in human endometrium. HPLC elution profiles of post-labelled DNA from tamoxifen-treated women were indistinguishable from those obtained with DNA from 14 untreated women and from six women taking toremifene, an analogue of tamoxifen.

  10. Comparison of Comparative Genomic Hybridization Technologies across Microarray Platforms

    Science.gov (United States)

    In the 2007 Association of Biomolecular Resource Facilities (ABRF) Microarray Research Group (MARG) project, we analyzed HL-60 DNA with five platforms: Agilent, Affymetrix 500K, Affymetrix U133 Plus 2.0, Illumina, and RPCI 19K BAC arrays. Copy number variation (CNV) was analyzed ...

  11. Microarray-Based Identification of Transcription Factor Target Genes

    NARCIS (Netherlands)

    Gorte, M.; Horstman, A.; Page, R.B.; Heidstra, R.; Stromberg, A.; Boutilier, K.A.

    2011-01-01

    Microarray analysis is widely used to identify transcriptional changes associated with genetic perturbation or signaling events. Here we describe its application in the identification of plant transcription factor target genes with emphasis on the design of suitable DNA constructs for controlling TF

  12. Biochemical evidence for deficient DNA repair leading to enhanced G2 chromatid radiosensitivity and susceptibility to cancer

    International Nuclear Information System (INIS)

    Gantt, R.; Parshad, R.; Price, F.M.; Sanford, K.K.

    1986-01-01

    Human tumor cells and cells from cancer-prone individuals, compared with those from normal individuals, show a significantly higher incidence of chromatid breaks and gaps seen in metaphase cells immediately after G2 X irradiation. Previous studies with DNA repair-deficient mutants and DNA repair inhibitors strongly indicate that the enhancement results from a G2 deficiency(ies) in DNA repair. We report here biochemical evidence for a DNA repair deficiency that correlates with the cytogenetic studies. In the alkaline elution technique, after a pulse label with radioactive thymidine in the presence of 3-acetylaminobenzamide (a G2-phase blocker) and X irradiation, DNA from tumor or cancer-prone cells elutes more rapidly during the postirradiation period than that from normal cells. These results indicate that the DNA of tumor and cancer-prone cells either repairs more slowly or acquires more breaks than that of normal cells; breaks can accumulate during incomplete or deficient repair processes. The kinetic difference between normal and tumor or cancer-prone cells in DNA strand-break repair reaches a maximum within 2 h, and this maximum corresponds to the kinetic difference in chromatid aberration incidence following X irradiation reported previously. These findings support the concept that cells showing enhanced G2 chromatid radiosensitivity are deficient in DNA repair. The findings could also lead to a biochemical assay for cancer susceptibility

  13. Evidence of pervasive biologically functional secondary structures within the genomes of eukaryotic single-stranded DNA viruses.

    Science.gov (United States)

    Muhire, Brejnev Muhizi; Golden, Michael; Murrell, Ben; Lefeuvre, Pierre; Lett, Jean-Michel; Gray, Alistair; Poon, Art Y F; Ngandu, Nobubelo Kwanele; Semegni, Yves; Tanov, Emil Pavlov; Monjane, Adérito Luis; Harkins, Gordon William; Varsani, Arvind; Shepherd, Dionne Natalie; Martin, Darren Patrick

    2014-02-01

    Single-stranded DNA (ssDNA) viruses have genomes that are potentially capable of forming complex secondary structures through Watson-Crick base pairing between their constituent nucleotides. A few of the structural elements formed by such base pairings are, in fact, known to have important functions during the replication of many ssDNA viruses. Unknown, however, are (i) whether numerous additional ssDNA virus genomic structural elements predicted to exist by computational DNA folding methods actually exist and (ii) whether those structures that do exist have any biological relevance. We therefore computationally inferred lists of the most evolutionarily conserved structures within a diverse selection of animal- and plant-infecting ssDNA viruses drawn from the families Circoviridae, Anelloviridae, Parvoviridae, Nanoviridae, and Geminiviridae and analyzed these for evidence of natural selection favoring the maintenance of these structures. While we find evidence that is consistent with purifying selection being stronger at nucleotide sites that are predicted to be base paired than at sites predicted to be unpaired, we also find strong associations between sites that are predicted to pair with one another and site pairs that are apparently coevolving in a complementary fashion. Collectively, these results indicate that natural selection actively preserves much of the pervasive secondary structure that is evident within eukaryote-infecting ssDNA virus genomes and, therefore, that much of this structure is biologically functional. Lastly, we provide examples of various highly conserved but completely uncharacterized structural elements that likely have important functions within some of the ssDNA virus genomes analyzed here.

  14. Evaluation of forensic DNA mixture evidence: protocol for evaluation, interpretation, and statistical calculations using the combined probability of inclusion.

    Science.gov (United States)

    Bieber, Frederick R; Buckleton, John S; Budowle, Bruce; Butler, John M; Coble, Michael D

    2016-08-31

    The evaluation and interpretation of forensic DNA mixture evidence faces greater interpretational challenges due to increasingly complex mixture evidence. Such challenges include: casework involving low quantity or degraded evidence leading to allele and locus dropout; allele sharing of contributors leading to allele stacking; and differentiation of PCR stutter artifacts from true alleles. There is variation in statistical approaches used to evaluate the strength of the evidence when inclusion of a specific known individual(s) is determined, and the approaches used must be supportable. There are concerns that methods utilized for interpretation of complex forensic DNA mixtures may not be implemented properly in some casework. Similar questions are being raised in a number of U.S. jurisdictions, leading to some confusion about mixture interpretation for current and previous casework. Key elements necessary for the interpretation and statistical evaluation of forensic DNA mixtures are described. Given the most common method for statistical evaluation of DNA mixtures in many parts of the world, including the USA, is the Combined Probability of Inclusion/Exclusion (CPI/CPE). Exposition and elucidation of this method and a protocol for use is the focus of this article. Formulae and other supporting materials are provided. Guidance and details of a DNA mixture interpretation protocol is provided for application of the CPI/CPE method in the analysis of more complex forensic DNA mixtures. This description, in turn, should help reduce the variability of interpretation with application of this methodology and thereby improve the quality of DNA mixture interpretation throughout the forensic community.

  15. AN IMPROVED FUZZY CLUSTERING ALGORITHM FOR MICROARRAY IMAGE SPOTS SEGMENTATION

    Directory of Open Access Journals (Sweden)

    V.G. Biju

    2015-11-01

    Full Text Available An automatic cDNA microarray image processing using an improved fuzzy clustering algorithm is presented in this paper. The spot segmentation algorithm proposed uses the gridding technique developed by the authors earlier, for finding the co-ordinates of each spot in an image. Automatic cropping of spots from microarray image is done using these co-ordinates. The present paper proposes an improved fuzzy clustering algorithm Possibility fuzzy local information c means (PFLICM to segment the spot foreground (FG from background (BG. The PFLICM improves fuzzy local information c means (FLICM algorithm by incorporating typicality of a pixel along with gray level information and local spatial information. The performance of the algorithm is validated using a set of simulated cDNA microarray images added with different levels of AWGN noise. The strength of the algorithm is tested by computing the parameters such as the Segmentation matching factor (SMF, Probability of error (pe, Discrepancy distance (D and Normal mean square error (NMSE. SMF value obtained for PFLICM algorithm shows an improvement of 0.9 % and 0.7 % for high noise and low noise microarray images respectively compared to FLICM algorithm. The PFLICM algorithm is also applied on real microarray images and gene expression values are computed.

  16. TrueAllele casework on Virginia DNA mixture evidence: computer and manual interpretation in 72 reported criminal cases.

    Directory of Open Access Journals (Sweden)

    Mark W Perlin

    Full Text Available Mixtures are a commonly encountered form of biological evidence that contain DNA from two or more contributors. Laboratory analysis of mixtures produces data signals that usually cannot be separated into distinct contributor genotypes. Computer modeling can resolve the genotypes up to probability, reflecting the uncertainty inherent in the data. Human analysts address the problem by simplifying the quantitative data in a threshold process that discards considerable identification information. Elevated stochastic threshold levels potentially discard more information. This study examines three different mixture interpretation methods. In 72 criminal cases, 111 genotype comparisons were made between 92 mixture items and relevant reference samples. TrueAllele computer modeling was done on all the evidence samples, and documented in DNA match reports that were provided as evidence for each case. Threshold-based Combined Probability of Inclusion (CPI and stochastically modified CPI (mCPI analyses were performed as well. TrueAllele's identification information in 101 positive matches was used to assess the reliability of its modeling approach. Comparison was made with 81 CPI and 53 mCPI DNA match statistics that were manually derived from the same data. There were statistically significant differences between the DNA interpretation methods. TrueAllele gave an average match statistic of 113 billion, CPI averaged 6.68 million, and mCPI averaged 140. The computer was highly specific, with a false positive rate under 0.005%. The modeling approach was precise, having a factor of two within-group standard deviation. TrueAllele accuracy was indicated by having uniformly distributed match statistics over the data set. The computer could make genotype comparisons that were impossible or impractical using manual methods. TrueAllele computer interpretation of DNA mixture evidence is sensitive, specific, precise, accurate and more informative than manual

  17. Emerging use of gene expression microarrays in plant physiology.

    Science.gov (United States)

    Wullschleger, Stan D; Difazio, Stephen P

    2003-01-01

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

  18. Emerging Use of Gene Expression Microarrays in Plant Physiology

    Directory of Open Access Journals (Sweden)

    Stephen P. Difazio

    2006-04-01

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

  19. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

    OpenAIRE

    Rao, Archana N.; Grainger, David W.

    2014-01-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surface...

  20. Silent witness, articulate collective: DNA evidence and the inference of visible traits

    NARCIS (Netherlands)

    M'charek, A.

    2008-01-01

    DNA profiling is a well-established technology for use in the criminal justice system, both in courtrooms and elsewhere. The fact that DNA profiles are based on non-coding DNA and do not reveal details about the physical appearance of an individual has contributed to the acceptability of this type

  1. Evidence for DNA repair after ultraviolet irradiation of Petunia hybrida pollen

    International Nuclear Information System (INIS)

    Jackson, J.F.; Linskens, H.F.; Katholieke Universiteit Nijmegen; Katholike Universiteit Nijmegen

    1978-01-01

    Ultraviolet irradiation of Petunia hybrida pollen led to an unscheduled labelling of pollen DNA by 3 H-thymidine during the early stages of germination. Hydroxyurea increased this DNA labelling, while added boron, required absolutely for pollen germination, tube elongation and tube generative cell mitosis, was not needed for this repair-like DNA synthesis. (orig.) [de

  2. Forgotten evidence: A mixed methods study of why sexual assault kits (SAKs) are not submitted for DNA forensic testing.

    Science.gov (United States)

    Campbell, Rebecca; Fehler-Cabral, Giannina; Bybee, Deborah; Shaw, Jessica

    2017-10-01

    Throughout the United States, hundreds of thousands of sexual assault kits (SAKs) (also termed "rape kits") have not been submitted by the police for forensic DNA testing. DNA evidence can help sexual assault investigations and prosecutions by identifying offenders, revealing serial offenders through DNA matches across cases, and exonerating those who have been wrongly accused. In this article, we describe a 5-year action research project conducted with 1 city that had large numbers of untested SAKs-Detroit, Michigan-and our examination into why thousands of rape kits in this city were never submitted for forensic DNA testing. This mixed methods study combined ethnographic observations and qualitative interviews to identify stakeholders' perspectives as to why rape kits were not routinely submitted for testing. Then, we quantitatively examined whether these factors may have affected police practices regarding SAK testing, as evidenced by predictable changes in SAK submission rates over time. Chronic resource scarcity only partially explained why the organizations that serve rape victims-the police, crime lab, prosecution, and victim advocacy-could not test all rape kits, investigate all reported sexual assaults, and support all rape survivors. SAK submission rates significantly increased once criminal justice professionals in this city had full access to the FBI DNA forensic database Combined DNA Index System (CODIS), but even then, most SAKs were still not submitted for DNA testing. Building crime laboratories' capacities for DNA testing and training police on the utility of forensic evidence and best practices in sexual assault investigations can help remedy, and possibly prevent, the problem of untested rape kits. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  3. A Java-based tool for the design of classification microarrays.

    Science.gov (United States)

    Meng, Da; Broschat, Shira L; Call, Douglas R

    2008-08-04

    Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays-and mixed-plasmid microarrays in particular-it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm), several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text), and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff). Weights generated using stepwise discriminant analysis can be stored for

  4. Transfer and persistence of non-self DNA on hands over time: Using empirical data to evaluate DNA evidence given activity level propositions.

    Science.gov (United States)

    Szkuta, Bianca; Ballantyne, Kaye N; Kokshoorn, Bas; van Oorschot, Roland A H

    2018-03-01

    complexity of activity level assessments concerning DNA evidence, and the power of Bayesian networks to visualise and explore the issues of interest for a given case. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Morphological mutants of Neurospora crassa: possible evidence of abnormal morphology due to changes in DNA composition

    Energy Technology Data Exchange (ETDEWEB)

    Chaudhuri, R K; Dutta, S.K. Ojha, M.

    1973-01-01

    DNA from seven experimentally induced morphological mutants and the wild type strain 74A of Neurospora crassa showed typical bimodal denaturation profiles in a Gilford 2400 spectrophotometer. The ''slime'' and ''ropy'' mutants showed a comparatively high proportion of A + T rich DNA sequences. Studies on thermal denaturation, percent hybridization, and thermal stability indicate the DNA sequences of the slime mutant were distinctly different from the normal genomes of parental DNA as well as other wild type DNAs. No such difference was noticed in any other mutant and natural isolate of the species N. crassa tested. These studies indicate possible correlation between a change in DNA nucleotide sequences and abnormal morphogenesis.

  6. Direct evidence for sequence-dependent attraction between double-stranded DNA controlled by methylation.

    Science.gov (United States)

    Yoo, Jejoong; Kim, Hajin; Aksimentiev, Aleksei; Ha, Taekjip

    2016-03-22

    Although proteins mediate highly ordered DNA organization in vivo, theoretical studies suggest that homologous DNA duplexes can preferentially associate with one another even in the absence of proteins. Here we combine molecular dynamics simulations with single-molecule fluorescence resonance energy transfer experiments to examine the interactions between duplex DNA in the presence of spermine, a biological polycation. We find that AT-rich DNA duplexes associate more strongly than GC-rich duplexes, regardless of the sequence homology. Methyl groups of thymine acts as a steric block, relocating spermine from major grooves to interhelical regions, thereby increasing DNA-DNA attraction. Indeed, methylation of cytosines makes attraction between GC-rich DNA as strong as that between AT-rich DNA. Recent genome-wide chromosome organization studies showed that remote contact frequencies are higher for AT-rich and methylated DNA, suggesting that direct DNA-DNA interactions that we report here may play a role in the chromosome organization and gene regulation.

  7. Evidence of disrupted high-risk human papillomavirus DNA in morphologically normal cervices of older women.

    Science.gov (United States)

    Leonard, Sarah M; Pereira, Merlin; Roberts, Sally; Cuschieri, Kate; Nuovo, Gerard; Athavale, Ramanand; Young, Lawrence; Ganesan, Raji; Woodman, Ciarán B

    2016-02-15

    High-risk human papillomavirus (HR-HPV) causes nearly 100% of cervical carcinoma. However, it remains unclear whether HPV can establish a latent infection, one which may be responsible for the second peak in incidence of cervical carcinoma seen in older women. Therefore, using Ventana in situ hybridisation (ISH), quantitative PCR assays and biomarkers of productive and transforming viral infection, we set out to provide the first robust estimate of the prevalence and characteristics of HPV genomes in FFPE tissue from the cervices of 99 women undergoing hysterectomy for reasons unrelated to epithelial abnormality. Our ISH assay detected HR-HPV in 42% of our study population. The majority of ISH positive samples also tested HPV16 positive using sensitive PCR based assays and were more likely to have a history of preceding cytological abnormality. Analysis of subsets of this population revealed HR-HPV to be transcriptionally inactive as there was no evidence of a productive or transforming infection. Critically, the E2 gene was always disrupted in those HPV16 positive cases which were assessed. These findings point to a reservoir of transcriptionally silent, disrupted HPV16 DNA in morphologically normal cervices, re-expression of which could explain the increase in incidence of cervical cancer observed in later life.

  8. Evidence for nuclear internalization of exogenous DNA into mammalian sperm cells

    International Nuclear Information System (INIS)

    Francolini, M.; Lavitrano, M.; Lamia, C.L.; French, D.; Frati, L.; Cotelli, F.; Spadafora, C.

    1993-01-01

    Mature sperm cells have the spontaneous capacity to take up exogenous DNA. Such DNA specifically interacts with the subacrosomal segment of the sperm head corresponding to the nuclear area. Part of the sperm-bound foreign DNA is further internalized into nuclei. Using end-labelled plasmid DNA we have found that 15-22% of the total sperm bound DNA is associated with nuclei as determined on isolated nuclei. On the basis of autoradiographic analysis, nuclear permeability to exogenous DNA seems to be a wide phenomenon involving the majority of the sperm nuclei. In fact, the foreign DNA, incubated with sperm cells for different lengths of time, is found in 45% (10 min) to 65% (2 hr) of the sperm nuclei. Ultrastructural autoradiography on thin sections of mammalian spermatozoa, preincubated with end-labelled plasmid DNA, shows that the exogenous DNA is internalized into the nucleus. This conclusion is further supported by ultrastructural autoradiographic analysis on thin sections of nuclei isolated from spermatozoa preincubated with end-labelled DNA

  9. Evidence for roles of the Escherichia coli Hda protein beyond regulatory inactivation of DnaA.

    Science.gov (United States)

    Baxter, Jamie C; Sutton, Mark D

    2012-08-01

    The ATP-bound form of the Escherichia coli DnaA protein binds 'DnaA boxes' present in the origin of replication (oriC) and operator sites of several genes, including dnaA, to co-ordinate their transcription with initiation of replication. The Hda protein, together with the β sliding clamp, stimulates the ATPase activity of DnaA via a process termed regulatory inactivation of DnaA (RIDA), to regulate the activity of DnaA in DNA replication. Here, we used the mutant dnaN159 strain, which expresses the β159 clamp protein, to gain insight into how the actions of Hda are co-ordinated with replication. Elevated expression of Hda impeded growth of the dnaN159 strain in a Pol II- and Pol IV-dependent manner, suggesting a role for Hda managing the actions of these Pols. In a wild-type strain, elevated levels of Hda conferred sensitivity to nitrofurazone, and suppressed the frequency of -1 frameshift mutations characteristic of Pol IV, while loss of hda conferred cold sensitivity. Using the dnaN159 strain, we identified 24 novel hda alleles, four of which supported E. coli viability despite their RIDA defect. Taken together, these findings suggest that although one or more Hda functions are essential for cell viability, RIDA may be dispensable. © 2012 Blackwell Publishing Ltd.

  10. Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years.

    Directory of Open Access Journals (Sweden)

    Linea Melchior

    Full Text Available BACKGROUND: Given the relative abundance of modern human DNA and the inherent impossibility for incontestable proof of authenticity, results obtained on ancient human DNA have often been questioned. The widely accepted rules regarding ancient DNA work mainly affect laboratory procedures, however, pre-laboratory contamination occurring during excavation and archaeological-/anthropological handling of human remains as well as rapid degradation of authentic DNA after excavation are major obstacles. METHODOLOGY/PRINCIPAL FINDINGS: We avoided some of these obstacles by analyzing DNA from ten Viking Age subjects that at the time of sampling were untouched by humans for 1,000 years. We removed teeth from the subjects prior to handling by archaeologists and anthropologists using protective equipment. An additional tooth was removed after standard archaeological and anthropological handling. All pre-PCR work was carried out in a "clean- laboratory" dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained with the "unhandled" teeth and there was no indication of contamination, while the latter was the case with half of the "handled" teeth. The results allowed the unequivocal assignment of a specific haplotype to each of the subjects, all haplotypes being compatible in their character states with a phylogenetic tree drawn from present day European populations. Several of the haplotypes are either infrequent or have not been observed in modern Scandinavians. The observation of haplogroup I in the present study (<2% in modern Scandinavians supports our previous findings of a pronounced frequency of this haplogroup in Viking and Iron Age Danes. CONCLUSION: The present work provides further evidence that retrieval of ancient human DNA is a possible task provided adequate

  11. Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years.

    Science.gov (United States)

    Melchior, Linea; Kivisild, Toomas; Lynnerup, Niels; Dissing, Jørgen

    2008-05-28

    Given the relative abundance of modern human DNA and the inherent impossibility for incontestable proof of authenticity, results obtained on ancient human DNA have often been questioned. The widely accepted rules regarding ancient DNA work mainly affect laboratory procedures, however, pre-laboratory contamination occurring during excavation and archaeological-/anthropological handling of human remains as well as rapid degradation of authentic DNA after excavation are major obstacles. We avoided some of these obstacles by analyzing DNA from ten Viking Age subjects that at the time of sampling were untouched by humans for 1,000 years. We removed teeth from the subjects prior to handling by archaeologists and anthropologists using protective equipment. An additional tooth was removed after standard archaeological and anthropological handling. All pre-PCR work was carried out in a "clean- laboratory" dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained with the "unhandled" teeth and there was no indication of contamination, while the latter was the case with half of the "handled" teeth. The results allowed the unequivocal assignment of a specific haplotype to each of the subjects, all haplotypes being compatible in their character states with a phylogenetic tree drawn from present day European populations. Several of the haplotypes are either infrequent or have not been observed in modern Scandinavians. The observation of haplogroup I in the present study (Viking and Iron Age Danes. The present work provides further evidence that retrieval of ancient human DNA is a possible task provided adequate precautions are taken and well-considered sampling is applied.

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

    Directory of Open Access Journals (Sweden)

    Eils Roland

    2005-11-01

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

  13. DNA sequence analyses reveal abundant diversity, endemism and evidence for Asian origin of the porcini mushrooms.

    Directory of Open Access Journals (Sweden)

    Bang Feng

    Full Text Available The wild gourmet mushroom Boletus edulis and its close allies are of significant ecological and economic importance. They are found throughout the Northern Hemisphere, but despite their ubiquity there are still many unresolved issues with regard to the taxonomy, systematics and biogeography of this group of mushrooms. Most phylogenetic studies of Boletus so far have characterized samples from North America and Europe and little information is available on samples from other areas, including the ecologically and geographically diverse regions of China. Here we analyzed DNA sequence variation in three gene markers from samples of these mushrooms from across China and compared our findings with those from other representative regions. Our results revealed fifteen novel phylogenetic species (about one-third of the known species and a newly identified lineage represented by Boletus sp. HKAS71346 from tropical Asia. The phylogenetic analyses support eastern Asia as the center of diversity for the porcini sensu stricto clade. Within this clade, B. edulis is the only known holarctic species. The majority of the other phylogenetic species are geographically restricted in their distributions. Furthermore, molecular dating and geological evidence suggest that this group of mushrooms originated during the Eocene in eastern Asia, followed by dispersal to and subsequent speciation in other parts of Asia, Europe, and the Americas from the middle Miocene through the early Pliocene. In contrast to the ancient dispersal of porcini in the strict sense in the Northern Hemisphere, the occurrence of B. reticulatus and B. edulis sensu lato in the Southern Hemisphere was probably due to recent human-mediated introductions.

  14. DNA Sequence Analyses Reveal Abundant Diversity, Endemism and Evidence for Asian Origin of the Porcini Mushrooms

    Science.gov (United States)

    Feng, Bang; Xu, Jianping; Wu, Gang; Zeng, Nian-Kai; Li, Yan-Chun; Tolgor, Bau; Kost, Gerhard W.; Yang, Zhu L.

    2012-01-01

    The wild gourmet mushroom Boletus edulis and its close allies are of significant ecological and economic importance. They are found throughout the Northern Hemisphere, but despite their ubiquity there are still many unresolved issues with regard to the taxonomy, systematics and biogeography of this group of mushrooms. Most phylogenetic studies of Boletus so far have characterized samples from North America and Europe and little information is available on samples from other areas, including the ecologically and geographically diverse regions of China. Here we analyzed DNA sequence variation in three gene markers from samples of these mushrooms from across China and compared our findings with those from other representative regions. Our results revealed fifteen novel phylogenetic species (about one-third of the known species) and a newly identified lineage represented by Boletus sp. HKAS71346 from tropical Asia. The phylogenetic analyses support eastern Asia as the center of diversity for the porcini sensu stricto clade. Within this clade, B. edulis is the only known holarctic species. The majority of the other phylogenetic species are geographically restricted in their distributions. Furthermore, molecular dating and geological evidence suggest that this group of mushrooms originated during the Eocene in eastern Asia, followed by dispersal to and subsequent speciation in other parts of Asia, Europe, and the Americas from the middle Miocene through the early Pliocene. In contrast to the ancient dispersal of porcini in the strict sense in the Northern Hemisphere, the occurrence of B. reticulatus and B. edulis sensu lato in the Southern Hemisphere was probably due to recent human-mediated introductions. PMID:22629418

  15. No evidence of Neandertal mtDNA contribution to early modern humans.

    Directory of Open Access Journals (Sweden)

    David Serre

    2004-03-01

    Full Text Available The retrieval of mitochondrial DNA (mtDNA sequences from four Neandertal fossils from Germany, Russia, and Croatia has demonstrated that these individuals carried closely related mtDNAs that are not found among current humans. However, these results do not definitively resolve the question of a possible Neandertal contribution to the gene pool of modern humans since such a contribution might have been erased by genetic drift or by the continuous influx of modern human DNA into the Neandertal gene pool. A further concern is that if some Neandertals carried mtDNA sequences similar to contemporaneous humans, such sequences may be erroneously regarded as modern contaminations when retrieved from fossils. Here we address these issues by the analysis of 24 Neandertal and 40 early modern human remains. The biomolecular preservation of four Neandertals and of five early modern humans was good enough to suggest the preservation of DNA. All four Neandertals yielded mtDNA sequences similar to those previously determined from Neandertal individuals, whereas none of the five early modern humans contained such mtDNA sequences. In combination with current mtDNA data, this excludes any large genetic contribution by Neandertals to early modern humans, but does not rule out the possibility of a smaller contribution.

  16. Application of plant DNA markers in forensic botany: genetic comparison of Quercus evidence leaves to crime scene trees using microsatellites.

    Science.gov (United States)

    Craft, Kathleen J; Owens, Jeffrey D; Ashley, Mary V

    2007-01-05

    As highly polymorphic DNA markers become increasingly available for a wide range of plant and animal species, there will be increasing opportunities for applications to forensic investigations. To date, however, relatively few studies have reported using DNA profiles of non-human species to place suspects at or near crime scenes. Here we describe an investigation of a double homicide of a female and her near-term fetus. Leaf material taken from a suspect's vehicle was identified to be that of sand live oak, Quercus geminata, the same tree species that occurred near a shallow grave where the victims were found. Quercus-specific DNA microsatellites were used to genotype both dried and fresh material from trees located near the burial site and from the material taken from the suspect's car. Samples from the local population of Q. geminata were also collected and genotyped in order to demonstrate that genetic variation at four microsatellite loci was sufficient to assign leaves to an individual tree with high statistical certainty. The cumulative average probability of identity for these four loci was 2.06x10(-6). DNA was successfully obtained from the dried leaf material although PCR amplification was more difficult than amplification of DNA from fresh leaves. The DNA profiles of the dried leaves from the suspect's car did not match those of the trees near the crime scene. Although this investigation did not provide evidence that could be used against the suspect, it does demonstrate the potential for plant microsatellite markers providing physical evidence that links plant materials to live plants at or near crime scenes.

  17. See what you eat--broad GMO screening with microarrays.

    Science.gov (United States)

    von Götz, Franz

    2010-03-01

    Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative.

  18. Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

    International Nuclear Information System (INIS)

    Bennetts, L.E.; De Iuliis, G.N.; Nixon, B.; Kime, M.; Zelski, K.; McVicar, C.M.; Lewis, S.E.; Aitken, R.J.

    2008-01-01

    A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear (β-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17β-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of male

  19. Impact of estrogenic compounds on DNA integrity in human spermatozoa: Evidence for cross-linking and redox cycling activities

    Energy Technology Data Exchange (ETDEWEB)

    Bennetts, L.E.; De Iuliis, G.N.; Nixon, B.; Kime, M.; Zelski, K. [ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW (Australia); McVicar, C.M.; Lewis, S.E. [Obstetrics and Gynaecology, Queen' s University, Belfast (United Kingdom); Aitken, R.J. [ARC Centre of Excellence in Biotechnology and Development and Discipline of Biological Sciences, University of Newcastle, NSW (Australia)], E-mail: jaitken@mail.newcastle.edu.au

    2008-05-10

    A great deal of circumstantial evidence has linked DNA damage in human spermatozoa with adverse reproductive outcomes including reduced fertility and high rates of miscarriage. Although oxidative stress is thought to make a significant contribution to DNA damage in the male germ line, the factors responsible for creating this stress have not been elucidated. One group of compounds that are thought to be active in this context are the estrogens, either generated as a result of the endogenous metabolism of androgens within the male reproductive tract or gaining access to the latter as a consequence of environmental exposure. In this study, a wide variety of estrogenic compounds were assessed for their direct effects on human spermatozoa in vitro. DNA integrity was assessed using the Comet and TUNEL assays, lesion frequencies were quantified by QPCR using targets within the mitochondrial and nuclear ({beta}-globin) genomes, DNA adducts were characterized by mass spectrometry and redox activity was monitored using dihydroethidium (DHE) as the probe. Of the estrogenic and estrogen analogue compounds evaluated, catechol estrogens, quercetin, diethylstilbestrol and pyrocatechol stimulated intense redox activity while genistein was only active at the highest doses tested. Other estrogens and estrogen analogues, such as 17{beta}-estradiol, nonylphenol, bisphenol A and 2,3-dihydroxynaphthalene were inactive. Estrogen-induced redox activity was associated with a dramatic loss of motility and, in the case of 2-hydroxyestradiol, the induction of significant DNA fragmentation. Mass spectrometry also indicated that catechol estrogens were capable of forming dimers that can cross-link the densely packed DNA strands in sperm chromatin, impairing nuclear decondensation. These results highlight the potential importance of estrogenic compounds in creating oxidative stress and DNA damage in the male germ line and suggest that further exploration of these compounds in the aetiology of

  20. Introgression evidence and phylogenetic relationships among three (ParaMisgurnus species as revealed by mitochondrial and nuclear DNA markers

    Directory of Open Access Journals (Sweden)

    Jakovlić I.

    2013-01-01

    Full Text Available The taxonomy of (ParaMisgurnus genera is still debated. We therefore used mitochondrial and nuclear DNA markers to analyze the phylogenetic relationships among Misgurnus anguillicaudatus, Paramisgurnus dabryanus and Misgurnus fossilis. Differing phylogenetic signals from mitochondrial and nuclear marker data suggest an introgression event in the history of M. anguillicaudatus and M. mohoity. No substantial genetic evidence was found that Paramisgurnus dabryanus should be classified as a separate genus.

  1. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE.

    Science.gov (United States)

    Rao, Archana N; Grainger, David W

    2014-04-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA's persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools.

  2. Polyadenylation state microarray (PASTA) analysis.

    Science.gov (United States)

    Beilharz, Traude H; Preiss, Thomas

    2011-01-01

    Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

  3. Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations

    Science.gov (United States)

    Zuo, Zhicheng; Liu, Jin

    2016-11-01

    The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg2+ ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg2+ bound. The simulation results showed that binding of two Mg2+ ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

  4. Internucleotide correlations and nucleotide periodicity in Drosophila mtDNA: New evidence for panselective evolution

    Directory of Open Access Journals (Sweden)

    Carlos Y Valenzuela

    2010-01-01

    Full Text Available Analysis for the homogeneity of the distribution of the second base of dinucleotides in relation to the first, whose bases are separated by 0, 1, 2,... 21 nucleotide sites, was performed with the VIH-1 genome (cDNA, the Drosophila mtDNA, the Drosophila Torso gene and the human p-globin gene. These four DNA segments showed highly significant heterogeneities of base distributions that cannot be accounted for by neutral or nearly neutral evolution or by the "neighbor influence" of nucleotides on mutation rates. High correlations are found in the bases of dinucleotides separated by 0, 1 and more number of sites. A periodicity of three consecutive significance values (measured by the x²9 was found only in Drosophila mtDNA. This periodicity may be due to an unknown structure or organization of mtDNA. This non-random distribution of the two bases of dinucleotides widespread throughout these DNA segments is rather compatible with panselective evolution and generalized internucleotide co-adaptation.

  5. Effect of DNA polymerase inhibitors on DNA repair in intact and permeable human fibroblasts: Evidence that DNA polymerases δ and β are involved in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine

    International Nuclear Information System (INIS)

    Hammond, R.A.; Miller, M.R.; McClung, J.K.

    1990-01-01

    The involvement of DNA polymerases α, β, and δ in DNA repair synthesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was investigated in human fibroblasts (HF). The effects of anti-(DNA polymerase α) monoclonal antibody, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), dideoxythymidine triphosphate (ddTTP), and aphidicolin on MNNG-induced DNA repair synthesis were investigated to dissect the roles of the different DNA polymerases. A subcellular system (permeable cells), in which DNA repair synthesis and DNA replication were differentiated by CsCl gradient centrifugation of BrdUMP density-labeled DNA, was used to examine the effects of the polymerase inhibitors. Another approach investigated the effects of several of these inhibitors of MNNG-induced DNA repair synthesis in intact cells by measuring the amount of [ 3 H]thymidine incorporated into repair DNA as determined by autoradiography and quantitation with an automated video image analysis system. In permeable cells, MNNG-induced DNA repair synthesis was inhibited 56% by 50 μg of aphidicolin/mL, 6% by 10 μM BuPdGTP, 13% by anti-(DNA polymerse α) monoclonal antibodies, and 29% by ddTTP. In intact cells, MNNG-induced DNA repair synthesis was inhibited 57% by 50 μg of aphidicolin/mL and was not significantly inhibited by microinjecting anti-(DNA polymerase α) antibodies into HF nuclei. These results indicate that both DNA polymerase δ and β are involved in repairing DNA damage caused by MNNG

  6. NF2 tumor suppressor gene: a comprehensive and efficient detection of somatic mutations by denaturing HPLC and microarray-CGH.

    Science.gov (United States)

    Szijan, Irene; Rochefort, Daniel; Bruder, Carl; Surace, Ezequiel; Machiavelli, Gloria; Dalamon, Viviana; Cotignola, Javier; Ferreiro, Veronica; Campero, Alvaro; Basso, Armando; Dumanski, Jan P; Rouleau, Guy A

    2003-01-01

    The NF2 tumor suppressor gene, located in chromosome 22q12, is involved in the development of multiple tumors of the nervous system, either associated with neurofibromatosis 2 or sporadic ones, mainly schwannomas and meningiomas. In order to evaluate the role of the NF2 gene in sporadic central nervous system (CNS) tumors, we analyzed NF2 mutations in 26 specimens: 14 meningiomas, 4 schwannomas, 4 metastases, and 4 other histopathological types of neoplasms. Denaturing high performance liquid chromatography (denaturing HPLC) and comparative genomic hybridization on a DNA microarray (microarray- CGH) were used as scanning methods for small mutations and gross rearrangements respectively. Small mutations were identified in six out of seventeen meningiomas and schwannomas, one mutation was novel. Large deletions were detected in six meningiomas. All mutations were predicted to result in truncated protein or in the absence of a large protein domain. No NF2 mutations were found in other histopathological types of CNS tumors. These results provide additional evidence that mutations in the NF2 gene play an important role in the development of sporadic meningiomas and schwannomas. Denaturing HPLC analysis of small mutations and microarray-CGH of large deletions are complementary, fast, and efficient methods for the detection of mutations in tumor tissues.

  7. Evidence for the role of Mycobacterium tuberculosis RecG helicase in DNA repair and recombination.

    Science.gov (United States)

    Thakur, Roshan S; Basavaraju, Shivakumar; Somyajit, Kumar; Jain, Akshatha; Subramanya, Shreelakshmi; Muniyappa, Kalappa; Nagaraju, Ganesh

    2013-04-01

    In order to survive and replicate in a variety of stressful conditions during its life cycle, Mycobacterium tuberculosis must possess mechanisms to safeguard the integrity of the genome. Although DNA repair and recombination related genes are thought to play key roles in the repair of damaged DNA in all organisms, so far only a few of them have been functionally characterized in the tubercle bacillus. In this study, we show that M. tuberculosis RecG (MtRecG) expression was induced in response to different genotoxic agents. Strikingly, expression of MtRecG in Escherichia coli ∆recG mutant strain provided protection against mitomycin C, methyl methane sulfonate and UV induced cell death. Purified MtRecG exhibited higher binding affinity for the Holliday junction (HJ) compared with a number of canonical recombinational DNA repair intermediates. Notably, although MtRecG binds at the core of the mobile and immobile HJs, and with higher binding affinity for the immobile HJ, branch migration was evident only in the case of the mobile HJ. Furthermore, immobile HJs stimulate MtRecG ATPase activity less efficiently than mobile HJs. In addition to HJ substrates, MtRecG exhibited binding affinity for a variety of branched DNA structures including three-way junctions, replication forks, flap structures, forked duplex and a D-loop structure, but demonstrated strong unwinding activity on replication fork and flap DNA structures. Together, these results support that MtRecG plays an important role in processes related to DNA metabolism under normal as well as stress conditions. © 2013 The Authors Journal compilation © 2013 FEBS.

  8. ACCELERATED EVOLUTION OF LAND SNAILS MANDARINA IN THE OCEANIC BONIN ISLANDS: EVIDENCE FROM MITOCHONDRIAL DNA SEQUENCES.

    Science.gov (United States)

    Chiba, Satoshi

    1999-04-01

    An endemic land snail genus Mandarina of the oceanic Bonin (Ogasawara) Islands shows exceptionally rapid evolution not only of morphological and ecological traits, but of DNA sequence. A phylogenetic relationship based on mitochondrial DNA (mtDNA) sequences suggests that morphological differences equivalent to the differences between families were produced between Mandarina and its ancestor during the Pleistocene. The inferred phylogeny shows that species with similar morphologies and life habitats appeared repeatedly and independently in different lineages and islands at different times. Sequential adaptive radiations occurred in different islands of the Bonin Islands and species occupying arboreal, semiarboreal, and terrestrial habitat arose independently in each island. Because of a close relationship between shell morphology and life habitat, independent evolution of the same life habitat in different islands created species possesing the same shell morphology in different islands and lineages. This rapid evolution produced some incongruences between phylogenetic relationship and species taxonomy. Levels of sequence divergence of mtDNA among the species of Mandarina is extremely high. The maximum level of sequence divergence at 16S and 12S ribosomal RNA sequence within Mandarina are 18.7% and 17.7%, respectively, and this suggests that evolution of mtDNA of Mandarina is extremely rapid, more than 20 times faster than the standard rate in other animals. The present examination reveals that evolution of morphological and ecological traits occurs at extremely high rates in the time of adaptive radiation, especially in fragmented environments. © 1999 The Society for the Study of Evolution.

  9. The intrinsic role of nanoconfinement in chemical equilibrium: evidence from DNA hybridization.

    Science.gov (United States)

    Rubinovich, Leonid; Polak, Micha

    2013-05-08

    Recently we predicted that when a reaction involving a small number of molecules occurs in a nanometric-scale domain entirely segregated from the surrounding media, the nanoconfinement can shift the position of equilibrium toward products via reactant-product reduced mixing. In this Letter, we demonstrate how most-recently reported single-molecule fluorescence measurements of partial hybridization of ssDNA confined within nanofabricated chambers provide the first experimental confirmation of this entropic nanoconfinement effect. Thus, focusing separately on each occupancy-specific equilibrium constant, quantitatively reveals extra stabilization of the product upon decreasing the chamber occupancy or size. Namely, the DNA hybridization under nanoconfined conditions is significantly favored over the identical reaction occurring in bulk media with the same reactant concentrations. This effect, now directly verified for DNA, can be relevant to actual biological processes, as well as to diverse reactions occurring within molecular capsules, nanotubes, and other functional nanospaces.

  10. CDNA Microarray Based Comparative Gene Expression Analysis of Primary Breast Tumors Versus In Vitro Transformed Neoplastic Breast Epithelium

    National Research Council Canada - National Science Library

    Szallasi, Zoltan

    2001-01-01

    .... The first group of clones is being sorted by their ability to form tumors. We are currently performing cDNA microarray analysis quantifying the expression level of about 15,000 genes in these cell lines...

  11. BIOPHYSICAL PROPERTIES OF NUCLEIC ACIDS AT SURFACES RELEVANT TO MICROARRAY PERFORMANCE

    Science.gov (United States)

    Rao, Archana N.; Grainger, David W.

    2014-01-01

    Both clinical and analytical metrics produced by microarray-based assay technology have recognized problems in reproducibility, reliability and analytical sensitivity. These issues are often attributed to poor understanding and control of nucleic acid behaviors and properties at solid-liquid interfaces. Nucleic acid hybridization, central to DNA and RNA microarray formats, depends on the properties and behaviors of single strand (ss) nucleic acids (e.g., probe oligomeric DNA) bound to surfaces. ssDNA’s persistence length, radius of gyration, electrostatics, conformations on different surfaces and under various assay conditions, its chain flexibility and curvature, charging effects in ionic solutions, and fluorescent labeling all influence its physical chemistry and hybridization under assay conditions. Nucleic acid (e.g., both RNA and DNA) target interactions with immobilized ssDNA strands are highly impacted by these biophysical states. Furthermore, the kinetics, thermodynamics, and enthalpic and entropic contributions to DNA hybridization reflect global probe/target structures and interaction dynamics. Here we review several biophysical issues relevant to oligomeric nucleic acid molecular behaviors at surfaces and their influences on duplex formation that influence microarray assay performance. Correlation of biophysical aspects of single and double-stranded nucleic acids with their complexes in bulk solution is common. Such analysis at surfaces is not commonly reported, despite its importance to microarray assays. We seek to provide further insight into nucleic acid-surface challenges facing microarray diagnostic formats that have hindered their clinical adoption and compromise their research quality and value as genomics tools. PMID:24765522

  12. Normalization and gene p-value estimation: issues in microarray data processing.

    Science.gov (United States)

    Fundel, Katrin; Küffner, Robert; Aigner, Thomas; Zimmer, Ralf

    2008-05-28

    Numerous methods exist for basic processing, e.g. normalization, of microarray gene expression data. These methods have an important effect on the final analysis outcome. Therefore, it is crucial to select methods appropriate for a given dataset in order to assure the validity and reliability of expression data analysis. Furthermore, biological interpretation requires expression values for genes, which are often represented by several spots or probe sets on a microarray. How to best integrate spot/probe set values into gene values has so far been a somewhat neglected problem. We present a case study comparing different between-array normalization methods with respect to the identification of differentially expressed genes. Our results show that it is feasible and necessary to use prior knowledge on gene expression measurements to select an adequate normalization method for the given data. Furthermore, we provide evidence that combining spot/probe set p-values into gene p-values for detecting differentially expressed genes has advantages compared to combining expression values for spots/probe sets into gene expression values. The comparison of different methods suggests to use Stouffer's method for this purpose. The study has been conducted on gene expression experiments investigating human joint cartilage samples of osteoarthritis related groups: a cDNA microarray (83 samples, four groups) and an Affymetrix (26 samples, two groups) data set. The apparently straight forward steps of gene expression data analysis, e.g. between-array normalization and detection of differentially regulated genes, can be accomplished by numerous different methods. We analyzed multiple methods and the possible effects and thereby demonstrate the importance of the single decisions taken during data processing. We give guidelines for evaluating normalization outcomes. An overview of these effects via appropriate measures and plots compared to prior knowledge is essential for the biological

  13. Human Parvovirus B19 Utilizes Cellular DNA Replication Machinery for Viral DNA Replication.

    Science.gov (United States)

    Zou, Wei; Wang, Zekun; Xiong, Min; Chen, Aaron Yun; Xu, Peng; Ganaie, Safder S; Badawi, Yomna; Kleiboeker, Steve; Nishimune, Hiroshi; Ye, Shui Qing; Qiu, Jianming

    2018-03-01

    Human parvovirus B19 (B19V) infection of human erythroid progenitor cells (EPCs) induces a DNA damage response and cell cycle arrest at late S phase, which facilitates viral DNA replication. However, it is not clear exactly which cellular factors are employed by this single-stranded DNA virus. Here, we used microarrays to systematically analyze the dynamic transcriptome of EPCs infected with B19V. We found that DNA metabolism, DNA replication, DNA repair, DNA damage response, cell cycle, and cell cycle arrest pathways were significantly regulated after B19V infection. Confocal microscopy analyses revealed that most cellular DNA replication proteins were recruited to the centers of viral DNA replication, but not the DNA repair DNA polymerases. Our results suggest that DNA replication polymerase δ and polymerase α are responsible for B19V DNA replication by knocking down its expression in EPCs. We further showed that although RPA32 is essential for B19V DNA replication and the phosphorylated forms of RPA32 colocalized with the replicating viral genomes, RPA32 phosphorylation was not necessary for B19V DNA replication. Thus, this report provides evidence that B19V uses the cellular DNA replication machinery for viral DNA replication. IMPORTANCE Human parvovirus B19 (B19V) infection can cause transient aplastic crisis, persistent viremia, and pure red cell aplasia. In fetuses, B19V infection can result in nonimmune hydrops fetalis and fetal death. These clinical manifestations of B19V infection are a direct outcome of the death of human erythroid progenitors that host B19V replication. B19V infection induces a DNA damage response that is important for cell cycle arrest at late S phase. Here, we analyzed dynamic changes in cellular gene expression and found that DNA metabolic processes are tightly regulated during B19V infection. Although genes involved in cellular DNA replication were downregulated overall, the cellular DNA replication machinery was tightly

  14. Current Knowledge on Microarray Technology - An Overview

    African Journals Online (AJOL)

    Erah

    This paper reviews basics and updates of each microarray technology and serves to .... through protein microarrays. Protein microarrays also known as protein chips are nothing but grids that ... conditioned media, patient sera, plasma and urine. Clontech ... based antibody arrays) is similar to membrane-based antibody ...

  15. Botanical DNA evidence in criminal cases: Knotgrass (Polygonum aviculare L.) as a model species

    NARCIS (Netherlands)

    Koopman, W.J.M.; Kuiper, I.; Klein Geltink, D.J.A.; Sabatino, G.J.H.; Smulders, M.J.M.

    2012-01-01

    The possibilities and strategies for using DNA characteristics to link a botanical sample to a specific source plant or location vary with its breeding system. For inbreeding species, which often form small patches of identical genotypes, knotgrass (Polygonum aviculare L.) is a suitable model

  16. Natural hybridization in tropical spikerushes of Eleocharis subgenus Limnochloa (Cyperaceae): Evidence from morphology and DNA markers

    Czech Academy of Sciences Publication Activity Database

    Košnar, J.; Košnar, Ji.; Macek, Petr; Herbstová, Miroslava; Rejmánková, E.; Stech, M.

    2010-01-01

    Roč. 97, č. 7 (2010), s. 1229-1240 ISSN 0002-9122 Institutional research plan: CEZ:AV0Z60050516; CEZ:AV0Z50510513 Keywords : Belize * Cyperaceae * DNA markers * hybridization Subject RIV: EB - Genetics ; Molecular Biology; EB - Genetics ; Molecular Biology (BU-J) Impact factor: 3.052, year: 2010

  17. mtDNA variation in the Yanomami: evidence for additional New World founding lineages.

    Science.gov (United States)

    Easton, R D; Merriwether, D A; Crews, D E; Ferrell, R E

    1996-07-01

    Native Americans have been classified into four founding haplogroups with as many as seven founding lineages based on mtDNA RFLPs and DNA sequence data. mtDNA analysis was completed for 83 Yanomami from eight villages in the Surucucu and Catrimani Plateau regions of Roraima in northwestern Brazil. Samples were typed for 15 polymorphic mtDNA sites (14 RFLP sites and 1 deletion site), and a subset was sequenced for both hypervariable regions of the mitochondrial D-loop. Substantial mitochondrial diversity was detected among the Yanomami, five of seven accepted founding haplotypes and three others were observed. Of the 83 samples, 4 (4.8%) were lineage B1, 1 (1.2%) was lineage B2, 31 (37.4%) were lineage C1, 29 (34.9%) were lineage C2, 2 (2.4%) were lineage D1, 6 (7.2%) were lineage D2, 7 (8.4%) were a haplotype we designated "X6," and 3 (3.6%) were a haplotype we designated "X7." Sequence analysis found 43 haplotypes in 50 samples. B2, X6, and X7 are previously unrecognized mitochondrial founding lineage types of Native Americans. The widespread distribution of these haplotypes in the New World and Asia provides support for declaring these lineages to be New World founding types.

  18. Mitochondrial DNA variability among eight Tikúna villages: evidence for an intratribal genetic heterogeneity pattern.

    Science.gov (United States)

    Mendes-Junior, Celso Teixeira; Simões, Aguinaldo Luiz

    2009-11-01

    To study the genetic structure of the Tikúna tribe, four major Native American mitochondrial DNA (mtDNA) founder haplogroups were analyzed in 187 Amerindians from eight Tikúna villages located in the Brazilian Amazon. The central position of these villages in the continent makes them relevant for attempts to reconstruct population movements in South America. In this geographic region, there is particular concern regarding the genetic structure of the Tikúna tribe, formerly designated "enigmatic" due to its remarkable degree of intratribal homogeneity and the scarcity of private protein variants. In spite of its large population size and geographic distribution, the Tikúna tribe presents marked genetic and linguistic isolation. All individuals presented indigenous mtDNA haplogroups. An intratribal genetic heterogeneity pattern characterized by two highly homogeneous Tikúna groups that differ considerably from each other was observed. Such a finding was unexpected, since the Tikúna tribe is characterized by a social system that favors intratribal exogamy and patrilocality that would lead to a higher female migration rate and homogenization of the mtDNA gene pool. Demographic explosions and religious events, which significantly changed the sizes and compositions of many Tikúna villages, may be reflected in the genetic results presented here.

  19. Kernel Based Nonlinear Dimensionality Reduction and Classification for Genomic Microarray

    Directory of Open Access Journals (Sweden)

    Lan Shu

    2008-07-01

    Full Text Available Genomic microarrays are powerful research tools in bioinformatics and modern medicinal research because they enable massively-parallel assays and simultaneous monitoring of thousands of gene expression of biological samples. However, a simple microarray experiment often leads to very high-dimensional data and a huge amount of information, the vast amount of data challenges researchers into extracting the important features and reducing the high dimensionality. In this paper, a nonlinear dimensionality reduction kernel method based locally linear embedding(LLE is proposed, and fuzzy K-nearest neighbors algorithm which denoises datasets will be introduced as a replacement to the classical LLE’s KNN algorithm. In addition, kernel method based support vector machine (SVM will be used to classify genomic microarray data sets in this paper. We demonstrate the application of the techniques to two published DNA microarray data sets. The experimental results confirm the superiority and high success rates of the presented method.

  20. Microarrays: Molecular allergology and nanotechnology for personalised medicine (II).

    Science.gov (United States)

    Lucas, J M

    2010-01-01

    Progress in nanotechnology and DNA recombination techniques have produced tools for the diagnosis and investigation of allergy at molecular level. The most advanced examples of such progress are the microarray techniques, which have been expanded not only in research in the field of proteomics but also in application to the clinical setting. Microarrays of allergic components offer results relating to hundreds of allergenic components in a single test, and using a small amount of serum which can be obtained from capillary blood. The availability of new molecules will allow the development of panels including new allergenic components and sources, which will require evaluation for clinical use. Their application opens the door to component-based diagnosis, to the holistic perception of sensitisation as represented by molecular allergy, and to patient-centred medical practice by allowing great diagnostic accuracy and the definition of individualised immunotherapy for each patient. The present article reviews the application of allergenic component microarrays to allergology for diagnosis, management in the form of specific immunotherapy, and epidemiological studies. A review is also made of the use of protein and gene microarray techniques in basic research and in allergological diseases. Lastly, an evaluation is made of the challenges we face in introducing such techniques to clinical practice, and of the future perspectives of this new technology. Copyright 2010 SEICAP. Published by Elsevier Espana. All rights reserved.

  1. Broad spectrum microarray for fingerprint-based bacterial species identification

    Directory of Open Access Journals (Sweden)

    Frey Jürg E

    2010-02-01

    Full Text Available Abstract Background Microarrays are powerful tools for DNA-based molecular diagnostics and identification of pathogens. Most target a limited range of organisms and are based on only one or a very few genes for specific identification. Such microarrays are limited to organisms for which specific probes are available, and often have difficulty discriminating closely related taxa. We have developed an alternative broad-spectrum microarray that employs hybridisation fingerprints generated by high-density anonymous markers distributed over the entire genome for identification based on comparison to a reference database. Results A high-density microarray carrying 95,000 unique 13-mer probes was designed. Optimized methods were developed to deliver reproducible hybridisation patterns that enabled confident discrimination of bacteria at the species, subspecies, and strain levels. High correlation coefficients were achieved between replicates. A sub-selection of 12,071 probes, determined by ANOVA and class prediction analysis, enabled the discrimination of all samples in our panel. Mismatch probe hybridisation was observed but was found to have no effect on the discriminatory capacity of our system. Conclusions These results indicate the potential of our genome chip for reliable identification of a wide range of bacterial taxa at the subspecies level without laborious prior sequencing and probe design. With its high resolution capacity, our proof-of-principle chip demonstrates great potential as a tool for molecular diagnostics of broad taxonomic groups.

  2. Exploiting fluorescence for multiplex immunoassays on protein microarrays

    International Nuclear Information System (INIS)

    Herbáth, Melinda; Balogh, Andrea; Matkó, János; Papp, Krisztián; Prechl, József

    2014-01-01

    Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications. (topical review)

  3. Fuzzy support vector machine for microarray imbalanced data classification

    Science.gov (United States)

    Ladayya, Faroh; Purnami, Santi Wulan; Irhamah

    2017-11-01

    DNA microarrays are data containing gene expression with small sample sizes and high number of features. Furthermore, imbalanced classes is a common problem in microarray data. This occurs when a dataset is dominated by a class which have significantly more instances than the other minority classes. Therefore, it is needed a classification method that solve the problem of high dimensional and imbalanced data. Support Vector Machine (SVM) is one of the classification methods that is capable of handling large or small samples, nonlinear, high dimensional, over learning and local minimum issues. SVM has been widely applied to DNA microarray data classification and it has been shown that SVM provides the best performance among other machine learning methods. However, imbalanced data will be a problem because SVM treats all samples in the same importance thus the results is bias for minority class. To overcome the imbalanced data, Fuzzy SVM (FSVM) is proposed. This method apply a fuzzy membership to each input point and reformulate the SVM such that different input points provide different contributions to the classifier. The minority classes have large fuzzy membership so FSVM can pay more attention to the samples with larger fuzzy membership. Given DNA microarray data is a high dimensional data with a very large number of features, it is necessary to do feature selection first using Fast Correlation based Filter (FCBF). In this study will be analyzed by SVM, FSVM and both methods by applying FCBF and get the classification performance of them. Based on the overall results, FSVM on selected features has the best classification performance compared to SVM.

  4. Sea cucumber species identification of family Caudinidae from Surabaya based on morphological and mitochondrial DNA evidence

    Science.gov (United States)

    Amin, Muhammad Hilman Fu'adil; Pidada, Ida Bagus Rai; Sugiharto, Widyatmoko, Johan Nuari; Irawan, Bambang

    2016-03-01

    Species identification and taxonomy of sea cucumber remains a challenge problem in some taxa. Caudinidae family of sea cucumber was comerciallized in Surabaya, and it was used as sea cucumber chips. Members of Caudinid sea cucumber have similiar morphology, so it is hard to identify this sea cucumber only from morphological appearance. DNA barcoding is useful method to overcome this problem. The aim of this study was to determine Caudinid specimen of sea cucumber in East Java by morphological and molecular approach. Sample was collected from east coast of Surabaya, then preserved in absolute ethanol. After DNA isolation, Cytochrome Oxydase I (COI) gene amplification was performed using Echinoderm universal primer and PCR product was sequenced. Sequencing result was analyzed and identified in NCBI database using BLAST. Results showed that Caudinid specimen in have closely related to Acaudina molpadioides sequence in GenBank with 86% identity. Morphological data, especially based on ossicle, also showed that the specimen is Acaudina molpadioides.

  5. DNA hybridization evidence for the Australasian affinity of the American marsupial Dromiciops australis.

    OpenAIRE

    Kirsch, J A; Dickerman, A W; Reig, O A; Springer, M S

    1991-01-01

    DNA hybridization was used to compare representatives of the major groups of marsupials and a eutherian outgroup. Because of the large genetic distances separating marsupial families, trees were calculated from normalized percentages of hybridization; thermal-melting statistics, however, gave identical topologies for the well-supported clades. The most notable results were the association of the only extant microbiotheriid, Dromiciops australis, an American marsupial, with the Australasian Di...

  6. Evidence for multiple repair pathways of double-strand DNA breaks in Chinese hamster cells

    International Nuclear Information System (INIS)

    Giaccia, A.J.; Weistein, R.; Stamato, T.D.; Roosa, R.

    1984-01-01

    XR-1 is a mutant of the Chinese hamster cell (CHO-K1) which is abnormally sensitive to killing by gamma rays in G/sub 1/ (D37 = 27 rads vs. 318 for parent) and early S phases of the cell cycle but has near normal resistance in late S and early G/sub 2/ (Somatic Cell Genetics, 9:165-173, 1983). Complementation studies between XR-1 and its parent indicate that this sensitivity to gamma rays is a recessive phenotype. Both the XR-1 and its parent cell are able to repair single strand DNA breaks. However, in comparison to its parental cell, the XR-1 cell is markedly deficient in the repair of double strand DNA breaks introduced by gamma irradiation during the sensitive G/sub 1/-early S period, while in the late S-G/sub 2/ resistant period the repair is similar in both cells. This correlation suggests that an unrepaired double strand DNA break is the lethal lesion and that at least two pathways for the repair of these lesions exist in mammalian cells

  7. Genetic evidence from mitochondrial DNA corroborates the origin of Tibetan chickens.

    Directory of Open Access Journals (Sweden)

    Long Zhang

    Full Text Available Chicken is the most common poultry species and is important to human societies. Tibetan chicken (Gallus gallus domesticus is a breed endemic to China that is distributed mainly on the Qinghai-Tibet Plateau. However, its origin has not been well characterized. In the present study, we sequenced partial mitochondrial DNA (mtDNA control region of 239 and 283 samples from Tibetan and Sichuan indigenous chickens, respectively. Incorporating 1091 published sequences, we constructed the matrilineal genealogy of Tibetan chickens to further document their domestication history. We found that the genetic structure of the mtDNA haplotypes of Tibetan chickens are dominated by seven major haplogroups (A-G. In addition, phylogenetic and network analyses showed that Tibetan chickens are not distinguishable from the indigenous chickens in surrounding areas. Furthermore, some clades of Tibetan chickens may have originated from game fowls. In summary, our results collectively indicated that Tibetan chickens may have diverged from indigenous chickens in the adjacent regions and hybridized with various chickens.

  8. Identifying source populations for the reintroduction of the Eurasian beaver, Castor fiber L. 1758, into Britain: evidence from ancient DNA.

    Science.gov (United States)

    Marr, Melissa M; Brace, Selina; Schreve, Danielle C; Barnes, Ian

    2018-02-09

    Establishing true phylogenetic relationships between populations is a critical consideration when sourcing individuals for translocation. This presents huge difficulties with threatened and endangered species that have become extirpated from large areas of their former range. We utilise ancient DNA (aDNA) to reconstruct the phylogenetic relationships of a keystone species which has become extinct in Britain, the Eurasian beaver Castor fiber. We sequenced seventeen 492 bp partial tRNAPro and control region sequences from Late Pleistocene and Holocene age beavers and included these in network, demographic and genealogy analyses. The mode of postglacial population expansion from refugia was investigated by employing tests of neutrality and a pairwise mismatch distribution analysis. We found evidence of a pre-Late Glacial Maximum ancestor for the Western C. fiber clade which experienced a rapid demographic expansion during the terminal Pleistocene to early Holocene period. Ancient British beavers were found to originate from the Western phylogroup but showed no phylogenetic affinity to any one modern relict population over another. Instead, we find that they formed part of a large, continuous, pan-Western European clade that harbored little internal substructure. Our study highlights the utility of aDNA in reconstructing population histories of extirpated species which has real-world implications for conservation planning.

  9. Large-scale chromatin immunoprecipitation with promoter sequence microarray analysis of the interaction of the NSs protein of Rift Valley fever virus with regulatory DNA regions of the host genome.

    Science.gov (United States)

    Benferhat, Rima; Josse, Thibaut; Albaud, Benoit; Gentien, David; Mansuroglu, Zeyni; Marcato, Vasco; Souès, Sylvie; Le Bonniec, Bernard; Bouloy, Michèle; Bonnefoy, Eliette

    2012-10-01

    Rift Valley fever virus (RVFV) is a highly pathogenic Phlebovirus that infects humans and ruminants. Initially confined to Africa, RVFV has spread outside Africa and presently represents a high risk to other geographic regions. It is responsible for high fatality rates in sheep and cattle. In humans, RVFV can induce hepatitis, encephalitis, retinitis, or fatal hemorrhagic fever. The nonstructural NSs protein that is the major virulence factor is found in the nuclei of infected cells where it associates with cellular transcription factors and cofactors. In previous work, we have shown that NSs interacts with the promoter region of the beta interferon gene abnormally maintaining the promoter in a repressed state. In this work, we performed a genome-wide analysis of the interactions between NSs and the host genome using a genome-wide chromatin immunoprecipitation combined with promoter sequence microarray, the ChIP-on-chip technique. Several cellular promoter regions were identified as significantly interacting with NSs, and the establishment of NSs interactions with these regions was often found linked to deregulation of expression of the corresponding genes. Among annotated NSs-interacting genes were present not only genes regulating innate immunity and inflammation but also genes regulating cellular pathways that have not yet been identified as targeted by RVFV. Several of these pathways, such as cell adhesion, axonal guidance, development, and coagulation were closely related to RVFV-induced disorders. In particular, we show in this work that NSs targeted and modified the expression of genes coding for coagulation factors, demonstrating for the first time that this hemorrhagic virus impairs the host coagulation cascade at the transcriptional level.

  10. DNA evidence of bowhead whale exploitation by Greenlandic Paleo-Inuit 4,000 years ago

    DEFF Research Database (Denmark)

    Seersholm, Frederik Valeur; Pedersen, Mikkel Winther; Søe, Martin Jensen

    2016-01-01

    -described midden deposits. Our results confirm that the species found in the fossil record, like harp seal and ringed seal, were a vital part of Inuit subsistence, but also add a new dimension with evidence that caribou, walrus and whale species played a more prominent role for the survival of Paleo-Inuit cultures...... than previously reported. Most notably, we report evidence of bowhead whale exploitation by the Saqqaq culture 4,000 years ago....

  11. Major Population Expansion of East Asians Began before Neolithic Time: Evidence of mtDNA Genomes

    Science.gov (United States)

    Qin, Zhen-Dong; Wang, Yi; Tan, Jing-Ze; Li, Hui; Jin, Li

    2011-01-01

    It is a major question in archaeology and anthropology whether human populations started to grow primarily after the advent of agriculture, i.e., the Neolithic time, especially in East Asia, which was one of the centers of ancient agricultural civilization. To answer this question requires an accurate estimation of the time of lineage expansion as well as that of population expansion in a population sample without ascertainment bias. In this study, we analyzed all available mtDNA genomes of East Asians ascertained by random sampling, a total of 367 complete mtDNA sequences generated by the 1000 Genome Project, including 249 Chinese (CHB, CHD, and CHS) and 118 Japanese (JPT). We found that major mtDNA lineages underwent expansions, all of which, except for two JPT-specific lineages, including D4, D4b2b, D4a, D4j, D5a2a, A, N9a, F1a1'4, F2, B4, B4a, G2a1 and M7b1'2'4, occurred before 10 kya, i.e., before the Neolithic time (symbolized by Dadiwan Culture at 7.9 kya) in East Asia. Consistent to this observation, the further analysis showed that the population expansion in East Asia started at 13 kya and lasted until 4 kya. The results suggest that the population growth in East Asia constituted a need for the introduction of agriculture and might be one of the driving forces that led to the further development of agriculture. PMID:21998705

  12. X linked neonatal centronuclear/myotubular myopathy: evidence for linkage to Xq28 DNA marker loci.

    OpenAIRE

    Thomas, N S; Williams, H; Cole, G; Roberts, K; Clarke, A; Liechti-Gallati, S; Braga, S; Gerber, A; Meier, C; Moser, H

    1990-01-01

    We have studied the inheritance of several polymorphic Xq27/28 DNA marker loci in two three generation families with the X linked neonatal lethal form of centronuclear/myotubular myopathy (XL MTM). We found complete linkage of XLMTM to all four informative Xq28 markers analysed, with GCP/RCP (Z = 3.876, theta = 0.00), with DXS15 (Z = 3.737, theta = 0.00), with DXS52 (Z = 2.709, theta = 0.00), and with F8C (Z = 1.020, theta = 0.00). In the absence of any observable recombination, we are unable...

  13. Evidence of a genetic instability induced by the incorporation of a DNA precursor marked with tritium

    International Nuclear Information System (INIS)

    Saintigny, Y.; Laurent, D.; Lahayel, J.B.; Roche, St.; Meynard, D.; Lopez, B.S.

    2009-01-01

    The authors report a molecular geno-toxicology investigation which allowed molecular events induced par intracellular incorporation of tritium to be studied, and the genetic instability resulting from a chronic exposure even at low dose to be analysed. For this purpose, they developed cell models (hamster tumorous cells and human fibroblasts) in which they know how to incorporate given quantities of marked nucleotides in the DNA. They show that the incorporation of tritium, even with doses which are said to be non toxic, causes a prolonged exposure of the cell to a genotoxic stress, and maybe a genetic instability due to a too great number of recombination events

  14. Seeded Bayesian Networks: Constructing genetic networks from microarray data

    Directory of Open Access Journals (Sweden)

    Quackenbush John

    2008-07-01

    Full Text Available Abstract Background DNA microarrays and other genomics-inspired technologies provide large datasets that often include hidden patterns of correlation between genes reflecting the complex processes that underlie cellular metabolism and physiology. The challenge in analyzing large-scale expression data has been to extract biologically meaningful inferences regarding these processes – often represented as networks – in an environment where the datasets are often imperfect and biological noise can obscure the actual signal. Although many techniques have been developed in an attempt to address these issues, to date their ability to extract meaningful and predictive network relationships has been limited. Here we describe a method that draws on prior information about gene-gene interactions to infer biologically relevant pathways from microarray data. Our approach consists of using preliminary networks derived from the literature and/or protein-protein interaction data as seeds for a Bayesian network analysis of microarray results. Results Through a bootstrap analysis of gene expression data derived from a number of leukemia studies, we demonstrate that seeded Bayesian Networks have the ability to identify high-confidence gene-gene interactions which can then be validated by comparison to other sources of pathway data. Conclusion The use of network seeds greatly improves the ability of Bayesian Network analysis to learn gene interaction networks from gene expression data. We demonstrate that the use of seeds derived from the biomedical literature or high-throughput protein-protein interaction data, or the combination, provides improvement over a standard Bayesian Network analysis, allowing networks involving dynamic processes to be deduced from the static snapshots of biological systems that represent the most common source of microarray data. Software implementing these methods has been included in the widely used TM4 microarray analysis package.

  15. Printing Proteins as Microarrays for High-Throughput Function Determination

    Science.gov (United States)

    MacBeath, Gavin; Schreiber, Stuart L.

    2000-09-01

    Systematic efforts are currently under way to construct defined sets of cloned genes for high-throughput expression and purification of recombinant proteins. To facilitate subsequent studies of protein function, we have developed miniaturized assays that accommodate extremely low sample volumes and enable the rapid, simultaneous processing of thousands of proteins. A high-precision robot designed to manufacture complementary DNA microarrays was used to spot proteins onto chemically derivatized glass slides at extremely high spatial densities. The proteins attached covalently to the slide surface yet retained their ability to interact specifically with other proteins, or with small molecules, in solution. Three applications for protein microarrays were demonstrated: screening for protein-protein interactions, identifying the substrates of protein kinases, and identifying the protein targets of small molecules.

  16. Evidence of ancient DNA reveals the first European lineage in Iron Age Central China.

    Science.gov (United States)

    Xie, C Z; Li, C X; Cui, Y Q; Zhang, Q C; Fu, Y Q; Zhu, H; Zhou, H

    2007-07-07

    Various studies on ancient DNA have attempted to reconstruct population movement in Asia, with much interest focused on determining the arrival of European lineages in ancient East Asia. Here, we discuss our analysis of the mitochondrial DNA of human remains excavated from the Yu Hong tomb in Taiyuan, China, dated 1400 years ago. The burial style of this tomb is characteristic of Central Asia at that time. Our analysis shows that Yu Hong belonged to the haplogroup U5, one of the oldest western Eurasian-specific haplogroups, while his wife can be classified as haplogroup G, the type prevalent in East Asia. Our findings show that this man with European lineage arrived in Taiyuan approximately 1400 years ago, and most probably married a local woman. Haplogroup U5 was the first west Eurasian-specific lineage to be found in the central part of ancient China, and Taiyuan may be the easternmost location of the discovered remains of European lineage in ancient China.

  17. Evidence that transferrin supports cell proliferation by supplying iron for DNA synthesis

    International Nuclear Information System (INIS)

    Laskey, J.; Webb, I.; Schulman, H.M.; Ponka, P.

    1988-01-01

    Transferrin is essential for cell proliferation and it was suggested that it may trigger a proliferative response following its interaction with receptors, serving as a growth factor. However, since the only clearly defined function of transferrin is iron transport, it may merely serve as an iron donor. To further clarify this issue, the authors took advantage of an iron chelate, ferric salicylaldehyde isonicotinoyl hydrazone (Fe-SIH), which they developed and previously demonstrated to efficiently supply iron to cells without using physiological transferrin receptor pathway. As expected, they observed that blocking monoclonal antibodies against transferrin receptors inhibited proliferation of both Raji and murine erythroleukemia cells. This inhibited cell growth was rescued upon the addition of Fe-SIH which was also shown to deliver iron to Raji cells in the presence of blocking anti-transferrin receptor antibodies. Moreover, blocking anti-transferrin receptor antibodies inhibited [ 3 H]thymidine incorporation into DNA and this inhibition could be overcome by added Fe-SIH. In addition, Fe-SIH slightly stimulated, while SIH (an iron chelator) significantly inhibited, DNA synthesis in phytohemagglutinin-stimulated peripheral blood lymphocytes. Taken together, these results indicate that the only function of transferrin supporting cell proliferation is to supply cells with iron

  18. Antimicrobial histones and DNA traps in invertebrate immunity: evidences in Crassostrea gigas.

    Science.gov (United States)

    Poirier, Aurore C; Schmitt, Paulina; Rosa, Rafael D; Vanhove, Audrey S; Kieffer-Jaquinod, Sylvie; Rubio, Tristan P; Charrière, Guillaume M; Destoumieux-Garzón, Delphine

    2014-09-05

    Although antimicrobial histones have been isolated from multiple metazoan species, their role in host defense has long remained unanswered. We found here that the hemocytes of the oyster Crassostrea gigas release antimicrobial H1-like and H5-like histones in response to tissue damage and infection. These antimicrobial histones were shown to be associated with extracellular DNA networks released by hemocytes, the circulating immune cells of invertebrates, in response to immune challenge. The hemocyte-released DNA was found to surround and entangle vibrios. This defense mechanism is reminiscent of the neutrophil extracellular traps (ETs) recently described in vertebrates. Importantly, oyster ETs were evidenced in vivo in hemocyte-infiltrated interstitial tissues surrounding wounds, whereas they were absent from tissues of unchallenged oysters. Consistently, antimicrobial histones were found to accumulate in oyster tissues following injury or infection with vibrios. Finally, oyster ET formation was highly dependent on the production of reactive oxygen species by hemocytes. This shows that ET formation relies on common cellular and molecular mechanisms from vertebrates to invertebrates. Altogether, our data reveal that ET formation is a defense mechanism triggered by infection and tissue damage, which is shared by relatively distant species suggesting either evolutionary conservation or convergent evolution within Bilateria. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Universal ligation-detection-reaction microarray applied for compost microbes

    Directory of Open Access Journals (Sweden)

    Romantschuk Martin

    2008-12-01

    Full Text Available Abstract Background Composting is one of the methods utilised in recycling organic communal waste. The composting process is dependent on aerobic microbial activity and proceeds through a succession of different phases each dominated by certain microorganisms. In this study, a ligation-detection-reaction (LDR based microarray method was adapted for species-level detection of compost microbes characteristic of each stage of the composting process. LDR utilises the specificity of the ligase enzyme to covalently join two adjacently hybridised probes. A zip-oligo is attached to the 3'-end of one probe and fluorescent label to the 5'-end of the other probe. Upon ligation, the probes are combined in the same molecule and can be detected in a specific location on a universal microarray with complementary zip-oligos enabling equivalent hybridisation conditions for all probes. The method was applied to samples from Nordic composting facilities after testing and optimisation with fungal pure cultures and environmental clones. Results Probes targeted for fungi were able to detect 0.1 fmol of target ribosomal PCR product in an artificial reaction mixture containing 100 ng competing fungal ribosomal internal transcribed spacer (ITS area or herring sperm DNA. The detection level was therefore approximately 0.04% of total DNA. Clone libraries were constructed from eight compost samples. The LDR microarray results were in concordance with the clone library sequencing results. In addition a control probe was used to monitor the per-spot hybridisation efficiency on the array. Conclusion This study demonstrates that the LDR microarray method is capable of sensitive and accurate species-level detection from a complex microbial community. The method can detect key species from compost samples, making it a basis for a tool for compost process monitoring in industrial facilities.

  20. Comparison of gene coverage of mouse oligonucleotide microarray platforms

    Directory of Open Access Journals (Sweden)

    Medrano Juan F

    2006-03-01

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

  1. Unraveling the Rat Intestine, Spleen and Liver Genome-Wide Transcriptome after the Oral Administration of Lavender Oil by a Two-Color Dye-Swap DNA Microarray Approach.

    Science.gov (United States)

    Kubo, Hiroko; Shibato, Junko; Saito, Tomomi; Ogawa, Tetsuo; Rakwal, Randeep; Shioda, Seiji

    2015-01-01

    The use of lavender oil (LO)--a commonly, used oil in aromatherapy, with well-defined volatile components linalool and linalyl acetate--in non-traditional medicine is increasing globally. To understand and demonstrate the potential positive effects of LO on the body, we have established an animal model in this current study, investigating the orally administered LO effects genome wide in the rat small intestine, spleen, and liver. The rats were administered LO at 5 mg/kg (usual therapeutic dose in humans) followed by the screening of differentially expressed genes in the tissues, using a 4×44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA) in conjunction with a dye-swap approach, a novelty of this study. Fourteen days after LO treatment and compared with a control group (sham), a total of 156 and 154 up (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes, 174 and 66 up- (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes, and 222 and 322 up- (≧ 1.5-fold)- and down (≦ 0.75-fold)-regulated genes showed differential expression at the mRNA level in the small intestine, spleen and liver, respectively. The reverse transcription-polymerase chain reaction (RT-PCR) validation of highly up- and down-regulated genes confirmed the regulation of the Papd4, Lrp1b, Alb, Cyr61, Cyp2c, and Cxcl1 genes by LO as examples in these tissues. Using bioinformatics, including Ingenuity Pathway Analysis (IPA), differentially expressed genes were functionally categorized by their Gene Ontology (GO) and biological function and network analysis, revealing their diverse functions and potential roles in LO-mediated effects in rat. Further IPA analysis in particular unraveled the presence of novel genes, such as Papd4, Or8k5, Gprc5b, Taar5, Trpc6, Pld2 and Onecut3 (up-regulated top molecules) and Tnf, Slc45a4, Slc25a23 and Samt4 (down-regulated top molecules), to be influenced by LO treatment in the small intestine, spleen and liver

  2. Unraveling the Rat Intestine, Spleen and Liver Genome-Wide Transcriptome after the Oral Administration of Lavender Oil by a Two-Color Dye-Swap DNA Microarray Approach.

    Directory of Open Access Journals (Sweden)

    Hiroko Kubo

    Full Text Available The use of lavender oil (LO--a commonly, used oil in aromatherapy, with well-defined volatile components linalool and linalyl acetate--in non-traditional medicine is increasing globally. To understand and demonstrate the potential positive effects of LO on the body, we have established an animal model in this current study, investigating the orally administered LO effects genome wide in the rat small intestine, spleen, and liver. The rats were administered LO at 5 mg/kg (usual therapeutic dose in humans followed by the screening of differentially expressed genes in the tissues, using a 4×44-K whole-genome rat chip (Agilent microarray platform; Agilent Technologies, Palo Alto, CA, USA in conjunction with a dye-swap approach, a novelty of this study. Fourteen days after LO treatment and compared with a control group (sham, a total of 156 and 154 up (≧ 1.5-fold- and down (≦ 0.75-fold-regulated genes, 174 and 66 up- (≧ 1.5-fold- and down (≦ 0.75-fold-regulated genes, and 222 and 322 up- (≧ 1.5-fold- and down (≦ 0.75-fold-regulated genes showed differential expression at the mRNA level in the small intestine, spleen and liver, respectively. The reverse transcription-polymerase chain reaction (RT-PCR validation of highly up- and down-regulated genes confirmed the regulation of the Papd4, Lrp1b, Alb, Cyr61, Cyp2c, and Cxcl1 genes by LO as examples in these tissues. Using bioinformatics, including Ingenuity Pathway Analysis (IPA, differentially expressed genes were functionally categorized by their Gene Ontology (GO and biological function and network analysis, revealing their diverse functions and potential roles in LO-mediated effects in rat. Further IPA analysis in particular unraveled the presence of novel genes, such as Papd4, Or8k5, Gprc5b, Taar5, Trpc6, Pld2 and Onecut3 (up-regulated top molecules and Tnf, Slc45a4, Slc25a23 and Samt4 (down-regulated top molecules, to be influenced by LO treatment in the small intestine, spleen and

  3. Quantification of histoautoradiographic evidence of DNA repair synthesis in the liver

    International Nuclear Information System (INIS)

    Hochmann, J.; Stambergova, H.

    1988-01-01

    Histoautoradiography was used to detect dimethylnitrosamine-induced 3 H-thymidine incorporation in vivo into G phase hepatocytes. The description of the standard procedure for counting the grains and the mode of mathematical evaluation are presented. The results exhibited higher sensitivity than those in the investigation of the DNA repair synthesis by means of a scintillation counter using the method of detecting hydroxyurea-resistant incorporation of 3 H-thymidine. Thus, it was possible to simplify the investigation by lowering the number of evaluated cells. A suitable compromise between precision and laboriousness will probably be achieved by counting 20 hepatocytes per animal. In case of striking differences between the experimental and the control groups a qualitative conclusion may be drawn even without counting the grains. (author). 5 tabs., 10 refs

  4. DNA hybridization evidence for the Australasian affinity of the American marsupial Dromiciops australis.

    Science.gov (United States)

    Kirsch, J A; Dickerman, A W; Reig, O A; Springer, M S

    1991-01-01

    DNA hybridization was used to compare representatives of the major groups of marsupials and a eutherian outgroup. Because of the large genetic distances separating marsupial families, trees were calculated from normalized percentages of hybridization; thermal-melting statistics, however, gave identical topologies for the well-supported clades. The most notable results were the association of the only extant microbiotheriid, Dromiciops australis, an American marsupial, with the Australasian Diprotodontia, and of both together with the Dasyuridae. Estimates of the rate of divergence among marsupial genomes suggest that the Dromiciops-Diprotodontia split occurred approximately 50 million years ago, well after the establishment of the major clades of marsupials but before deep oceanic barriers prohibited dispersal among Australia, Antarctica, and South America. Because Dromiciops is nested within an Australasian group, it seems likely that dispersal from Australia accounts for its present distribution. Images PMID:1961710

  5. Stable isotope and DNA evidence for ritual sequences in Inca child sacrifice

    DEFF Research Database (Denmark)

    Wilson, Andrew S; Taylor, Timothy; Ceruti, Maria Constanza

    2007-01-01

    Four recently discovered frozen child mummies from two of the highest peaks in the south central Andes now yield tantalizing evidence of the preparatory stages leading to Inca ritual killing as represented by the unique capacocha rite. Our interdisciplinary study examined hair from the mummies to...

  6. The IronChip evaluation package: a package of perl modules for robust analysis of custom microarrays

    Directory of Open Access Journals (Sweden)

    Brazma Alvis

    2010-03-01

    Full Text Available Abstract Background Gene expression studies greatly contribute to our understanding of complex relationships in gene regulatory networks. However, the complexity of array design, production and manipulations are limiting factors, affecting data quality. The use of customized DNA microarrays improves overall data quality in many situations, however, only if for these specifically designed microarrays analysis tools are available. Results The IronChip Evaluation Package (ICEP is a collection of Perl utilities and an easy to use data evaluation pipeline for the analysis of microarray data with a focus on data quality of custom-designed microarrays. The package has been developed for the statistical and bioinformatical analysis of the custom cDNA microarray IronChip but can be easily adapted for other cDNA or oligonucleotide-based designed microarray platforms. ICEP uses decision tree-based algorithms to assign quality flags and performs robust analysis based on chip design properties regarding multiple repetitions, ratio cut-off, background and negative controls. Conclusions ICEP is a stand-alone Windows application to obtain optimal data quality from custom-designed microarrays and is freely available here (see "Additional Files" section and at: http://www.alice-dsl.net/evgeniy.vainshtein/ICEP/

  7. Replication of UV-irradiated single-stranded DNA by DNA polymerase III holoenzyme of Escherichia coli: evidence for bypass of pyrimidine photodimers

    International Nuclear Information System (INIS)

    Livneh, Z.

    1986-01-01

    Replication of UV-irradiated circular single-stranded phage M13 DNA by Escherichia coli RNA polymerase (EC 2.7.7.6) and DNA polymerase III holoenzyme (EC 2.7.7.7) in the presence of single-stranded DNA binding protein yielded full-length as well as partially replicated products. A similar result was obtained with phage G4 DNA primed with E. coli DNA primase, and phage phi X174 DNA primed with a synthetic oligonucleotide. The fraction of full-length DNA was several orders of magnitude higher than predicted if pyrimidine photodimers were to constitute absolute blocks to DNA replication. Recent models have suggested that pyrimidine photodimers are absolute blocks to DNA replication and that SOS-induced proteins are required to allow their bypass. Our results demonstrate that, under in vitro replication conditions, E. coli DNA polymerase III holoenzyme can insert nucleotides opposite pyrimidine dimers to a significant extent, even in the absence of SOS-induced proteins

  8. Evidence for a Higher Number of Species of Odontotermes (Isoptera) than Currently Known from Peninsular Malaysia from Mitochondrial DNA Phylogenies

    Science.gov (United States)

    Cheng, Shawn; Kirton, Laurence G.; Panandam, Jothi M.; Siraj, Siti S.; Ng, Kevin Kit-Siong; Tan, Soon-Guan

    2011-01-01

    Termites of the genus Odontotermes are important decomposers in the Old World tropics and are sometimes important pests of crops, timber and trees. The species within the genus often have overlapping size ranges and are difficult to differentiate based on morphology. As a result, the taxonomy of Odontotermes in Peninsular Malaysia has not been adequately worked out. In this study, we examined the phylogeny of 40 samples of Odontotermes from Peninsular Malaysia using two mitochondrial DNA regions, that is, the 16S ribosomal RNA and cytochrome oxidase subunit I genes, to aid in elucidating the number of species in the peninsula. Phylogenies were reconstructed from the individual gene and combined gene data sets using parsimony and likelihood criteria. The phylogenies supported the presence of up to eleven species in Peninsular Malaysia, which were identified as O. escherichi, O. hainanensis, O. javanicus, O. longignathus, O. malaccensis, O. oblongatus, O. paraoblongatus, O. sarawakensis, and three possibly new species. Additionally, some of our taxa are thought to comprise a complex of two or more species. The number of species found in this study using DNA methods was more than the initial nine species thought to occur in Peninsular Malaysia. The support values for the clades and morphology of the soldiers provided further evidence for the existence of eleven or more species. Higher resolution genetic markers such as microsatellites would be required to confirm the presence of cryptic species in some taxa. PMID:21687629

  9. Tissue Microarray Analysis Applied to Bone Diagenesis.

    Science.gov (United States)

    Mello, Rafael Barrios; Silva, Maria Regina Regis; Alves, Maria Teresa Seixas; Evison, Martin Paul; Guimarães, Marco Aurelio; Francisco, Rafaella Arrabaca; Astolphi, Rafael Dias; Iwamura, Edna Sadayo Miazato

    2017-01-04

    Taphonomic processes affecting bone post mortem are important in forensic, archaeological and palaeontological investigations. In this study, the application of tissue microarray (TMA) analysis to a sample of femoral bone specimens from 20 exhumed individuals of known period of burial and age at death is described. TMA allows multiplexing of subsamples, permitting standardized comparative analysis of adjacent sections in 3-D and of representative cross-sections of a large number of specimens. Standard hematoxylin and eosin, periodic acid-Schiff and silver methenamine, and picrosirius red staining, and CD31 and CD34 immunohistochemistry were applied to TMA sections. Osteocyte and osteocyte lacuna counts, percent bone matrix loss, and fungal spheroid element counts could be measured and collagen fibre bundles observed in all specimens. Decalcification with 7% nitric acid proceeded more rapidly than with 0.5 M EDTA and may offer better preservation of histological and cellular structure. No endothelial cells could be detected using CD31 and CD34 immunohistochemistry. Correlation between osteocytes per lacuna and age at death may reflect reported age-related responses to microdamage. Methodological limitations and caveats, and results of the TMA analysis of post mortem diagenesis in bone are discussed, and implications for DNA survival and recovery considered.

  10. Evident?

    DEFF Research Database (Denmark)

    Plant, Peter

    2012-01-01

    Quality assurance and evidence in career guidance in Europe are often seen as self-evident approaches, but particular interests lie behind......Quality assurance and evidence in career guidance in Europe are often seen as self-evident approaches, but particular interests lie behind...

  11. Stable isotope and DNA evidence for ritual sequences in Inca child sacrifice

    Science.gov (United States)

    Wilson, Andrew S.; Taylor, Timothy; Ceruti, Maria Constanza; Chavez, Jose Antonio; Reinhard, Johan; Grimes, Vaughan; Meier-Augenstein, Wolfram; Cartmell, Larry; Stern, Ben; Richards, Michael P.; Worobey, Michael; Barnes, Ian; Gilbert, M. Thomas P.

    2007-01-01

    Four recently discovered frozen child mummies from two of the highest peaks in the south central Andes now yield tantalizing evidence of the preparatory stages leading to Inca ritual killing as represented by the unique capacocha rite. Our interdisciplinary study examined hair from the mummies to obtain detailed genetic and diachronic isotopic information. This approach has allowed us to reconstruct aspects of individual identity and diet, make inferences concerning social background, and gain insight on the hitherto unknown processes by which victims were selected, elevated in social status, prepared for a high-altitude pilgrimage, and killed. Such direct information amplifies, yet also partly contrasts with, Spanish historical accounts. PMID:17923675

  12. DNA fingerprinting of sugarcane for detecting molecular evidence of somaclonal variation

    International Nuclear Information System (INIS)

    Ramos Leal, M.; Canales, E.; Michel, R.; Coto, O.; Cornide, M.T.

    1998-01-01

    Molecular characterisation of eight sugarcane mutants which tolerate 1% NaCl, was performed by means of restriction fragment length polymorphism (RFLP) analysis at the genomic level, using a group of molecular probes for ribosomal DNAs and two others, Δ-pyrroline-5-carboxylate reductase (ΔP5CR) and ATPase, probably related to salinity stress tolerance. Results showed the molecular evidence of genetic changes in comparison to the susceptible donor variety and the possible presence of more that one mechanism to tolerate salt stress. (author)

  13. Microarray-based genotyping of Salmonella: Inter-laboratory evaluation of reproducibility and standardization potential

    DEFF Research Database (Denmark)

    Grønlund, Hugo Ahlm; Riber, Leise; Vigre, Håkan

    2011-01-01

    Bacterial food-borne infections in humans caused by Salmonella spp. are considered a crucial food safety issue. Therefore, it is important for the risk assessments of Salmonella to consider the genomic variationamong different isolates in order to control pathogen-induced infections. Microarray...... critical methodology parameters that differed between the two labs were identified. These related to printing facilities, choice of hybridization buffer,wash buffers used following the hybridization and choice of procedure for purifying genomic DNA. Critical parameters were randomized in a four......DNA and different wash buffers. However, less agreement (Kappa=0.2–0.6) between microarray results were observed when using different hybridization buffers, indicating this parameter as being highly criticalwhen transferring a standard microarray assay between laboratories. In conclusion, this study indicates...

  14. Evidence that DNA excision-repair in xeroderma pigmentosum group A is limited but biologically significant

    International Nuclear Information System (INIS)

    Hull, D.R.; Kantor, G.J.

    1983-01-01

    The loss of pyrimidine dimers in nondividing populations of an excision-repair deficient xeroderma pigmentosum group. A strain (XP12BE) was measured throughout long periods (up to 5 months) following exposure to low doses of ultraviolet light (UV, 254 nm) using a UV endonuclease-alkaline sedimentation assay. Excision of about 90% of the dimers induced by 1 J/m 2 occurred during the first 50 days. The rate curve has some similarities with that of normal excision-repair proficient cultures that may not be coincidental. Rate curves for both XP12BE and normal cultures are characterized by a fast and slow component, with both rate constants for the XP12BE cultures (0.15 day -1 and 0.025 day -1 ) a factor of 10 smaller than those observed for the respective components of normal cell cultures. The slow components for both XP12BE and normal cultures extrapolate to about 30% of the initial number of dimers. No further excision was detected throughout an additional 90-day period even though the cultures were capable of excision-repair of other newly-introduced pyrimidine dimers. We conclude that nondividing XP12BE cells in addition to having a slower repair rate, cannot repair some of the UV-induced DNA damage. The repair in XP12BE is shown to have biological significance as detected by a cell-survival assay and dose-fractionation techniques. Nondividing XP12BE cells are more resistant to UV when irradiated chronically than when irradiated acutely with the same total dose. (orig.)

  15. Archaeal community changes in Lateglacial lake sediments: Evidence from ancient DNA

    Science.gov (United States)

    Ahmed, Engy; Parducci, Laura; Unneberg, Per; Ågren, Rasmus; Schenk, Frederik; Rattray, Jayne E.; Han, Lu; Muschitiello, Francesco; Pedersen, Mikkel W.; Smittenberg, Rienk H.; Yamoah, Kweku Afrifa; Slotte, Tanja; Wohlfarth, Barbara

    2018-02-01

    The Lateglacial/early Holocene sediments from the ancient lake at Hässeldala Port, southern Sweden provide an important archive for the environmental and climatic shifts at the end of the last ice age and the transition into the present Interglacial. The existing multi-proxy data set highlights the complex interplay of physical and ecological changes in response to climatic shifts and lake status changes. Yet, it remains unclear how microorganisms, such as Archaea, which do not leave microscopic features in the sedimentary record, were affected by these climatic shifts. Here we present the metagenomic data set of Hässeldala Port with a special focus on the abundance and biodiversity of Archaea. This allows reconstructing for the first time the temporal succession of major Archaea groups between 13.9 and 10.8 ka BP by using ancient environmental DNA metagenomics and fossil archaeal cell membrane lipids. We then evaluate to which extent these findings reflect physical changes of the lake system, due to changes in lake-water summer temperature and seasonal lake-ice cover. We show that variations in archaeal composition and diversity were related to a variety of factors (e.g., changes in lake water temperature, duration of lake ice cover, rapid sediment infilling), which influenced bottom water conditions and the sediment-water interface. Methanogenic Archaea dominated during the Allerød and Younger Dryas pollen zones, when the ancient lake was likely stratified and anoxic for large parts of the year. The increase in archaeal diversity at the Younger Dryas/Holocene transition is explained by sediment infilling and formation of a mire/peatbog.

  16. A Java-based tool for the design of classification microarrays

    Directory of Open Access Journals (Sweden)

    Broschat Shira L

    2008-08-01

    Full Text Available Abstract Background Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. Results The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. Conclusion In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays–and mixed-plasmid microarrays in particular–it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm, several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text, and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff. Weights

  17. A label-free, fluorescence based assay for microarray

    Science.gov (United States)

    Niu, Sanjun

    DNA chip technology has drawn tremendous attention since it emerged in the mid 90's as a method that expedites gene sequencing by over 100-fold. DNA chip, also called DNA microarray, is a combinatorial technology in which different single-stranded DNA (ssDNA) molecules of known sequences are immobilized at specific spots. The immobilized ssDNA strands are called probes. In application, the chip is exposed to a solution containing ssDNA of unknown sequence, called targets, which are labeled with fluorescent dyes. Due to specific molecular recognition among the base pairs in the DNA, the binding or hybridization occurs only when the probe and target sequences are complementary. The nucleotide sequence of the target is determined by imaging the fluorescence from the spots. The uncertainty of background in signal detection and statistical error in data analysis, primarily due to the error in the DNA amplification process and statistical distribution of the tags in the target DNA, have become the fundamental barriers in bringing the technology into application for clinical diagnostics. Furthermore, the dye and tagging process are expensive, making the cost of DNA chips inhibitive for clinical testing. These limitations and challenges make it difficult to implement DNA chip methods as a diagnostic tool in a pathology laboratory. The objective of this dissertation research is to provide an alternative approach that will address the above challenges. In this research, a label-free assay is designed and studied. Polystyrene (PS), a commonly used polymeric material, serves as the fluorescence agent. Probe ssDNA is covalently immobilized on polystyrene thin film that is supported by a reflecting substrate. When this chip is exposed to excitation light, fluorescence light intensity from PS is detected as the signal. Since the optical constants and conformations of ssDNA and dsDNA (double stranded DNA) are different, the measured fluorescence from PS changes for the same

  18. Comparative genomics of Streptomyces avermitilis, Streptomyces cattleya, Streptomyces maritimus and Kitasatospora aureofaciens using a Streptomyces coelicolor microarray system

    NARCIS (Netherlands)

    Hsiao, Nai-hua; Kirby, Ralph

    DNA/DNA microarray hybridization was used to compare the genome content of Streptomyces avermitilis, Streptomyces cattleya, Streptomyces maritimus and Kitasatospora aureofaciens with that of Streptomyces coelicolor A3(2). The array data showed an about 93% agreement with the genome sequence data

  19. Assessment of evidence for nanosized titanium dioxide-generated DNA strand breaks and oxidatively damaged DNA in cells and animal models

    DEFF Research Database (Denmark)

    Møller, Peter; Jensen, Ditte Marie; Wils, Regitze Sølling

    2017-01-01

    Nanosized titanium dioxide (TiO2) has been investigated in numerous studies on genotoxicity, including comet assay endpoints and oxidatively damaged DNA in cell cultures and animal models. The results have been surprisingly mixed, which might be attributed to physico-chemical differences...... culture studies also demonstrate increased levels of oxidatively damaged DNA after exposure to TiO2. There are relatively few studies on animal models where DNA strand breaks and oxidatively damaged DNA have been tested with reliable methods. Collectively, this review shows that exposure to nanosized TiO2...... of the tested TiO2. In the present review, we assess the role of certain methodological issues and publication bias. The analysis shows that studies on DNA strand breaks without proper assay controls or very low intra-group variation tend to show statistically significant effects. Levels of oxidatively damaged...

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

    Science.gov (United States)

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

  1. APPLICATION OF CDNA MICROARRAY TO THE STUDY OF ARSENIC TOXICOLOGY AND CARCINOGENESIS

    Science.gov (United States)

    Arsenic (As) is a common environmental toxicant and known human carcinogen. Epidemiological studies link As exposure to various disorders and cancers. However, the molecular mechanisms for As toxicity and carcinogenicity are not completely known. The cDNA microarray, a high-th...

  2. Diagnostic Yield of Chromosomal Microarray Analysis in an Autism Primary Care Practice: Which Guidelines to Implement?

    Science.gov (United States)

    McGrew, Susan G.; Peters, Brittany R.; Crittendon, Julie A.; Veenstra-VanderWeele, Jeremy

    2012-01-01

    Genetic testing is recommended for patients with ASD; however specific recommendations vary by specialty. American Academy of Pediatrics and American Academy of Neurology guidelines recommend G-banded karyotype and Fragile X DNA. The American College of Medical Genetics recommends Chromosomal Microarray Analysis (CMA). We determined the yield of…

  3. Microarray-based identification of clinically relevant vaginal bacteria in relation to bacterial vaginosis

    NARCIS (Netherlands)

    Dols, J.A.M.; Smit, P.W.; Kort, R.; Reid, G.; Schuren, F.H.J.; Tempelman, H.; Bontekoe, T.R.; Korporaal, H.; Boon, M.E.

    2011-01-01

    Objective: The objective was to examine the use of a tailor-made DNA microarray containing probes representing the vaginal microbiota to examine bacterial vaginosis. Study Design: One hundred one women attending a health center for HIV testing in South Africa were enrolled. Stained, liquid-based

  4. Microarray-based identification of clinically relevant vaginal bacteria in relation to bacterial vaginosis

    NARCIS (Netherlands)

    Dols, Joke A M; Smit, Pieter W; Kort, Remco; Reid, Gregor; Schuren, Frank H J; Tempelman, Hugo; Bontekoe, Tj Romke; Korporaal, Hans; Boon, Mathilde E

    OBJECTIVE: The objective was to examine the use of a tailor-made DNA microarray containing probes representing the vaginal microbiota to examine bacterial vaginosis. STUDY DESIGN: One hundred one women attending a health center for HIV testing in South Africa were enrolled. Stained, liquid-based

  5. "On-chip magnetic bead microarray using hydrodynamic focusing in a passive magnetic separator"

    DEFF Research Database (Denmark)

    Smistrup, Kristian; Kjeldsen, B.; Reimers, R.L.

    2005-01-01

    Implementing DNA and protein microarrays into lab-on-a-chip systems can be problematic since these are sensitive to heat and strong chemicals. Here, we describe the functionalization of a microchannel with two types of magnetic beads using hydrodynamic focusing combined with a passive magnetic...

  6. Microarray Analysis of Late Response to Boron Toxicity in Barley (Hordeum vulgare L.) Leaves

    NARCIS (Netherlands)

    Oz, M.T.; Yilmaz, R.; Eyidogan, F.; Graaff, de L.H.; Yucel, M.; Oktem, H.A.

    2009-01-01

    DNA microarrays, being high-density and high-throughput, allow quantitative analyses of thousands of genes and their expression patterns in parallel. In this study, Barley1 GereChip was used to investigate transcriptome changes associated with boron (B) toxicity in a sensitive barley cultivar

  7. GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN CDNA MICROARRAY ANALYSES

    Science.gov (United States)

    GENE EXPRESSION IN THE TESTES OF NORMOSPERMIC VERSUS TERATOSPERMIC DOMESTIC CATS USING HUMAN cDNA MICROARRAY ANALYSESB.S. Pukazhenthi1, J. C. Rockett2, M. Ouyang3, D.J. Dix2, J.G. Howard1, P. Georgopoulos4, W.J. J. Welsh3 and D. E. Wildt11Department of Reproductiv...

  8. Discrimination of phytoplasmas using an oligonucleotide microarray targeting rps3, rpl22, and rps19 genes

    Czech Academy of Sciences Publication Activity Database

    Lenz, Ondřej; Marková, J.; Sarkisova, Tatiana; Fránová, Jana; Přibylová, Jaroslava

    2015-01-01

    Roč. 70, January 2015 (2015), s. 47-52 ISSN 0261-2194 Institutional support: RVO:60077344 Keywords : DNA microarray * rpl22 gene * rps19 gene * rps3 gene Subject RIV: EE - Microbiology, Virology Impact factor: 1.652, year: 2015

  9. Carbohydrate Microarrays in Plant Science

    DEFF Research Database (Denmark)

    Fangel, Jonatan Ulrik; Pedersen, H.L.; Vidal-Melgosa, S.

    2012-01-01

    Almost all plant cells are surrounded by glycan-rich cell walls, which form much of the plant body and collectively are the largest source of biomass on earth. Plants use polysaccharides for support, defense, signaling, cell adhesion, and as energy storage, and many plant glycans are also important...... industrially and nutritionally. Understanding the biological roles of plant glycans and the effective exploitation of their useful properties requires a detailed understanding of their structures, occurrence, and molecular interactions. Microarray technology has revolutionized the massively high...... for plant research and can be used to map glycan populations across large numbers of samples to screen antibodies, carbohydrate binding proteins, and carbohydrate binding modules and to investigate enzyme activities....

  10. The EADGENE Microarray Data Analysis Workshop

    DEFF Research Database (Denmark)

    de Koning, Dirk-Jan; Jaffrézic, Florence; Lund, Mogens Sandø

    2007-01-01

    Microarray analyses have become an important tool in animal genomics. While their use is becoming widespread, there is still a lot of ongoing research regarding the analysis of microarray data. In the context of a European Network of Excellence, 31 researchers representing 14 research groups from...... 10 countries performed and discussed the statistical analyses of real and simulated 2-colour microarray data that were distributed among participants. The real data consisted of 48 microarrays from a disease challenge experiment in dairy cattle, while the simulated data consisted of 10 microarrays...... statistical weights, to omitting a large number of spots or omitting entire slides. Surprisingly, these very different approaches gave quite similar results when applied to the simulated data, although not all participating groups analysed both real and simulated data. The workshop was very successful...

  11. Microarray-based genomic surveying of gene polymorphisms in Chlamydia trachomatis

    OpenAIRE

    Brunelle, Brian W; Nicholson, Tracy L; Stephens, Richard S

    2004-01-01

    By comparing two fully sequenced genomes of Chlamydia trachomatis using competitive hybridization on DNA microarrays, a logarithmic correlation was demonstrated between the signal ratio of the arrays and the 75-99% range of nucleotide identities of the genes. Variable genes within 14 uncharacterized strains of C. trachomatis were identified by array analysis and verified by DNA sequencing. These genes may be crucial for understanding chlamydial virulence and pathogenesis.

  12. Gene selection for microarray data classification via subspace learning and manifold regularization.

    Science.gov (United States)

    Tang, Chang; Cao, Lijuan; Zheng, Xiao; Wang, Minhui

    2017-12-19

    With the rapid development of DNA microarray technology, large amount of genomic data has been generated. Classification of these microarray data is a challenge task since gene expression data are often with thousands of genes but a small number of samples. In this paper, an effective gene selection method is proposed to select the best subset of genes for microarray data with the irrelevant and redundant genes removed. Compared with original data, the selected gene subset can benefit the classification task. We formulate the gene selection task as a manifold regularized subspace learning problem. In detail, a projection matrix is used to project the original high dimensional microarray data into a lower dimensional subspace, with the constraint that the original genes can be well represented by the selected genes. Meanwhile, the local manifold structure of original data is preserved by a Laplacian graph regularization term on the low-dimensional data space. The projection matrix can serve as an importance indicator of different genes. An iterative update algorithm is developed for solving the problem. Experimental results on six publicly available microarray datasets and one clinical dataset demonstrate that the proposed method performs better when compared with other state-of-the-art methods in terms of microarray data classification. Graphical Abstract The graphical abstract of this work.

  13. Multisegment one-step RT-PCR fluorescent labeling of influenza A virus genome for use in diagnostic microarray applications

    Energy Technology Data Exchange (ETDEWEB)

    Vasin, A V; Plotnikova, M A; Klotchenko, S A; Elpaeva, E A; Komissarov, A B; Egorov, V V; Kiselev, O I [Research Institute of Influenza of the Ministry of Health and Social Development of the Russian Federation, 15/17 Prof. Popova St., St. Petersburg (Russian Federation); Sandybaev, N T; Chervyakova, O V; Strochkov, V M; Taylakova, E T; Koshemetov, J K; Mamadaliev, S M, E-mail: vasin@influenza.spb.ru [Research Institute for Biological Safety Problems of the RK NBC/SC ME and S RK, Gvardeiskiy (Kazakhstan)

    2011-04-01

    Microarray technology is one of the most challenging methods of influenza A virus subtyping, which is based on the antigenic properties of viral surface glycoproteins - hemagglutinin and neuraminidase. On the example of biochip for detection of influenza A/H5N1 virus we showed the possibility of using multisegment RTPCR method for amplification of fluorescently labeled cDNA of all possible influenza A virus subtypes with a single pair of primers in influenza diagnostic microarrays.

  14. DNA effects upon the reaction between acetonitrile pentacyanoferrate (II) and ruthenium pentammine pyrazine: Kinetic and thermodynamic evidence of the interaction of DNA with anionic species

    International Nuclear Information System (INIS)

    Grueso, E.; Prado-Gotor, R.; Lopez, M.; Gomez-Herrera, C.; Sanchez, F.

    2005-01-01

    The kinetics of the reaction between ruthenium pentaammine pyrazine and acetonitrile pentacyanoferrate (II) to obtain the binuclear anionic complex [Fe(CN) 5 pzRu(NH 3 ) 5 ] - , and the reverse (dissociation) process, have been studied in solutions containing DNA. The results corresponding to this reaction and those corresponding to the reverse (dissociation) process show a clear influence of DNA on their kinetics. The results can be interpreted using a modified Pseudophase Model. From the results obtained for the dissociation reaction one can conclude that the binuclear anionic complex [Fe(CN) 5 pzRu(NH 3 ) 5 ] - interacts with DNA

  15. Utility of the pooling approach as applied to whole genome association scans with high-density Affymetrix microarrays

    Directory of Open Access Journals (Sweden)

    Gray Joanna

    2010-11-01

    Full Text Available Abstract Background We report an attempt to extend the previously successful approach of combining SNP (single nucleotide polymorphism microarrays and DNA pooling (SNP-MaP employing high-density microarrays. Whereas earlier studies employed a range of Affymetrix SNP microarrays comprising from 10 K to 500 K SNPs, this most recent investigation used the 6.0 chip which displays 906,600 SNP probes and 946,000 probes for the interrogation of CNVs (copy number variations. The genotyping assay using the Affymetrix SNP 6.0 array is highly demanding on sample quality due to the small feature size, low redundancy, and lack of mismatch probes. Findings In the first study published so far using this microarray on pooled DNA, we found that pooled cheek swab DNA could not accurately predict real allele frequencies of the samples that comprised the pools. In contrast, the allele frequency estimates using blood DNA pools were reasonable, although inferior compared to those obtained with previously employed Affymetrix microarrays. However, it might be possible to improve performance by developing improved analysis methods. Conclusions Despite the decreasing costs of genome-wide individual genotyping, the pooling approach may have applications in very large-scale case-control association studies. In such cases, our study suggests that high-quality DNA preparations and lower density platforms should be preferred.

  16. Comprehensive analysis of preeclampsia-associated DNA methylation in the placenta.

    Directory of Open Access Journals (Sweden)

    Tianjiao Chu

    Full Text Available A small number of recent reports have suggested that altered placental DNA methylation may be associated with early onset preeclampsia. It is important that further studies be undertaken to confirm and develop these findings. We therefore undertook a systematic analysis of DNA methylation patterns in placental tissue from 24 women with preeclampsia and 24 with uncomplicated pregnancy outcome.We analyzed the DNA methylation status of approximately 27,000 CpG sites in placental tissues in a massively parallel fashion using an oligonucleotide microarray. Follow up analysis of DNA methylation at specific CpG loci was performed using the Epityper MassArray approach and high-throughput bisulfite sequencing.Preeclampsia-specific DNA methylation changes were identified in placental tissue samples irrespective of gestational age of delivery. In addition, we identified a group of CpG sites within specific gene sequences that were only altered in early onset-preeclampsia (EOPET although these DNA methylation changes did not correlate with altered mRNA transcription. We found evidence that fetal gender influences DNA methylation at autosomal loci but could find no clear association between DNA methylation and gestational age.Preeclampsia is associated with altered placental DNA methylation. Fetal gender should be carefully considered during the design of future studies in which placental DNA is analyzed at the level of DNA methylation. Further large-scale analyses of preeclampsia-associated DNA methylation are necessary.

  17. A probabilistic framework for microarray data analysis: fundamental probability models and statistical inference.

    Science.gov (United States)

    Ogunnaike, Babatunde A; Gelmi, Claudio A; Edwards, Jeremy S

    2010-05-21

    Gene expression studies generate large quantities of data with the defining characteristic that the number of genes (whose expression profiles are to be determined) exceed the number of available replicates by several orders of magnitude. Standard spot-by-spot analysis still seeks to extract useful information for each gene on the basis of the number of available replicates, and thus plays to the weakness of microarrays. On the other hand, because of the data volume, treating the entire data set as an ensemble, and developing theoretical distributions for these ensembles provides a framework that plays instead to the strength of microarrays. We present theoretical results that under reasonable assumptions, the distribution of microarray intensities follows the Gamma model, with the biological interpretations of the model parameters emerging naturally. We subsequently establish that for each microarray data set, the fractional intensities can be represented as a mixture of Beta densities, and develop a procedure for using these results to draw statistical inference regarding differential gene expression. We illustrate the results with experimental data from gene expression studies on Deinococcus radiodurans following DNA damage using cDNA microarrays. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  18. Microarray profile of seizure damage-refractory hippocampal CA3 in a mouse model of epileptic preconditioning.

    Science.gov (United States)

    Hatazaki, S; Bellver-Estelles, C; Jimenez-Mateos, E M; Meller, R; Bonner, C; Murphy, N; Matsushima, S; Taki, W; Prehn, J H M; Simon, R P; Henshall, D C

    2007-12-05

    A neuroprotected state can be acquired by preconditioning brain with a stimulus that is subthreshold for damage (tolerance). Acquisition of tolerance involves coordinate, bi-directional changes to gene expression levels and the re-programmed phenotype is determined by the preconditioning stimulus. While best studied in ischemic brain there is evidence brief seizures can confer tolerance against prolonged seizures (status epilepticus). Presently, we developed a model of epileptic preconditioning in mice and used microarrays to gain insight into the transcriptional phenotype within the target hippocampus at the time tolerance had been acquired. Epileptic tolerance was induced by an episode of non-damaging seizures in adult C57Bl/6 mice using a systemic injection of kainic acid. Neuron and DNA damage-positive cell counts 24 h after status epilepticus induced by intraamygdala microinjection of kainic acid revealed preconditioning given 24 h prior reduced CA3 neuronal death by approximately 45% compared with non-tolerant seizure mice. Microarray analysis of over 39,000 transcripts (Affymetrix 430 2.0 chip) from microdissected CA3 subfields was undertaken at the point at which tolerance was acquired. Results revealed a unique profile of small numbers of equivalently up- and down-regulated genes with biological functions that included transport and localization, ubiquitin metabolism, apoptosis and cell cycle control. Select microarray findings were validated post hoc by real-time polymerase chain reaction and Western blotting. The present study defines a paradigm for inducing epileptic preconditioning in mice and first insight into the global transcriptome of the seizure-damage refractory brain.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Young [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada)], E-mail: daeyoung.lee@ec.gc.ca; Lauder, Heather; Cruwys, Heather; Falletta, Patricia [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada); Beaudette, Lee A. [Environmental Science and Technology Centre, Environment Canada, 335 River Road South, Ottawa, Ontario, K1A 0H3 (Canada)], E-mail: lee.beaudette@ec.gc.ca

    2008-07-15

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

  20. Improving comparability between microarray probe signals by thermodynamic intensity correction

    DEFF Research Database (Denmark)

    Bruun, G. M.; Wernersson, Rasmus; Juncker, Agnieszka

    2007-01-01

    different probes. It is therefore of great interest to correct for the variation between probes. Much of this variation is sequence dependent. We demonstrate that a thermodynamic model for hybridization of either DNA or RNA to a DNA microarray, which takes the sequence-dependent probe affinities...... determination of transcription start sites for a subset of yeast genes. In another application, we identify present/absent calls for probes hybridized to the sequenced Escherichia coli strain O157:H7 EDL933. The model improves the correct calls from 85 to 95% relative to raw intensity measures. The model thus...... makes applications which depend on comparisons between probes aimed at different sections of the same target more reliable....

  1. MARS: Microarray analysis, retrieval, and storage system

    Directory of Open Access Journals (Sweden)

    Scheideler Marcel

    2005-04-01

    Full Text Available Abstract Background Microarray analysis has become a widely used technique for the study of gene-expression patterns on a genomic scale. As more and more laboratories are adopting microarray technology, there is a need for powerful and easy to use microarray databases facilitating array fabrication, labeling, hybridization, and data analysis. The wealth of data generated by this high throughput approach renders adequate database and analysis tools crucial for the pursuit of insights into the transcriptomic behavior of cells. Results MARS (Microarray Analysis and Retrieval System provides a comprehensive MIAME supportive suite for storing, retrieving, and analyzing multi color microarray data. The system comprises a laboratory information management system (LIMS, a quality control management, as well as a sophisticated user management system. MARS is fully integrated into an analytical pipeline of microarray image analysis, normalization, gene expression clustering, and mapping of gene expression data onto biological pathways. The incorporation of ontologies and the use of MAGE-ML enables an export of studies stored in MARS to public repositories and other databases accepting these documents. Conclusion We have developed an integrated system tailored to serve the specific needs of microarray based research projects using a unique fusion of Web based and standalone applications connected to the latest J2EE application server technology. The presented system is freely available for academic and non-profit institutions. More information can be found at http://genome.tugraz.at.

  2. Annotating breast cancer microarray samples using ontologies

    Science.gov (United States)

    Liu, Hongfang; Li, Xin; Yoon, Victoria; Clarke, Robert

    2008-01-01

    As the most common cancer among women, breast cancer results from the accumulation of mutations in essential genes. Recent advance in high-throughput gene expression microarray technology has inspired researchers to use the technology to assist breast cancer diagnosis, prognosis, and treatment prediction. However, the high dimensionality of microarray experiments and public access of data from many experiments have caused inconsistencies which initiated the development of controlled terminologies and ontologies for annotating microarray experiments, such as the standard microarray Gene Expression Data (MGED) ontology (MO). In this paper, we developed BCM-CO, an ontology tailored specifically for indexing clinical annotations of breast cancer microarray samples from the NCI Thesaurus. Our research showed that the coverage of NCI Thesaurus is very limited with respect to i) terms used by researchers to describe breast cancer histology (covering 22 out of 48 histology terms); ii) breast cancer cell lines (covering one out of 12 cell lines); and iii) classes corresponding to the breast cancer grading and staging. By incorporating a wider range of those terms into BCM-CO, we were able to indexed breast cancer microarray samples from GEO using BCM-CO and MGED ontology and developed a prototype system with web interface that allows the retrieval of microarray data based on the ontology annotations. PMID:18999108

  3. Simulation of microarray data with realistic characteristics

    Directory of Open Access Journals (Sweden)

    Lehmussola Antti

    2006-07-01

    Full Text Available Abstract Background Microarray technologies have become common tools in biological research. As a result, a need for effective computational methods for data analysis has emerged. Numerous different algorithms have been proposed for analyzing the data. However, an objective evaluation of the proposed algorithms is not possible due to the lack of biological ground truth information. To overcome this fundamental problem, the use of simulated microarray data for algorithm validation has been proposed. Results We present a microarray simulation model which can be used to validate different kinds of data analysis algorithms. The proposed model is unique in the sense that it includes all the steps that affect the quality of real microarray data. These steps include the simulation of biological ground truth data, applying biological and measurement technology specific error models, and finally simulating the microarray slide manufacturing and hybridization. After all these steps are taken into account, the simulated data has realistic biological and statistical characteristics. The applicability of the proposed model is demonstrated by several examples. Conclusion The proposed microarray simulation model is modular and can be used in different kinds of applications. It includes several error models that have been proposed earlier and it can be used with different types of input data. The model can be used to simulate both spotted two-channel and oligonucleotide based single-channel microarrays. All this makes the model a valuable tool for example in validation of data analysis algorithms.

  4. Reconstructing the temporal ordering of biological samples using microarray data.

    Science.gov (United States)

    Magwene, Paul M; Lizardi, Paul; Kim, Junhyong

    2003-05-01

    Accurate time series for biological processes are difficult to estimate due to problems of synchronization, temporal sampling and rate heterogeneity. Methods are needed that can utilize multi-dimensional data, such as those resulting from DNA microarray experiments, in order to reconstruct time series from unordered or poorly ordered sets of observations. We present a set of algorithms for estimating temporal orderings from unordered sets of sample elements. The techniques we describe are based on modifications of a minimum-spanning tree calculated from a weighted, undirected graph. We demonstrate the efficacy of our approach by applying these techniques to an artificial data set as well as several gene expression data sets derived from DNA microarray experiments. In addition to estimating orderings, the techniques we describe also provide useful heuristics for assessing relevant properties of sample datasets such as noise and sampling intensity, and we show how a data structure called a PQ-tree can be used to represent uncertainty in a reconstructed ordering. Academic implementations of the ordering algorithms are available as source code (in the programming language Python) on our web site, along with documentation on their use. The artificial 'jelly roll' data set upon which the algorithm was tested is also available from this web site. The publicly available gene expression data may be found at http://genome-www.stanford.edu/cellcycle/ and http://caulobacter.stanford.edu/CellCycle/.

  5. Investigation of cellular signalling responses to non-ionising radiation in melanocytes by microarray analysis

    International Nuclear Information System (INIS)

    Boyle, G.M.; Pedley, J.; Martyn, A.C.; Fraser, L.M.; Banducci, K.J.; Parsons, P.G.; Breit, S.N.

    2003-01-01

    Melanoma is a highly aggressive cancer resulting from the abnormal proliferation and spread of specialised pigment cells in the skin, known as melanocytes. Extensive epidemiological and molecular evidence suggests that a major risk factor for melanoma formation is exposure to non-ionising radiation in the form of solar ultra-violet (UV) light. However, the exact role of solar UV in the development of melanoma is unclear. To elucidate the molecular events that occur in melanocytes following solar UV exposure and determine how they lead to melanoma development, cDNA microarray analysis was used to analyse the gene expression profile of normal melanocytes, melanocytes exposed to simulated solar UV and melanoma cells. The development of cDNA microarray technology has allowed gene expression profiling at the mRNA level to be conducted for many thousands of genes simultaneously by hybridising an array of known sequences with labelled cDNA reverse transcribed form the sample RNA. Gene expression analysis was performed for over 13,000 genes. More than 500 genes were identified as differentially expressed in melanocytes following a single UV exposure, although overall there was a general suppression of transcription. Genes that were up-regulated included oncogenes and cytoskeletal genes; in contrast, genes encoding protein tyrosine kinases and apoptosis effectors were down-regulated. Many of the genes identified as being differentially expressed represent novel UV-regulated targets. Repeated exposure to solar UV resulted in the elevation in expression of a novel member of the transforming growth factor-b (TGF-b) superfamily, the Macrophage Inhibitory Cytokine-1 (MIC-1). Our results have shown that MIC-1 is up-regulated by solar UV in melanocytes, and is highly expressed (>3 fold) in a number of metastatic melanoma cell lines (31/61) in comparison to primary melanocytes. Furthermore functional, dimerised MIC-1 was found to be secreted by melanocytes, and secreted levels were

  6. Application of four dyes in gene expression analyses by microarrays

    Directory of Open Access Journals (Sweden)

    van Schooten Frederik J

    2005-07-01

    Full Text Available Abstract Background DNA microarrays are widely used in gene expression analyses. To increase throughput and minimize costs without reducing gene expression data obtained, we investigated whether four mRNA samples can be analyzed simultaneously by applying four different fluorescent dyes. Results Following tests for cross-talk of fluorescence signals, Alexa 488, Alexa 594, Cyanine 3 and Cyanine 5 were selected for hybridizations. For self-hybridizations, a single RNA sample was labelled with all dyes and hybridized on commercial cDNA arrays or on in-house spotted oligonucleotide arrays. Correlation coefficients for all combinations of dyes were above 0.9 on the cDNA array. On the oligonucleotide array they were above 0.8, except combinations with Alexa 488, which were approximately 0.5. Standard deviation of expression differences for replicate spots were similar on the cDNA array for all dye combinations, but on the oligonucleotide array combinations with Alexa 488 showed a higher variation. Conclusion In conclusion, the four dyes can be used simultaneously for gene expression experiments on the tested cDNA array, but only three dyes can be used on the tested oligonucleotide array. This was confirmed by hybridizations of control with test samples, as all combinations returned similar numbers of differentially expressed genes with comparable effects on gene expression.

  7. Generalization of DNA microarray dispersion properties: microarray equivalent of t-distribution

    Czech Academy of Sciences Publication Activity Database

    Novák, J.P.; Kim, S.Y.; Xu, J.; Modlich, O.; Volsky, D.J.; Honys, David; Slonczewski, J.L.; Bell, D.A.; Blattner, F.R.; Blumwald, E.; Boerma, M.; Cosio, M.; Gatalica, Z.; Hajduch, M.; Hidalgo, J.; McInnes, R.R.; Miller III, M.C.; Penkowa, M.; Rolph, M.S.; Sottosanto, J.; St-Arnaud, R.; Szego, M.J.; Twell, D.; Wang, Ch.

    2006-01-01

    Roč. 1, č. 27 (2006), s. 1-24 ISSN 1745-6150 R&D Projects: GA ČR GA522/06/0896 Institutional research plan: CEZ:AV0Z50380511 Keywords : GENE-EXPRESSION DATA * OLIGONUCLEOTIDE ARRAY EXPERIMENTS * ESCHERICHIA-COLI K-12 Subject RIV: EB - Genetics ; Molecular Biology

  8. Biosensors for DNA sequence detection

    Science.gov (United States)

    Vercoutere, Wenonah; Akeson, Mark

    2002-01-01

    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  9. cluML: A markup language for clustering and cluster validity assessment of microarray data.

    Science.gov (United States)

    Bolshakova, Nadia; Cunningham, Pádraig

    2005-01-01

    cluML is a new markup language for microarray data clustering and cluster validity assessment. The XML-based format has been designed to address some of the limitations observed in traditional formats, such as inability to store multiple clustering (including biclustering) and validation results within a dataset. cluML is an effective tool to support biomedical knowledge representation in gene expression data analysis. Although cluML was developed for DNA microarray analysis applications, it can be effectively used for the representation of clustering and for the validation of other biomedical and physical data that has no limitations.

  10. Gene Expression and Microarray Investigation of Dendrobium ...

    African Journals Online (AJOL)

    blood glucose > 16.7 mmol/L were used as the model group and treated with Dendrobium mixture. (DEN ... Keywords: Diabetes, Gene expression, Dendrobium mixture, Microarray testing ..... homeostasis in airway smooth muscle. Am J.

  11. SLIMarray: Lightweight software for microarray facility management

    Directory of Open Access Journals (Sweden)

    Marzolf Bruz

    2006-10-01

    Full Text Available Abstract Background Microarray core facilities are commonplace in biological research organizations, and need systems for accurately tracking various logistical aspects of their operation. Although these different needs could be handled separately, an integrated management system provides benefits in organization, automation and reduction in errors. Results We present SLIMarray (System for Lab Information Management of Microarrays, an open source, modular database web application capable of managing microarray inventories, sample processing and usage charges. The software allows modular configuration and is well suited for further development, providing users the flexibility to adapt it to their needs. SLIMarray Lite, a version of the software that is especially easy to install and run, is also available. Conclusion SLIMarray addresses the previously unmet need for free and open source software for managing the logistics of a microarray core facility.

  12. Evidence that OGG1 glycosylase protects neurons against oxidative DNA damage and cell death under ischemic conditions

    DEFF Research Database (Denmark)

    Liu, Dong; Croteau, Deborah L; Souza-Pinto, Nadja

    2011-01-01

    to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential......7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1(-/-)) mice to examine the possible roles of OGG1 in the vulnerability of neurons....... Cortical neurons isolated from OGG1(-/-) mice were more vulnerable to oxidative insults than were OGG1(+/+) neurons, and OGG1(-/-) mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1(+/+) mice. Accumulations of oxidative DNA...

  13. Recovery of Trace DNA on Clothing: A Comparison of Mini-tape Lifting and Three Other Forensic Evidence Collection Techniques.

    Science.gov (United States)

    Hess, Sabine; Haas, Cordula

    2017-01-01

    Trace DNA is often found in forensic science investigations. Experience has shown that it is difficult to retrieve a DNA profile when trace DNA is collected from clothing. The aim of this study was to compare four different DNA collection techniques on six different types of clothing in order to determine the best trace DNA recovery method. The classical stain recovery technique using a wet cotton swab was tested against dry swabbing, scraping and a new method, referred to as the mini-tape lifting technique. Physical contact was simulated with three different "perpetrators" on 18 machine-washed garments. DNA was collected with the four different DNA recovery methods and subjected to standard PCR-based DNA profiling. The comparison of STR results showed best results for the mini-tape lifting and scraping methods independent of the type of clothing. The new mini-tape lifting technique proved to be an easy and reliable DNA collection method for textiles. © 2016 American Academy of Forensic Sciences.

  14. PATMA: parser of archival tissue microarray

    Directory of Open Access Journals (Sweden)

    Lukasz Roszkowiak

    2016-12-01

    Full Text Available Tissue microarrays are commonly used in modern pathology for cancer tissue evaluation, as it is a very potent technique. Tissue microarray slides are often scanned to perform computer-aided histopathological analysis of the tissue cores. For processing the image, splitting the whole virtual slide into images of individual cores is required. The only way to distinguish cores corresponding to specimens in the tissue microarray is through their arrangement. Unfortunately, distinguishing the correct order of cores is not a trivial task as they are not labelled directly on the slide. The main aim of this study was to create a procedure capable of automatically finding and extracting cores from archival images of the tissue microarrays. This software supports the work of scientists who want to perform further image processing on single cores. The proposed method is an efficient and fast procedure, working in fully automatic or semi-automatic mode. A total of 89% of punches were correctly extracted with automatic selection. With an addition of manual correction, it is possible to fully prepare the whole slide image for extraction in 2 min per tissue microarray. The proposed technique requires minimum skill and time to parse big array of cores from tissue microarray whole slide image into individual core images.

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

    Science.gov (United States)

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

    2015-01-01

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

  16. Further evidence for population specific differences in the effect of DNA markers and gender on eye colour prediction in forensics.

    Science.gov (United States)

    Pośpiech, Ewelina; Karłowska-Pik, Joanna; Ziemkiewicz, Bartosz; Kukla, Magdalena; Skowron, Małgorzata; Wojas-Pelc, Anna; Branicki, Wojciech

    2016-07-01

    The genetics of eye colour has been extensively studied over the past few years, and the identified polymorphisms have been applied with marked success in the field of Forensic DNA Phenotyping. A picture that arises from evaluation of the currently available eye colour prediction markers shows that only the analysis of HERC2-OCA2 complex has similar effectiveness in different populations, while the predictive potential of other loci may vary significantly. Moreover, the role of gender in the explanation of human eye colour variation should not be neglected in some populations. In the present study, we re-investigated the data for 1020 Polish individuals and using neural networks and logistic regression methods explored predictive capacity of IrisPlex SNPs and gender in this population sample. In general, neural networks provided higher prediction accuracy comparing to logistic regression (AUC increase by 0.02-0.06). Four out of six IrisPlex SNPs were associated with eye colour in the studied population. HERC2 rs12913832, OCA2 rs1800407 and SLC24A4 rs12896399 were found to be the most important eye colour predictors (p Gender was found to be significantly associated with eye colour with males having ~1.5 higher odds for blue eye colour comparing to females (p = 0.002) and was ranked as the third most important factor in blue/non-blue eye colour determination. However, the implementation of gender into the developed prediction models had marginal and ambiguous impact on the overall accuracy of prediction confirming that the effect of gender on eye colour in this population is small. Our study indicated the advantage of neural networks in prediction modeling in forensics and provided additional evidence for population specific differences in the predictive importance of the IrisPlex SNPs and gender.

  17. Evidence of authentic DNA from Danish Viking Age skeletons untouched by humans for 1,000 years

    DEFF Research Database (Denmark)

    Melchior, Linea; Kivisild, Toomas; Lynnerup, Niels

    2008-01-01

    -PCR work was carried out in a "clean- laboratory" dedicated solely to ancient DNA work. Mitochondrial DNA was extracted and overlapping fragments spanning the HVR-1 region as well as diagnostic sites in the coding region were PCR amplified, cloned and sequenced. Consistent results were obtained...

  18. Microarray mRNA expression analysis of Fanconi anemia fibroblasts.

    Science.gov (United States)

    Galetzka, D; Weis, E; Rittner, G; Schindler, D; Haaf, T

    2008-01-01

    Fanconi anemia (FA) cells are generally hypersensitive to DNA cross-linking agents, implying that mutations in the different FANC genes cause a similar DNA repair defect(s). By using a customized cDNA microarray chip for DNA repair- and cell cycle-associated genes, we identified three genes, cathepsin B (CTSB), glutaredoxin (GLRX), and polo-like kinase 2 (PLK2), that were misregulated in untreated primary fibroblasts from three unrelated FA-D2 patients, compared to six controls. Quantitative real-time RT PCR was used to validate these results and to study possible molecular links between FA-D2 and other FA subtypes. GLRX was misregulated to opposite directions in a variety of different FA subtypes. Increased CTSB and decreased PLK2 expression was found in all or almost all of the analyzed complementation groups and, therefore, may be related to the defective FA pathway. Transcriptional upregulation of the CTSB proteinase appears to be a secondary phenomenon due to proliferation differences between FA and normal fibroblast cultures. In contrast, PLK2 is known to play a pivotal role in processes that are linked to FA defects and may contribute in multiple ways to the FA phenotype: PLK2 is a target gene for TP53, is likely to function as a tumor suppressor gene in hematologic neoplasia, and Plk2(-/-) mice are small because of defective embryonal development. (c) 2008 S. Karger AG, Basel.

  19. Microarray profiling of mononuclear peripheral blood cells identifies novel candidate genes related to chemoradiation response in rectal cancer.

    Directory of Open Access Journals (Sweden)

    Pablo Palma

    Full Text Available Preoperative chemoradiation significantly improves oncological outcome in locally advanced rectal cancer. However there is no effective method of predicting tumor response to chemoradiation in these patients. Peripheral blood mononuclear cells have emerged recently as pathology markers of cancer and other diseases, making possible their use as therapy predictors. Furthermore, the importance of the immune response in radiosensivity of solid organs led us to hypothesized that microarray gene expression profiling of peripheral blood mononuclear cells could identify patients with response to chemoradiation in rectal cancer. Thirty five 35 patients with locally advanced rectal cancer were recruited initially to perform the study. Peripheral blood samples were obtained before neaodjuvant treatment. RNA was extracted and purified to obtain cDNA and cRNA for hybridization of microarrays included in Human WG CodeLink bioarrays. Quantitative real time PCR was used to validate microarray experiment data. Results were correlated with pathological response, according to Mandard´s criteria and final UICC Stage (patients with tumor regression grade 1-2 and downstaging being defined as responders and patients with grade 3-5 and no downstaging as non-responders. Twenty seven out of 35 patients were finally included in the study. We performed a multiple t-test using Significance Analysis of Microarrays, to find those genes differing significantly in expression, between responders (n = 11 and non-responders (n = 16 to CRT. The differently expressed genes were: BC 035656.1, CIR, PRDM2, CAPG, FALZ, HLA-DPB2, NUPL2, and ZFP36. The measurement of FALZ (p = 0.029 gene expression level determined by qRT-PCR, showed statistically significant differences between the two groups. Gene expression profiling reveals novel genes in peripheral blood samples of mononuclear cells that could predict responders and non-responders to chemoradiation in patients with

  20. Development and evaluation of a high-throughput, low-cost genotyping platform based on oligonucleotide microarrays in rice

    Directory of Open Access Journals (Sweden)

    Liu Bin

    2008-05-01

    Full Text Available Abstract Background We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features. Results We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype. Conclusion We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals.

  1. Detecting imbalanced expression of SNP alleles by minisequencing on microarrays

    Directory of Open Access Journals (Sweden)

    Dahlgren Andreas

    2004-10-01

    Full Text Available Abstract Background Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system. Results The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R2 values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1–9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and

  2. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: evidence for oxidatively DNA damage generation.

    Science.gov (United States)

    Pinto, A Viviana; Deodato, Elder L; Cardoso, Janine S; Oliveira, Eliza F; Machado, Sérgio L; Toma, Helena K; Leitão, Alvaro C; de Pádula, Marcelo

    2010-06-01

    Although titanium dioxide (TiO(2)) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO(2) is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO(2)-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO(2) associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO(2) plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO(2) protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO(2) plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO(2) plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine. Copyright 2010 Elsevier B.V. All rights reserved.

  3. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, A. Viviana, E-mail: alicia.pinto@incqs.fiocruz.br [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Deodato, Elder L. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Cardoso, Janine S. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K. [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil); Leitao, Alvaro C. [Laboratorio de Radiobiologia Molecular, Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21949-900, Rio de Janeiro (Brazil); Padula, Marcelo de [Laboratorio de Diagnostico Molecular e Hematologia, Faculdade de Farmacia, Universidade Federal do Rio de Janeiro, Centro de Ciencias da Saude - Ilha do Fundao, CEP 21941-540, Rio de Janeiro (Brazil)

    2010-06-01

    Although titanium dioxide (TiO{sub 2}) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO{sub 2} is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO{sub 2}-UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO{sub 2} associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO{sub 2} plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO{sub 2} protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO{sub 2} plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO{sub 2} plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

  4. Enzymatic recognition of DNA damage induced by UVB-photosensitized titanium dioxide and biological consequences in Saccharomyces cerevisiae: Evidence for oxidatively DNA damage generation

    International Nuclear Information System (INIS)

    Pinto, A. Viviana; Deodato, Elder L.; Cardoso, Janine S.; Oliveira, Eliza F.; Machado, Sergio L.; Toma, Helena K.; Leitao, Alvaro C.; Padula, Marcelo de

    2010-01-01

    Although titanium dioxide (TiO 2 ) has been considered to be biologically inert, finding use in cosmetics, paints and food colorants, recent reports have demonstrated that when TiO 2 is attained by UVA radiation oxidative genotoxic and cytotoxic effects are observed in living cells. However, data concerning TiO 2 -UVB association is poor, even if UVB radiation represents a major environmental carcinogen. Herein, we investigated DNA damage, repair and mutagenesis induced by TiO 2 associated with UVB irradiation in vitro and in vivo using Saccharomyces cerevisiae model. It was found that TiO 2 plus UVB treatment in plasmid pUC18 generated, in addition to cyclobutane pyrimidine dimers (CPDs), specific damage to guanine residues, such as 8-oxo-7,8-dihydroguanine (8-oxoG) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG), which are characteristic oxidatively generated lesions. In vivo experiments showed that, although the presence of TiO 2 protects yeast cells from UVB cytotoxicity, high mutation frequencies are observed in the wild-type (WT) and in an ogg1 strain (deficient in 8-oxoG and FapyG repair). Indeed, after TiO 2 plus UVB treatment, induced mutagenesis was drastically enhanced in ogg1 cells, indicating that mutagenic DNA lesions are repaired by the Ogg1 protein. This effect could be attenuated by the presence of metallic ion chelators: neocuproine or dipyridyl, which partially block oxidatively generated damage occurring via Fenton reactions. Altogether, the results indicate that TiO 2 plus UVB potentates UVB oxidatively generated damage to DNA, possibly via Fenton reactions involving the production of DNA base damage, such as 8-oxo-7,8-dihydroguanine.

  5. A molecular beacon microarray based on a quantum dot label for detecting single nucleotide polymorphisms.

    Science.gov (United States)

    Guo, Qingsheng; Bai, Zhixiong; Liu, Yuqian; Sun, Qingjiang

    2016-03-15

    In this work, we report the application of streptavidin-coated quantum dot (strAV-QD) in molecular beacon (MB) microarray assays by using the strAV-QD to label the immobilized MB, avoiding target labeling and meanwhile obviating the use of amplification. The MBs are stem-loop structured oligodeoxynucleotides, modified with a thiol and a biotin at two terminals of the stem. With the strAV-QD labeling an "opened" MB rather than a "closed" MB via streptavidin-biotin reaction, a sensitive and specific detection of label-free target DNA sequence is demonstrated by the MB microarray, with a signal-to-background ratio of 8. The immobilized MBs can be perfectly regenerated, allowing the reuse of the microarray. The MB microarray also is able to detect single nucleotide polymorphisms, exhibiting genotype-dependent fluorescence signals. It is demonstrated that the MB microarray can perform as a 4-to-2 encoder, compressing the genotype information into two outputs. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Development of a genotyping microarray for Usher syndrome.

    Science.gov (United States)

    Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner-Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva-Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

    2007-02-01

    Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein-coding exons. To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele-specific oligonucleotides corresponding to all 298 Usher syndrome-associated sequence variants known to date, 76 of which are novel, were arrayed. Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first-pass screening tool.

  7. Development of a genotyping microarray for Usher syndrome

    Science.gov (United States)

    Cremers, Frans P M; Kimberling, William J; Külm, Maigi; de Brouwer, Arjan P; van Wijk, Erwin; te Brinke, Heleen; Cremers, Cor W R J; Hoefsloot, Lies H; Banfi, Sandro; Simonelli, Francesca; Fleischhauer, Johannes C; Berger, Wolfgang; Kelley, Phil M; Haralambous, Elene; Bitner‐Glindzicz, Maria; Webster, Andrew R; Saihan, Zubin; De Baere, Elfride; Leroy, Bart P; Silvestri, Giuliana; McKay, Gareth J; Koenekoop, Robert K; Millan, Jose M; Rosenberg, Thomas; Joensuu, Tarja; Sankila, Eeva‐Marja; Weil, Dominique; Weston, Mike D; Wissinger, Bernd; Kremer, Hannie

    2007-01-01

    Background Usher syndrome, a combination of retinitis pigmentosa (RP) and sensorineural hearing loss with or without vestibular dysfunction, displays a high degree of clinical and genetic heterogeneity. Three clinical subtypes can be distinguished, based on the age of onset and severity of the hearing impairment, and the presence or absence of vestibular abnormalities. Thus far, eight genes have been implicated in the syndrome, together comprising 347 protein‐coding exons. Methods: To improve DNA diagnostics for patients with Usher syndrome, we developed a genotyping microarray based on the arrayed primer extension (APEX) method. Allele‐specific oligonucleotides corresponding to all 298 Usher syndrome‐associated sequence variants known to date, 76 of which are novel, were arrayed. Results Approximately half of these variants were validated using original patient DNAs, which yielded an accuracy of >98%. The efficiency of the Usher genotyping microarray was tested using DNAs from 370 unrelated European and American patients with Usher syndrome. Sequence variants were identified in 64/140 (46%) patients with Usher syndrome type I, 45/189 (24%) patients with Usher syndrome type II, 6/21 (29%) patients with Usher syndrome type III and 6/20 (30%) patients with atypical Usher syndrome. The chip also identified two novel sequence variants, c.400C>T (p.R134X) in PCDH15 and c.1606T>C (p.C536S) in USH2A. Conclusion The Usher genotyping microarray is a versatile and affordable screening tool for Usher syndrome. Its efficiency will improve with the addition of novel sequence variants with minimal extra costs, making it a very useful first‐pass screening tool. PMID:16963483

  8. Replication of UV-irradiated DNA in human cell extracts: Evidence for mutagenic bypass of pyrimidine dimers

    International Nuclear Information System (INIS)

    Thomas, D.C.; Kunkel, T.A.

    1993-01-01

    The authors have examined the efficiency and fidelity of simian virus 40-origin-dependent replication of UV-irradiated double-stranded DNA in extracts of human cells. Using as a mutational target the α-complementation domain of the Escherichia coli lacZ gene in bacteriophage M13mp2DNA, replication of undamaged DNA in HeLa cell extracts was highly accurate, whereas replication of DNA irradiated with UV light (280-320 nm) was both less efficient and less accurate. Replication was inhibited by irradiation in a dose-dependent manner. Nonetheless, covalently closed, monomer-length circular products were generated that were resistant to digestion by Dpn I, showing that they resulted from semiconservative replication. These products were incised by T4 endonuclease V, whereas the undamaged replication products were not, suggesting that pyrimidine dimers were bypassed during replication. When replicated, UV-irradiated DNA was used to transfect an E. coli α-complementation host strain to score mutant M13mp2 plaques, the mutant plaque frequency was substantially higher than that obtained with either unirradiated, replicated DNA, or unreplicated, UV-irradiated DNA. Both the increased mutagenicity and the inhibition of replication associated with UV irradiation were reversed by treatment of the irradiated DNA with photolyase before replication. Sequence analysis of mutants resulting from replication of UV-irradiated DNA demonstrated that most mutants contained C → T transition errors at dipyrimidine sites. A few mutants contained 1-nt frameshift errors or tandem double CC → TT substitutions. The data are consistent with the interpretation that pyrimidine dimers are bypassed during replication by the multiprotein replication apparatus in human cell extracts and that this bypass is mutagenic primarily via misincorporation of dAMP opposite a cytosine (or uracil) in the dimer. 56 refs., 2 figs., 3 tabs

  9. Transcriptome sequencing of the Microarray Quality Control (MAQC RNA reference samples using next generation sequencing

    Directory of Open Access Journals (Sweden)

    Thierry-Mieg Danielle

    2009-06-01

    Full Text Available Abstract Background Transcriptome sequencing using next-generation sequencing platforms will soon be competing with DNA microarray technologies for global gene expression analysis. As a preliminary evaluation of these promising technologies, we performed deep sequencing of cDNA synthesized from the Microarray Quality Control (MAQC reference RNA samples using Roche's 454 Genome Sequencer FLX. Results We generated more that 3.6 million sequence reads of average length 250 bp for the MAQC A and B samples and introduced a data analysis pipeline for translating cDNA read counts into gene expression levels. Using BLAST, 90% of the reads mapped to the human genome and 64% of the reads mapped to the RefSeq database of well annotated genes with e-values ≤ 10-20. We measured gene expression levels in the A and B samples by counting the numbers of reads that mapped to individual RefSeq genes in multiple sequencing runs to evaluate the MAQC quality metrics for reproducibility, sensitivity, specificity, and accuracy and compared the results with DNA microarrays and Quantitative RT-PCR (QRTPCR from the MAQC studies. In addition, 88% of the reads were successfully aligned directly to the human genome using the AceView alignment programs with an average 90% sequence similarity to identify 137,899 unique exon junctions, including 22,193 new exon junctions not yet contained in the RefSeq database. Conclusion Using the MAQC metrics for evaluating the performance of gene expression platforms, the ExpressSeq results for gene expression levels showed excellent reproducibility, sensitivity, and specificity that improved systematically with increasing shotgun sequencing depth, and quantitative accuracy that was comparable to DNA microarrays and QRTPCR. In addition, a careful mapping of the reads to the genome using the AceView alignment programs shed new light on the complexity of the human transcriptome including the discovery of thousands of new splice variants.

  10. Transcriptional profiling of endocrine cerebro-osteodysplasia using microarray and next-generation sequencing.

    Directory of Open Access Journals (Sweden)

    Piya Lahiry

    Full Text Available BACKGROUND: Transcriptome profiling of patterns of RNA expression is a powerful approach to identify networks of genes that play a role in disease. To date, most mRNA profiling of tissues has been accomplished using microarrays, but next-generation sequencing can offer a richer and more comprehensive picture. METHODOLOGY/PRINCIPAL FINDINGS: ECO is a rare multi-system developmental disorder caused by a homozygous mutation in ICK encoding intestinal cell kinase. We performed gene expression profiling using both cDNA microarrays and next-generation mRNA sequencing (mRNA-seq of skin fibroblasts from ECO-affected subjects. We then validated a subset of differentially expressed transcripts identified by each method using quantitative reverse transcription-polymerase chain reaction (qRT-PCR. Finally, we used gene ontology (GO to identify critical pathways and processes that were abnormal according to each technical platform. Methodologically, mRNA-seq identifies a much larger number of differentially expressed genes with much better correlation to qRT-PCR results than the microarray (r² = 0.794 and 0.137, respectively. Biologically, cDNA microarray identified functional pathways focused on anatomical structure and development, while the mRNA-seq platform identified a higher proportion of genes involved in cell division and DNA replication pathways. CONCLUSIONS/SIGNIFICANCE: Transcriptome profiling with mRNA-seq had greater sensitivity, range and accuracy than the microarray. The two platforms generated different but complementary hypotheses for further evaluation.

  11. Fuzzy C-means method for clustering microarray data.

    Science.gov (United States)

    Dembélé, Doulaye; Kastner, Philippe

    2003-05-22

    Clustering analysis of data from DNA microarray hybridization studies is essential for identifying biologically relevant groups of genes. Partitional clustering methods such as K-means or self-organizing maps assign each gene to a single cluster. However, these methods do not provide information about the influence of a given gene for the overall shape of clusters. Here we apply a fuzzy partitioning method, Fuzzy C-means (FCM), to attribute cluster membership values to genes. A major problem in applying the FCM method for clustering microarray data is the choice of the fuzziness parameter m. We show that the commonly used value m = 2 is not appropriate for some data sets, and that optimal values for m vary widely from one data set to another. We propose an empirical method, based on the distribution of distances between genes in a given data set, to determine an adequate value for m. By setting threshold levels for the membership values, genes which are tigthly associated to a given cluster can be selected. Using a yeast cell cycle data set as an example, we show that this selection increases the overall biological significance of the genes within the cluster. Supplementary text and Matlab functions are available at http://www-igbmc.u-strasbg.fr/fcm/

  12. Nanotechnology: moving from microarrays toward nanoarrays.

    Science.gov (United States)

    Chen, Hua; Li, Jun

    2007-01-01

    Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

  13. Integrative missing value estimation for microarray data.

    Science.gov (United States)

    Hu, Jianjun; Li, Haifeng; Waterman, Michael S; Zhou, Xianghong Jasmine

    2006-10-12

    Missing value estimation is an important preprocessing step in microarray analysis. Although several methods have been developed to solve this problem, their performance is unsatisfactory for datasets with high rates of missing data, high measurement noise, or limited numbers of samples. In fact, more than 80% of the time-series datasets in Stanford Microarray Database contain less than eight samples. We present the integrative Missing Value Estimation method (iMISS) by incorporating information from multiple reference microarray datasets to improve missing value estimation. For each gene with missing data, we derive a consistent neighbor-gene list by taking reference data sets into consideration. To determine whether the given reference data sets are sufficiently informative for integration, we use a submatrix imputation approach. Our experiments showed that iMISS can significantly and consistently improve the accuracy of the state-of-the-art Local Least Square (LLS) imputation algorithm by up to 15% improvement in our benchmark tests. We demonstrated that the order-statistics-based integrative imputation algorithms can achieve significant improvements over the state-of-the-art missing value estimation approaches such as LLS and is especially good for imputing microarray datasets with a limited number of samples, high rates of missing data, or very noisy measurements. With the rapid accumulation of microarray datasets, the performance of our approach can be further improved by incorporating larger and more appropriate reference datasets.

  14. Integrative missing value estimation for microarray data

    Directory of Open Access Journals (Sweden)

    Zhou Xianghong

    2006-10-01

    Full Text Available Abstract Background Missing value estimation is an important preprocessing step in microarray analysis. Although several methods have been developed to solve this problem, their performance is unsatisfactory for datasets with high rates of missing data, high measurement noise, or limited numbers of samples. In fact, more than 80% of the time-series datasets in Stanford Microarray Database contain less than eight samples. Results We present the integrative Missing Value Estimation method (iMISS by incorporating information from multiple reference microarray datasets to improve missing value estimation. For each gene with missing data, we derive a consistent neighbor-gene list by taking reference data sets into consideration. To determine whether the given reference data sets are sufficiently informative for integration, we use a submatrix imputation approach. Our experiments showed that iMISS can significantly and consistently improve the accuracy of the state-of-the-art Local Least Square (LLS imputation algorithm by up to 15% improvement in our benchmark tests. Conclusion We demonstrated that the order-statistics-based integrative imputation algorithms can achieve significant improvements over the state-of-the-art missing value estimation approaches such as LLS and is especially good for imputing microarray datasets with a limited number of samples, high rates of missing data, or very noisy measurements. With the rapid accumulation of microarray datasets, the performance of our approach can be further improved by incorporating larger and more appropriate reference datasets.

  15. DNA arrays : methods and protocols [Methods in molecular biology, v. 170

    National Research Council Canada - National Science Library

    Rampal, Jang B

    2001-01-01

    "In DNA Arrays: Methods and Protocols, Jang Rampal and a authoritative panel of researchers, engineers, and technologists explain in detail how to design and construct DNA microarrays, as well as how to...

  16. SNPMClust: Bivariate Gaussian Genotype Clustering and Calling for Illumina Microarrays

    Directory of Open Access Journals (Sweden)

    Stephen W. Erickson

    2016-07-01

    Full Text Available SNPMClust is an R package for genotype clustering and calling with Illumina microarrays. It was originally developed for studies using the GoldenGate custom genotyping platform but can be used with other Illumina platforms, including Infinium BeadChip. The algorithm first rescales the fluorescent signal intensity data, adds empirically derived pseudo-data to minor allele genotype clusters, then uses the package mclust for bivariate Gaussian model fitting. We compared the accuracy and sensitivity of SNPMClust to that of GenCall, Illumina's proprietary algorithm, on a data set of 94 whole-genome amplified buccal (cheek swab DNA samples. These samples were genotyped on a custom panel which included 1064 SNPs for which the true genotype was known with high confidence. SNPMClust produced uniformly lower false call rates over a wide range of overall call rates.

  17. Discovering biological progression underlying microarray samples.

    Directory of Open Access Journals (Sweden)

    Peng Qiu

    2011-04-01

    Full Text Available In biological systems that undergo processes such as differentiation, a clear concept of progression exists. We present a novel computational approach, called Sample Progression Discovery (SPD, to discover patterns of biological progression underlying microarray gene expression data. SPD assumes that individual samples of a microarray dataset are related by an unknown biological process (i.e., differentiation, development, cell cycle, disease progression, and that each sample represents one unknown point along the progression of that process. SPD aims to organize the samples in a manner that reveals the underlying progression and to simultaneously identify subsets of genes that are responsible for that progression. We demonstrate the performance of SPD on a variety of microarray datasets that were generated by sampling a biological process at different points along its progression, without providing SPD any information of the underlying process. When applied to a cell cycle time series microarray dataset, SPD was not provided any prior knowledge of samples' time order or of which genes are cell-cycle regulated, yet SPD recovered the correct time order and identified many genes that have been associated with the cell cycle. When applied to B-cell differentiation data, SPD recovered the correct order of stages of normal B-cell differentiation and the linkage between preB-ALL tumor cells with their cell origin preB. When applied to mouse embryonic stem cell differentiation data, SPD uncovered a landscape of ESC differentiation into various lineages and genes that represent both generic and lineage specific processes. When applied to a prostate cancer microarray dataset, SPD identified gene modules that reflect a progression consistent with disease stages. SPD may be best viewed as a novel tool for synthesizing biological hypotheses because it provides a likely biological progression underlying a microarray dataset and, perhaps more importantly, the

  18. Analysis of DNA Methylation in Young People: Limited Evidence for an Association Between Victimization Stress and Epigenetic Variation in Blood.

    Science.gov (United States)

    Marzi, Sarah J; Sugden, Karen; Arseneault, Louise; Belsky, Daniel W; Burrage, Joe; Corcoran, David L; Danese, Andrea; Fisher, Helen L; Hannon, Eilis; Moffitt, Terrie E; Odgers, Candice L; Pariante, Carmine; Poulton, Richie; Williams, Benjamin S; Wong, Chloe C Y; Mill, Jonathan; Caspi, Avshalom

    2018-01-12

    DNA methylation has been proposed as an epigenetic mechanism by which early-life experiences become "embedded" in the genome and alter transcriptional processes to compromise health. The authors sought to investigate whether early-life victimization stress is associated with genome-wide DNA methylation. The authors tested the hypothesis that victimization is associated with DNA methylation in the Environmental Risk (E-Risk) Longitudinal Study, a nationally representative 1994-1995 birth cohort of 2,232 twins born in England and Wales and assessed at ages 5, 7, 10, 12, and 18 years. Multiple forms of victimization were ascertained in childhood and adolescence (including physical, sexual, and emotional abuse; neglect; exposure to intimate-partner violence; bullying; cyber-victimization; and crime). Epigenome-wide analyses of polyvictimization across childhood and adolescence revealed few significant associations with DNA methylation in peripheral blood at age 18, but these analyses were confounded by tobacco smoking and/or did not survive co-twin control tests. Secondary analyses of specific forms of victimization revealed sparse associations with DNA methylation that did not replicate across different operationalizations of the same putative victimization experience. Hypothesis-driven analyses of six candidate genes in the stress response (NR3C1, FKBP5, BDNF, AVP, CRHR1, SLC6A4) did not reveal predicted associations with DNA methylation in probes annotated to these genes. Findings from this epidemiological analysis of the epigenetic effects of early-life stress do not support the hypothesis of robust changes in DNA methylation in victimized young people. We need to come to terms with the possibility that epigenetic epidemiology is not yet well matched to experimental, nonhuman models in uncovering the biological embedding of stress.

  19. 3D Biomaterial Microarrays for Regenerative Medicine

    DEFF Research Database (Denmark)

    Gaharwar, Akhilesh K.; Arpanaei, Ayyoob; Andresen, Thomas Lars

    2015-01-01

    Three dimensional (3D) biomaterial microarrays hold enormous promise for regenerative medicine because of their ability to accelerate the design and fabrication of biomimetic materials. Such tissue-like biomaterials can provide an appropriate microenvironment for stimulating and controlling stem...... for tissue engineering and drug screening applications....... cell differentiation into tissue-specifi c lineages. The use of 3D biomaterial microarrays can, if optimized correctly, result in a more than 1000-fold reduction in biomaterials and cells consumption when engineering optimal materials combinations, which makes these miniaturized systems very attractive...

  20. Temporal aspects of DNA and RNA synthesis during human immunodeficiency virus infection: Evidence for differential gene expression

    International Nuclear Information System (INIS)

    Kim, Sunyoung; Baltimore, D.; Byrn, R.; Groopman, J.

    1989-01-01

    The kinetics of retroviral DNA and RNA synthesis are parameters vital to understanding viral growth, especially for human immunodeficiency virus (HIV), which encodes several of its own regulatory genes. The authors have established a single-cycle growth condition for HIV in H9 cells, a human CD4 + lymphocyte line. The full-length viral linear DNA is first detectable by 4 h postinfection. During a one-step growth of HIV, amounts of viral DNA gradually increase until 8 to 12 h postinfection and then decrease. The copy number of unintegrated viral DNA is not extraordinarily high even at its peak. Most strikingly, there is a temporal program of RNA accumulation: the earliest RNA is greatly enriched in the 2-kilobase subgenomic mRNA species, while the level of 9.2-kilobase RNA which is both genomic RNA and mRNA remains low until after 24 h of infection. Virus production begins at about 24 h postinfection. Thus, viral DNA synthesis is as rapid as for other retroviruses, but viral RNA synthesis involves temporal alteration in the species that accumulate, presumably as a consequence of viral regulatory genes

  1. Functional Characterization of Gibberellin-Regulated Genes in Rice Using Microarray System

    OpenAIRE

    Jan, Asad; Komatsu, Setsuko

    2006-01-01

    Gibberellin (GA) is collectively referred to a group of diterpenoid acids, some of which act as plant hormones and are essential for normal plant growth and development. DNA microarray technology has become the standard tool for the parallel quantification of large numbers of messenger RNA transcripts. The power of this approach has been demonstrated in dissecting plant physiology and development, and in unraveling the underlying cellular signaling pathways. To understand the molecular mechan...

  2. Comparison of small n statistical tests of differential expression applied to microarrays

    Directory of Open Access Journals (Sweden)

    Lee Anna Y

    2009-02-01

    Full Text Available Abstract Background DNA microarrays provide data for genome wide patterns of expression between observation classes. Microarray studies often have small samples sizes, however, due to cost constraints or specimen availability. This can lead to poor random error estimates and inaccurate statistical tests of differential expression. We compare the performance of the standard t-test, fold change, and four small n statistical test methods designed to circumvent these problems. We report results of various normalization methods for empirical microarray data and of various random error models for simulated data. Results Three Empirical Bayes methods (CyberT, BRB, and limma t-statistics were the most effective statistical tests across simulated and both 2-colour cDNA and Affymetrix experimental data. The CyberT regularized t-statistic in particular was able to maintain expected false positive rates with simulated data showing high variances at low gene intensities, although at the cost of low true positive rates. The Local Pooled Error (LPE test introduced a bias that lowered false positive rates below theoretically expected values and had lower power relative to the top performers. The standard two-sample t-test and fold change were also found to be sub-optimal for detecting differentially expressed genes. The generalized log transformation was shown to be beneficial in improving results with certain data sets, in particular high variance cDNA data. Conclusion Pre-processing of data influences performance and the proper combination of pre-processing and statistical testing is necessary for obtaining the best results. All three Empirical Bayes methods assessed in our study are good choices for statistical tests for small n microarray studies for both Affymetrix and cDNA data. Choice of method for a particular study will depend on software and normalization preferences.

  3. Spatial positioning of all 24 chromosomes in the lymphocytes of six subjects: evidence of reproducible positioning and spatial repositioning following DNA damage with hydrogen peroxide and ultraviolet B.

    Directory of Open Access Journals (Sweden)

    Dimitrios Ioannou

    Full Text Available The higher-order organization of chromatin is well-established, with chromosomes occupying distinct positions within the interphase nucleus. Chromatin is susceptible to, and constantly assaulted by both endogenous and exogenous threats. However, the effects of DNA damage on the spatial topology of chromosomes are hitherto, poorly understood. This study investigates the organization of all 24 human chromosomes in lymphocytes from six individuals prior to- and following in-vitro exposure to genotoxic agents: hydrogen peroxide and ultraviolet B. This study is the first to report reproducible distinct hierarchical radial organization of chromosomes with little inter-individual differences between subjects. Perturbed nuclear organization was observed following genotoxic exposure for both agents; however a greater effect was observed for hydrogen peroxide including: 1 More peripheral radial organization; 2 Alterations in the global distribution of chromosomes; and 3 More events of chromosome repositioning (18 events involving 10 chromosomes vs. 11 events involving 9 chromosomes for hydrogen peroxide and ultraviolet B respectively. Evidence is provided of chromosome repositioning and altered nuclear organization following in-vitro exposure to genotoxic agents, with notable differences observed between the two investigated agents. Repositioning of chromosomes following genotoxicity involved recurrent chromosomes and is most likely part of the genomes inherent response to DNA damage. The variances in nuclear organization observed between the two agents likely reflects differences in mobility and/or decondensation of chromatin as a result of differences in the type of DNA damage induced, chromatin regions targeted, and DNA repair mechanisms.

  4. A salmonid EST genomic study: genes, duplications, phylogeny and microarrays

    Directory of Open Access Journals (Sweden)

    Brahmbhatt Sonal

    2008-11-01

    Full Text Available Abstract Background Salmonids are of interest because of their relatively recent genome duplication, and their extensive use in wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different species provide valuable genomic information for one of the most widely studied groups of fish. Results 298,304 expressed sequence tags (ESTs from Atlantic salmon (69% of the total, 11,664 chinook, 10,813 sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in this study and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmon clones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, gene predictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbow trout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4, 92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchus and Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae. Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of the available EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-species hybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species. Conclusion An extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon, Atlantic salmon and charr are 94–96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and 86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is

  5. Microarray Я US: a user-friendly graphical interface to Bioconductor tools that enables accurate microarray data analysis and expedites comprehensive functional analysis of microarray results.

    Science.gov (United States)

    Dai, Yilin; Guo, Ling; Li, Meng; Chen, Yi-Bu

    2012-06-08

    Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results. We present Microarray Я US, an R-based graphical user interface that implements over a dozen popular Bioconductor packages to offer researchers a streamlined workflow for routine differential microarray expression data analysis without the need to learn R language. In order to enable a more accurate analysis and interpretation of microarray data, we incorporated the latest custom probe re-definition and re-annotation for Affymetrix and Illumina chips. A versatile microarray results output utility tool was also implemented for easy and fast generation of input files for over 20 of the most widely used functional analysis software programs. Coupled with a well-designed user interface, Microarray Я US leverages cutting edge Bioconductor packages for researchers with no knowledge in R language. It also enables a more reliable and accurate microarray data analysis and expedites downstream functional analysis of microarray results.

  6. Molecular phylogeny of grey mullets (Teleostei: Mugilidae) in Greece: evidence from sequence analysis of mtDNA segments.

    Science.gov (United States)

    Papasotiropoulos, Vasilis; Klossa-Kilia, Elena; Alahiotis, Stamatis N; Kilias, George

    2007-08-01

    Mitochondrial DNA sequence analysis has been used to explore genetic differentiation and phylogenetic relationships among five species of the Mugilidae family, Mugil cephalus, Chelon labrosus, Liza aurata, Liza ramada, and Liza saliens. DNA was isolated from samples originating from the Messolongi Lagoon in Greece. Three mtDNA segments (12s rRNA, 16s rRNA, and CO I) were PCR amplified and sequenced. Sequencing analysis revealed that the greatest genetic differentiation was observed between M. cephalus and all the other species studied, while C. labrosus and L. aurata were the closest taxa. Dendrograms obtained by the neighbor-joining method and Bayesian inference analysis exhibited the same topology. According to this topology, M. cephalus is the most distinct species and the remaining taxa are clustered together, with C. labrosus and L. aurata forming a single group. The latter result brings into question the monophyletic origin of the genus Liza.

  7. Evaluation of the performance of a p53 sequencing microarray chip using 140 previously sequenced bladder tumor samples

    DEFF Research Database (Denmark)

    Wikman, Friedrik; Lu, Ming-Lan; Andersen, Thomas Thykjær

    2000-01-01

    sensitivity, from 0.92 to 0.84, leading to a much better concordance (92%) with results obtained by traditional sequencing. The chip method detected as little as 1% mutated DNA. Conclusions: Microarray-based sequencing is a novel option to assess TP53 mutations, representing a fast and inexpensive method...

  8. Gene targeting associated with the radiation sensitivity in squamous cell carcinoma by using microarray analysis

    International Nuclear Information System (INIS)

    Nimura, Yoshinori; Kumagai, Ken; Kouzu, Yoshinao; Higo, Morihiro; Kato, Yoshikuni; Seki, Naohiko; Yamada, Shigeru

    2005-01-01

    In order to identify a set of genes related to radiation sensitivity of squamous cell carcinoma (SCC) and establish a predictive method, we compared expression profiles of radio-sensitive/radio-resistant SCC cell lines, using the in-house cDNA microarray consisting of 2,201 human genes derived from full-length enriched SCC cDNA libraries and the Human oligo chip 30 K (Hitachi Software Engineering). Surviving fractions (SF) after irradiation of heavy iron were calculated by colony formation assay. Three pairs (TE2-TE13, YES5-YES6, and HSC3-HSC2), sensitive (SF1 0.6), were selected for the microarray analysis. The results of cDNA microarray analysis showed that 20 genes in resistant cell lines and 5 genes in sensitive cell lines were up regulated more than 1.5-fold compared with sensitive and resistant cell lines respectively. Fourteen out of 25 genes were confirmed the gene expression profiles by real-time polymerase chain reaction (PCR). Twenty-seven genes identified by Human oligo chip 30 K are candidate for the markers to distinguish radio-sensitive from radio-resistant. These results suggest that the isolated 27 genes are the candidates that might be used as specific molecular markers to predict radiation sensitivity. (author)

  9. A non-parametric meta-analysis approach for combining independent microarray datasets: application using two microarray datasets pertaining to chronic allograft nephropathy

    Directory of Open Access Journals (Sweden)

    Archer Kellie J

    2008-02-01

    Full Text Available Abstract Background With the popularity of DNA microarray technology, multiple groups of researchers have studied the gene expression of similar biological conditions. Different methods have been developed to integrate the results from various microarray studies, though most of them rely on distributional assumptions, such as the t-statistic based, mixed-effects model, or Bayesian model methods. However, often the sample size for each individual microarray experiment is small. Therefore, in this paper we present a non-parametric meta-analysis approach for combining data from independent microarray studies, and illustrate its application on two independent Affymetrix GeneChip studies that compared the gene expression of biopsies from kidney transplant recipients with chronic allograft nephropathy (CAN to those with normal functioning allograft. Results The simulation study comparing the non-parametric meta-analysis approach to a commonly used t-statistic based approach shows that the non-parametric approach has better sensitivity and specificity. For the application on the two CAN studies, we identified 309 distinct genes that expressed differently in CAN. By applying Fisher's exact test to identify enriched KEGG pathways among those genes called differentially expressed, we found 6 KEGG pathways to be over-represented among the identified genes. We used the expression measurements of the identified genes as predictors to predict the class labels for 6 additional biopsy samples, and the predicted results all conformed to their pathologist diagnosed class labels. Conclusion We present a new approach for combining data from multiple independent microarray studies. This approach is non-parametric and does not rely on any distributional assumptions. The rationale behind the approach is logically intuitive and can be easily understood by researchers not having advanced training in statistics. Some of the identified genes and pathways have been

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

    Directory of Open Access Journals (Sweden)

    Nathan D Grubaugh

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

  11. In silico design and performance of peptide microarrays for breast cancer tumour-auto-antibody testing

    Directory of Open Access Journals (Sweden)

    Andreas Weinhäusel

    2012-06-01

    Full Text Available The simplicity and potential of minimally invasive testing using sera from patients makes auto-antibody based biomarkers a very promising tool for use in cancer diagnostics. Protein microarrays have been used for the identification of such auto-antibody signatures. Because high throughput protein expression and purification is laborious, synthetic peptides might be a good alternative for microarray generation and multiplexed analyses. In this study, we designed 1185 antigenic peptides, deduced from proteins expressed by 642 cDNA expression clones found to be sero-reactive in both breast tumour patients and controls. The sero-reactive proteins and the corresponding peptides were used for the production of protein and peptide microarrays. Serum samples from females with benign and malignant breast tumours and healthy control sera (n=16 per group were then analysed. Correct classification of the serum samples on peptide microarrays were 78% for discrimination of ‘malignant versus healthy controls’, 72% for ‘benign versus malignant’ and 94% for ‘benign versus controls’. On protein arrays, correct classification for these contrasts was 69%, 59% and 59%, respectively. The over-representation analysis of the classifiers derived from class prediction showed enrichment of genes associated with ribosomes, spliceosomes, endocytosis and the pentose phosphate pathway. Sequence analyses of the peptides with the highest sero-reactivity demonstrated enrichment of the zinc-finger domain. Peptides’ sero-reactivities were found negatively correlated with hydrophobicity and positively correlated with positive charge, high inter-residue protein contact energies and a secondary structure propensity bias. This study hints at the possibility of using in silico designed antigenic peptide microarrays as an alternative to protein microarrays for the improvement of tumour auto-antibody based diagnostics.

  12. AffyMiner: mining differentially expressed genes and biological knowledge in GeneChip microarray data

    Directory of Open Access Journals (Sweden)

    Xia Yuannan

    2006-12-01

    Full Text Available Abstract Background DNA microarrays are a powerful tool for monitoring the expression of tens of thousands of genes simultaneously. With the advance of microarray technology, the challenge issue becomes how to analyze a large amount of microarray data and make biological sense of them. Affymetrix GeneChips are widely used microarrays, where a variety of statistical algorithms have been explored and used for detecting significant genes in the experiment. These methods rely solely on the quantitative data, i.e., signal intensity; however, qualitative data are also important parameters in detecting differentially expressed genes. Results AffyMiner is a tool developed for detecting differentially expressed genes in Affymetrix GeneChip microarray data and for associating gene annotation and gene ontology information with the genes detected. AffyMiner consists of the functional modules, GeneFinder for detecting significant genes in a treatment versus control experiment and GOTree for mapping genes of interest onto the Gene Ontology (GO space; and interfaces to run Cluster, a program for clustering analysis, and GenMAPP, a program for pathway analysis. AffyMiner has been used for analyzing the GeneChip data and the results were presented in several publications. Conclusion AffyMiner fills an important gap in finding differentially expressed genes in Affymetrix GeneChip microarray data. AffyMiner effectively deals with multiple replicates in the experiment and takes into account both quantitative and qualitative data in identifying significant genes. AffyMiner reduces the time and effort needed to compare data from multiple arrays and to interpret the possible biological implications associated with significant changes in a gene's expression.

  13. SCK-CEN Genomic Platform: the microarray technology

    International Nuclear Information System (INIS)

    Benotmane, R.

    2006-01-01

    The human body contains approximately 10 14 cells, wherein each one is a nucleus. The nucleus contains 2x23 chromosomes, or two complete sets of the human genome, one set coming from the mother and the other from the father. In principle each set includes 30.000-40.000 genes. If the genome was a book, it would be twenty-three chapters, called chromosomes,each chapter with several thousand stories, called genes. Each story made up of paragraphs, called exons and introns. Each paragraph made up of 3 letter words, called codons. Each word is written with letters called bases (AGCT). But the whole is written in a single very long sentence, which is the DNA molecule or deoxy nucleic acid. The usual state of DNA is two complementary strands intertwined forming a double helix. In the cell, DNA is duplicated during each cell division to ensure the transmission of the genome to the daughter cells. For expression, the DNA is transcribed to messenger RNA. The RNA is edited and finally translated to a protein, each three bases coding for one amino acid. When the whole message is translated, the chain of amino acids folds itself up into a distinctive shape that depends on its sequence. Proteins are the effectors of the genes, and are responsible for all metabolic, hormonal and enzymatic reactions in the cells. The expressed RNA determines the amount of proteins to be produced and subsequently the desired effect (strong or weak) in the cell. The microarray technology aims at quantifying the amount of RNA present in the cell from each expressed gene, and at evaluating the changes of these amounts after exposure of the cell to toxic chemicals, ionising radiation or other stress components. The global picture of expressed genes helps to understand the affected genetic pathways in the cell at the molecular level. The microarray technology is used in the Radiobiology and Microbiology topics to study the effect of ionising radiation on human cells and mouse tissue, as well as the

  14. PERBANDINGAN QUANTUM CLUSTERING DAN SUPPORT VECTOR CLUSTERING UNTUK DATA MICROARRAY EXPRESSION YEAST CELL DALAM RUANG SINGULAR VALUE DECOMPOSITION (SVD)

    OpenAIRE

    ., Riwinoto

    2013-01-01

    Sekarang ini, metode clustering telah diimplementasikan dalam riset DNA. Data dari DNA didapat melalui teknik microarray. Dengan menggunakan metode teknik SVD, dimensi data dikurangi sehingga mempermudah proses komputasi. Dalam paper ini, ditampilkan hasil clustering tanpa pengarahan terhadap gen-gen dari data bakteri ragi dengan menggunakan metode quantum clustering. Sebagai pembanding, dilakukan juga clustering menggunakan metoda Support Vector Clustering. Selain itu juga ditampilkan data h...

  15. Detection of selected plant viruses by microarrays

    OpenAIRE

    HRABÁKOVÁ, Lenka

    2013-01-01

    The main aim of this master thesis was the simultaneous detection of four selected plant viruses ? Apple mosaic virus, Plum pox virus, Prunus necrotic ringspot virus and Prune harf virus, by microarrays. The intermediate step in the process of the detection was optimizing of multiplex polymerase chain reaction (PCR).

  16. Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

    Science.gov (United States)

    Sitaraman, Kalavathy; Chatterjee, Deb K

    2011-01-01

    In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.

  17. Intron loss from the NADH dehydrogenase subunit 4 gene of lettuce mitochondrial DNA: evidence for homologous recombination of a cDNA intermediate.

    Science.gov (United States)

    Geiss, K T; Abbas, G M; Makaroff, C A

    1994-04-01

    The mitochondrial gene coding for subunit 4 of the NADH dehydrogenase complex I (nad4) has been isolated and characterized from lettuce, Lactuca sativa. Analysis of nad4 genes in a number of plants by Southern hybridization had previously suggested that the intron content varied between species. Characterization of the lettuce gene confirms this observation. Lettuce nad4 contains two exons and one group IIA intron, whereas previously sequenced nad4 genes from turnip and wheat contain three group IIA introns. Northern analysis identified a transcript of 1600 nucleotides, which represents the mature nad4 mRNA and a primary transcript of 3200 nucleotides. Sequence analysis of lettuce and turnip nad4 cDNAs was used to confirm the intron/exon border sequences and to examine RNA editing patterns. Editing is observed at the 5' and 3' ends of the lettuce transcript, but is absent from sequences that correspond to exons two, three and the 5' end of exon four in turnip and wheat. In contrast, turnip transcripts are highly edited in this region, suggesting that homologous recombination of an edited and spliced cDNA intermediate was involved in the loss of introns two and three from an ancestral lettuce nad4 gene.

  18. Facilitating functional annotation of chicken microarray data

    Directory of Open Access Journals (Sweden)

    Gresham Cathy R

    2009-10-01

    Full Text Available Abstract Background Modeling results from chicken microarray studies is challenging for researchers due to little functional annotation associated with these arrays. The Affymetrix GenChip chicken genome array, one of the biggest arrays that serve as a key research tool for the study of chicken functional genomics, is among the few arrays that link gene products to Gene Ontology (GO. However the GO annotation data presented by Affymetrix is incomplete, for example, they do not show references linked to manually annotated functions. In addition, there is no tool that facilitates microarray researchers to directly retrieve functional annotations for their datasets from the annotated arrays. This costs researchers amount of time in searching multiple GO databases for functional information. Results We have improved the breadth of functional annotations of the gene products associated with probesets on the Affymetrix chicken genome array by 45% and the quality of annotation by 14%. We have also identified the most significant diseases and disorders, different types of genes, and known drug targets represented on Affymetrix chicken genome array. To facilitate functional annotation of other arrays and microarray experimental datasets we developed an Array GO Mapper (AGOM tool to help researchers to quickly retrieve corresponding functional information for their dataset. Conclusion Results from this study will directly facilitate annotation of other chicken arrays and microarray experimental datasets. Researchers will be able to quickly model their microarray dataset into more reliable biological functional information by using AGOM tool. The disease, disorders, gene types and drug targets revealed in the study will allow researchers to learn more about how genes function in complex biological systems and may lead to new drug discovery and development of therapies. The GO annotation data generated will be available for public use via AgBase website and

  19. Microarray Study of Pathway Analysis Expression Profile Associated with MicroRNA-29a with Regard to Murine Cholestatic Liver Injuries

    Directory of Open Access Journals (Sweden)

    Sung-Chou Li

    2016-03-01

    Full Text Available Accumulating evidence demonstrates that microRNA-29 (miR-29 expression is prominently decreased in patients with hepatic fibrosis, which consequently stimulates hepatic stellate cells’ (HSCs activation. We used a cDNA microarray study to gain a more comprehensive understanding of genome-wide gene expressions by adjusting miR-29a expression in a bile duct-ligation (BDL animal model. Methods: Using miR-29a transgenic mice and wild-type littermates and applying the BDL mouse model, we characterized the function of miR-29a with regard to cholestatic liver fibrosis. Pathway enrichment analysis and/or specific validation were performed for differentially expressed genes found within the comparisons. Results: Analysis of the microarray data identified a number of differentially expressed genes due to the miR-29a transgene, BDL, or both. Additional pathway enrichment analysis revealed that TGF-β signaling had a significantly differential activated pathway depending on the occurrence of miR-29a overexpression or the lack thereof. Furthermore, overexpression was found to elicit changes in Wnt/β-catenin after BDL. Conclusion: This study verified that an elevated miR-29a level could alleviate liver fibrosis caused by cholestasis. Furthermore, the protective effects of miR-29a correlate with the downregulation of TGF-β and associated with Wnt/β-catenin signal pathway following BDL.

  20. Evaluation by microarray of the potential safety of Sarracenia purpurea L. (Sarraceniaceae) a traditional medicine used by the Cree of Eeyou Istchee.

    Science.gov (United States)

    Cieniak, Carolina; McDonald, Charlotte; Nash, John; Muhammad, Asim; Badawi, Alaa; Haddad, Pierre S; Cuerrier, Alain; Bennett, Steffany A L; Foster, Brian C; Arnason, John T

    2015-01-01

    The purpose of this study was to assess safety of the traditional antidiabetic extracts of either S. purpurea or its lead active principle, morroniside at the transcriptional level. The overarching objective was to profile and validate transcriptional changes in the cytochrome P450 family of genes, in response to treatment with S. purpurea ethanolic extract or its lead active, morroniside. Transcriptional activity was profiled using a 19K human cDNA microarray in C2BBe1 cells, clone of Caco-2 intestinal cells, which are a model of first-pass metabolism (1, 2). Cells were treated with S. purpurea extract for 4 and 24 hrs, as well as the pure compound morroniside for 4 hrs, to determine their effects. No evidence of cytochrome P450 transcriptome regulation or of transcriptional activation of other diabetes relevant mRNA was detected after rigorous quantitative-PCR validation of microarray results. Our data do not support a transcriptional mechanism of action for either S. purpurea extract or its lead active, morroniside. This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.

  1. Liver ultrastructural morphology and mitochondrial DNA levels in HIV/hepatitis C virus coinfection: no evidence of mitochondrial damage with highly active antiretroviral therapy.

    Science.gov (United States)

    Matsukura, Motoi; Chu, Fanny F S; Au, May; Lu, Helen; Chen, Jennifer; Rietkerk, Sonja; Barrios, Rolando; Farley, John D; Montaner, Julio S; Montessori, Valentina C; Walker, David C; Côté, Hélène C F

    2008-06-19

    Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy.

  2. Preliminary study of mechanism of action of SN38 derivatives. Physicochemical data, evidence of interaction and alkylation of DNA octamer d(GCGATCGC)2.

    Science.gov (United States)

    Naumczuk, Beata; Kawęcki, Robert; Bocian, Wojciech; Bednarek, Elżbieta; Sitkowski, Jerzy; Kozerski, Lech

    2017-02-01

    The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC) 2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  3. Profiling cellular protein complexes by proximity ligation with dual tag microarray readout.

    Science.gov (United States)

    Hammond, Maria; Nong, Rachel Yuan; Ericsson, Olle; Pardali, Katerina; Landegren, Ulf

    2012-01-01

    Patterns of protein interactions provide important insights in basic biology, and their analysis plays an increasing role in drug development and diagnostics of disease. We have established a scalable technique to compare two biological samples for the levels of all pairwise interactions among a set of targeted protein molecules. The technique is a combination of the proximity ligation assay with readout via dual tag microarrays. In the proximity ligation assay protein identities are encoded as DNA sequences by attaching DNA oligonucleotides to antibodies directed against the proteins of interest. Upon binding by pairs of antibodies to proteins present in the same molecular complexes, ligation reactions give rise to reporter DNA molecules that contain the combined sequence information from the two DNA strands. The ligation reactions also serve to incorporate a sample barcode in the reporter molecules to allow for direct comparison between pairs of samples. The samples are evaluated using a dual tag microarray where information is decoded, revealing which pairs of tags that have become joined. As a proof-of-concept we demonstrate that this approach can be used to detect a set of five proteins and their pairwise interactions both in cellular lysates and in fixed tissue culture cells. This paper provides a general strategy to analyze the extent of any pairwise interactions in large sets of molecules by decoding reporter DNA strands that identify the interacting molecules.

  4. Micrococcus luteus correndonucleases. III. Evidence for involvement in repair in vivo of two endonucleases specific for DNA containing pyrimidine dimers

    International Nuclear Information System (INIS)

    Riazuddin, S.; Grossman, L.; Mahler, I.

    1977-01-01

    Involvement of Py--Py correndonucleases I and II in repair of ultraviolet radiation damage in vivo by Micrococcus luteus has been demonstrated by their absence in the ultraviolet-sensitive mutant DB-7 derived by treatment of the wild type parent with N-methyl-N'-nitro-N-nitrosoguanidine. The necessity for their combined action in DNA repair in M. luteus is shown by: (a) reactivation of ultraviolet-damaged phiX174 RFI DNA in incision-defective hosts after in vivo treatment with both enzymes, (b) correlation between survival after ultraviolet irradiation and the level of the two enzymes, and (c) increased levels of repair synthesis after ultraviolet irradiation of toluenized cells DB-400 with wild type correndonuclease levels when compared with the transformant DB-200 and the mutant DB-7, which lack one or both enzymes

  5. Contribution of sleep to the repair of neuronal DNA double-strand breaks: evidence from flies and mice.

    Science.gov (United States)

    Bellesi, Michele; Bushey, Daniel; Chini, Mattia; Tononi, Giulio; Cirelli, Chiara

    2016-11-10

    Exploration of a novel environment leads to neuronal DNA double-strand breaks (DSBs). These DSBs are generated by type 2 topoisomerase to relieve topological constrains that limit transcription of plasticity-related immediate early genes. If not promptly repaired, however, DSBs may lead to cell death. Since the induction of plasticity-related genes is higher in wake than in sleep, we asked whether it is specifically wake associated with synaptic plasticity that leads to DSBs, and whether sleep provides any selective advantage over wake in their repair. In flies and mice, we find that enriched wake, more than simply time spent awake, induces DSBs, and their repair in mice is delayed or prevented by subsequent wake. In both species the repair of irradiation-induced neuronal DSBs is also quicker during sleep, and mouse genes mediating the response to DNA damage are upregulated in sleep. Thus, sleep facilitates the repair of neuronal DSBs.

  6. Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants

    International Nuclear Information System (INIS)

    Kaefer, E.; Mayor, O.

    1986-01-01

    To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or γ-rays. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. (Auth.)

  7. Saw palmetto alters nuclear measurements reflecting DNA content in men with symptomatic BPH: evidence for a possible molecular mechanism.

    Science.gov (United States)

    Veltri, Robert W; Marks, Leonard S; Miller, M Craig; Bales, Wes D; Fan, John; Macairan, Maria Luz; Epstein, Jonathan I; Partin, Alan W

    2002-10-01

    To examine the nuclear chromatin characteristics of epithelial cells, looking for an SPHB-mediated effect on nuclear DNA structure and organization. Saw palmetto herbal blend (SPHB) causes contraction of prostate epithelial cells and suppression of tissue dihydrotestosterone levels in men with symptomatic benign prostatic hyperplasia, but a fundamental mechanism remains unknown. A 6-month randomized trial, comparing prostatic tissue of men treated with SPHB (n = 20) or placebo (n = 20), was performed. At baseline, the two groups were similar in age (65 versus 64 years), symptoms (International Prostate Symptom Score 18 versus 17), uroflow (maximal urinary flow rate 10 versus 11 mL/s), prostate volume (59 versus 58 cm(3)), prostate-specific antigen (4.2 versus 2.7 ng/mL), and percentage of epithelium (17% versus 16%). Prostatic tissue was obtained by sextant biopsy before and after treatment. Five-micron sections were Feulgen stained and quantitatively analyzed using the AutoCyte QUIC-DNA imaging system. Images were captured from 200 randomly selected epithelial cell nuclei, and 60 nuclear morphometric descriptors (NMDs) (eg, size, shape, DNA content, and textural features) were determined for each nucleus. Logistic regression analysis was used to assess the differences in the variances of the NMDs between the treated and untreated prostate epithelial cells. At baseline, the SPHB and placebo groups had similar NMD values. After 6 months of placebo, no significant change from baseline was found in the NMDs. However, after 6 months of SPHB, 25 of the 60 NMDs were significantly different compared with baseline, and a multivariate model for predicting treatment effect using 4 of the 25 was created (P <0.001). The multivariate model had an area under the receiver operating characteristic curve of 94% and an accuracy of 85%. Six months of SPHB treatment appears to alter the DNA chromatin structure and organization in prostate epithelial cells. Thus, a possible molecular

  8. Contribution of sleep to the repair of neuronal DNA double-strand breaks: evidence from flies and mice

    OpenAIRE

    Bellesi, Michele; Bushey, Daniel; Chini, Mattia; Tononi, Giulio; Cirelli, Chiara

    2016-01-01

    Exploration of a novel environment leads to neuronal DNA double-strand breaks (DSBs). These DSBs are generated by type 2 topoisomerase to relieve topological constrains that limit transcription of plasticity-related immediate early genes. If not promptly repaired, however, DSBs may lead to cell death. Since the induction of plasticity-related genes is higher in wake than in sleep, we asked whether it is specifically wake associated with synaptic plasticity that leads to DSBs, and whether slee...

  9. Tertiary montane origin of the Central Asian flora, evidence inferred from cpDNA sequences of Atraphaxis (Polygonaceae)

    Science.gov (United States)

    Ming-Li Zhang; Stewart C. Sanderson; Yan-Xia Sun; Byalt V. Vyacheslav; Xiao-Li Hao

    2014-01-01

    Atraphaxis has approximately 25 species and a distribution center in Central Asia. It has been previously used to hypothesize an origin from montane forest. We sampled 18 species covering three sections within the genus and sequenced five cpDNA spacers, atpB-rbcL, psbK-psbI, psbAtrnH, rbcL, and trnL-trnF. BEAST was used to reconstruct phylogenetic relationship and time...