96,054 research outputs found
Bridging Functional Genomics and Toxicogenomics Through DNA Microarrays in a Fish Model
In a case study of finding gene expression signatures for environmental stressors in Cyprinodon variegatus, this dissertation examines several important issues of applying DNA microarray technology to fish toxicogenomics. The most relevant disciplines, fish toxicogenomics and computational systems biology are reviewed in Chapter 1. Chapter 2 reviews major aspects of DNA microarray technology.
On DNA microarrays, even for probes that target the same transcript, large variations are seen in the probe signals. These variations are partly dependent and partly independent on probe sequences. Chapter 3 estimates the sequence independent variation by combining experimental and computational approaches. Chapter 4 and 5 take on the central problem of sequence dependent variations, that is, modeling the physiochemistry of microarray hybridization. I propose a new competitive hybridization model, which demonstrates good success on publically available benchmark data. This new model leads the way to quantification of absolute target concentration, and brings critical insights into probe design and data interpretation on DNA microarrays. Our model relies on the accuracy of computing duplexing energy, which does yet not take into account secondary structures of probes and targets. I further explore the structural effects in Chapter 6.
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Single-molecule super-resolution imaging of chromosomes and in situ haplotype visualization using Oligopaint FISH probes
Fluorescence in situ hybridization (FISH) is a powerful single-cell technique for studying nuclear structure and organization. Here we report two advances in FISH-based imaging. We first describe the in situ visualization of single-copy regions of the genome using two single-molecule super-resolution methodologies. We then introduce a robust and reliable system that harnesses single-nucleotide polymorphisms (SNPs) to visually distinguish the maternal and paternal homologous chromosomes in mammalian and insect systems. Both of these new technologies are enabled by renewable, bioinformatically designed, oligonucleotide-based Oligopaint probes, which we augment with a strategy that uses secondary oligonucleotides (oligos) to produce and enhance fluorescent signals. These advances should substantially expand the capability to query parent-of-origin-specific chromosome positioning and gene expression on a cell-by-cell basis
A novel cassette method for probe evaluation in the designed biochips
A critical step in biochip design is the selection of probes with identical hybridisation characteristics. In this article we describe a novel method for evaluating DNA hybridisation probes, allowing the fine-tuning of biochips, that uses cassettes with multiple probes. Each cassette contains probes in equimolar proportions so that their hybridisation performance can be assessed in a single reaction. The model used to demonstrate this method was a series of probes developed to detect TORCH pathogens. DNA probes were designed for Toxoplasma gondii, Chlamidia trachomatis, Rubella, Cytomegalovirus, and Herpes virus and these were used to construct the DNA cassettes. Five cassettes were constructed to detect TORCH pathogens using a variety of genes coding for membrane proteins, viral matrix protein, an early expressed viral protein, viral DNA polymerase and the repetitive gene B1 of Toxoplasma gondii. All of these probes, except that for the B1 gene, exhibited similar profiles under the same hybridisation conditions. The failure of the B1 gene probe to hybridise was not due to a position effect, and this indicated that the probe was unsuitable for inclusion in the biochip. The redesigned probe for the B1 gene exhibited identical hybridisation properties to the other probes, suitable for inclusion in a biochip
Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts
A microarray spotted with 369 different 16S rRNA gene probes specific to microorganisms involved in the degradation process of organic waste during composting was developed. The microarray was tested with pure cultures, and of the 30,258 individual probe-target hybridization reactions performed, there were only 188 false positive (0.62%) and 22 false negative signals (0.07%). Labeled target DNA was prepared by polymerase chain reaction amplification of 16S rRNA genes using a Cy5-labeled universal bacterial forward primer and a universal reverse primer. The COMPOCHIP microarray was applied to three different compost types (green compost, manure mix compost, and anaerobic digestate compost) of different maturity (2, 8, and 16 weeks), and differences in the microorganisms in the three compost types and maturity stages were observed. Multivariate analysis showed that the bacterial composition of the three composts was different at the beginning of the composting process and became more similar upon maturation. Certain probes (targeting Sphingobacterium, Actinomyces, Xylella/Xanthomonas/ Stenotrophomonas, Microbacterium, Verrucomicrobia, Planctomycetes, Low G + C and Alphaproteobacteria) were more influential in discriminating between different composts. Results from denaturing gradient gel electrophoresis supported those of microarray analysis. This study showed that the COMPOCHIP array is a suitable tool to study bacterial communities in composts
Specific discrimination of three pathogenic salmonella enterica subsp enterica serotypes using CarB-based oligonuceotide microarray
It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.open117Nsciescopu
Compressive Sensing DNA Microarrays
Compressive sensing microarrays (CSMs) are DNA-based sensors that operate using group testing and compressive sensing (CS) principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed
Nanostructured luminescently labeled nucleic acids
Important and emerging trends at the interface of luminescence, nucleic acids and nanotechnology
are: (i) the conventional luminescence labeling of nucleic acid nanostructures (e.g. DNA tetrahedron);
(ii) the labeling of bulk nucleic acids (e.g. single‐stranded DNA, double‐stranded DNA) with
nanostructured luminescent labels (e.g. copper nanoclusters); and (iii) the labeling of nucleic acid
nanostructures (e.g. origami DNA) with nanostructured luminescent labels (e.g. silver
nanoclusters). This review surveys recent advances in these three different approaches to the
generation of nanostructured luminescently labeled nucleic acids, and includes both direct and
indirect labeling methods
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Three-dimensional modeling of single stranded DNA hairpins for aptamer-based biosensors.
Aptamers consist of short oligonucleotides that bind specific targets. They provide advantages over antibodies, including robustness, low cost, and reusability. Their chemical structure allows the insertion of reporter molecules and surface-binding agents in specific locations, which have been recently exploited for the development of aptamer-based biosensors and direct detection strategies. Mainstream use of these devices, however, still requires significant improvements in optimization for consistency and reproducibility. DNA aptamers are more stable than their RNA counterparts for biomedical applications but have the disadvantage of lacking the wide array of computational tools for RNA structural prediction. Here, we present the first approach to predict from sequence the three-dimensional structures of single stranded (ss) DNA required for aptamer applications, focusing explicitly on ssDNA hairpins. The approach consists of a pipeline that integrates sequentially building ssDNA secondary structure from sequence, constructing equivalent 3D ssRNA models, transforming the 3D ssRNA models into ssDNA 3D structures, and refining the resulting ssDNA 3D structures. Through this pipeline, our approach faithfully predicts the representative structures available in the Nucleic Acid Database and Protein Data Bank databases. Our results, thus, open up a much-needed avenue for integrating DNA in the computational analysis and design of aptamer-based biosensors
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Comprehensive sequence-to-function mapping of cofactor-dependent RNA catalysis in the glmS ribozyme.
Massively parallel, quantitative measurements of biomolecular activity across sequence space can greatly expand our understanding of RNA sequence-function relationships. We report the development of an RNA-array assay to perform such measurements and its application to a model RNA: the core glmS ribozyme riboswitch, which performs a ligand-dependent self-cleavage reaction. We measure the cleavage rates for all possible single and double mutants of this ribozyme across a series of ligand concentrations, determining kcat and KM values for active variants. These systematic measurements suggest that evolutionary conservation in the consensus sequence is driven by maintenance of the cleavage rate. Analysis of double-mutant rates and associated mutational interactions produces a structural and functional mapping of the ribozyme sequence, revealing the catalytic consequences of specific tertiary interactions, and allowing us to infer structural rearrangements that permit certain sequence variants to maintain activity
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