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At least 667 records · Page 37Linked to original sources
PubMed2006

On-line hyphenation of flow injection, miniaturized capillary electrophoresis and atomic fluorescence spectrometry for high-throughput speciation analysis.

A hyphenated technique was developed for high-throughput speciation analysis by on-line coupling of flow injection (FI), miniaturized capillary electrophoresis (CE) and atomic fluorescence spectrometry (AFS). Two interfaces were used to couple all three systems: the first to couple FI and CE and the second to couple miniaturized CE and AFS. The first interface was a modified flow through chamber, connected to the FI valve with a piece of PTFE tube (0.1mm i.d.x 20 cm long). The capillary outlet was coupled to the AFS by using the second concentric "tube-in-tube" interface. Split sampling was achieved in the electrokinetic mode. Inorganic mercury (Hg(II)) and methylmercury (MeHg(I)) were taken as model analytes to demonstrate the performance of the developed hyphenated technique. A volatile species generation (VSG) technique was employed to convert the analytes from the CE effluent into their respective volatile species. Baseline separation of Hg(II) and MeHg(I) was achieved by CE in a 50 microm i.d.x 8 cm long capillary at 3.0 kV within 60s. The precisions (RSD, n=12) were in the range of 0.7-0.9% for migration time, 3.8-4.2% for peak area, and 2.1-3.5% for peak height. The detection limits were 0.1 and 0.2 microgmL(-1) (as Hg) for Hg(II) and MeHg(I) with a sample throughput of 60 samples h(-1). The recoveries of both mercury species in the water samples studied were in the range of 93-106%.

Electrophoresis, Capillary↗
PubMed2005

High throughput assay for the determination of lumefantrine in plasma.

A high throughput bioanalytical assay for the determination of lumefantrine in plasma has been developed and validated extensively. The within-day precisions for lumefantrine were 5.2, 3.5 and 2.5% at 200, 2000 and 15000 ng/mL, respectively. The between-day precisions were 4.0, 2.8 and 3.1% at 200, 2000 and 15000 ng/mL, respectively. The lower limits of quantification (LLOQ) and the limits of detection (LOD) were 25 and 10 ng/mL, respectively using 0.250 mL plasma. The average recovery of lumefantrine was 85% and independent upon concentration. The use of 96-well plate format and short chromatographic run has increased the daily sample throughput four times. The assay is particularly suitable for large therapeutic drug monitoring studies using day 7 sampling.

Antimalarials↗
PubMed2005

High throughput assay for the determination of piperaquine in plasma.

A high throughput assay for the determination of the antimalarial piperaquine in plasma has been developed and validated. The assay utilises 96-wellplate formats throughout the whole procedure, and easily enables a throughput of 192 samples a day using a single LC system. Buffer (pH 2.0; 0.05 M) containing internal standard was added to 0.25 mL plasma in a 96-wellplate (2 mL wells). The samples were extracted on a MPC solid phase extraction deep well 96-wellplate (3M Empore). Piperaquine and internal standard were analysed by liquid chromatography with UV detection on a Chromolith Performance (100 mm x 4.6 mm) column with a mobile phase containing acetonitrile-phosphate buffer (pH 2.5; 0.1 M) (8:92, v/v) at a flow rate of 3.0 mL/min. The within-day precisions for piperaquine were 3.3 and 2.3% at 40 and 1250 ng/mL, respectively. The between-day precisions for piperaquine were 5.8 and 1.3% at 40 and 1250 ng/mL, respectively. The total assay precisions using 29 replicates over 5 days were 6.7, 4.5 and 2.7% at 40, 200 and 1250 ng/mL, respectively. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 10 and 5 ng/mL, respectively using 0.25 mL plasma. Using 1 mL of plasma, it was possible to decrease LLOQ and LOD to 2.5 and 1.25 ng/mL, respectively.

Animals↗
PubMed2000

High-throughput single-molecule spectroscopy in free solution.

A high-speed high-throughput single-molecule imaging technique for identifying molecules in free solution based on differences in their fluorescence emission spectra is presented. Unlike previous reports, the entire spectrum, rather than selected wavelengths through optical filters, is recorded. Furthermore, the millisecond data acquisition time means that the molecules do not need to be immobilized or spatially confined. In one example, individual lambdaDNA molecules labeled with YOYO-I, POPO-III, or a combination of the two dyes can be distinguished from one another. In another example, biotinylated 2.1-kb DNA labeled with YOYO-I was reacted with avidin-conjugated R-phycoerythrin. The two different reactant molecules and the product molecule can be simultaneously imaged and identified by their spectroscopic characteristics. This technique can therefore be used for screening single molecules for disease markers and for monitoring individual molecular interactions at a rate of thousands of molecules per second.

Biotinylation↗
PubMedDate not supplied

Combinatorial materials research applied to the development of new surface coatings I: a multiwell plate screening method for the high-throughput assessment of bacterial biofilm retention on surfaces.

Combinatorial, high-throughput capabilities have been established to aid in the rapid development of new and effective antifouling marine coatings for naval applications. A biological screening process involving marine bacteria was developed that allows for rapid and effective quantification of bacterial biofilm growth and retention on large numbers of coating surfaces in parallel. The screening process involves (1) multiwell plate modifications for coating deposition, (2) deposition of combinatorial coating libraries via an automated liquid dispensing robot, (3) coating thickness measurements of cured coatings, (4) preconditioning of coatings via immersion in deionized water, (5) bacterial incubation, (6) plate processing, and (7) data analysis for identification of promising candidates. The details of the method developed are described in this document.

Automation↗
PubMed2006

Fluorescent cell barcoding in flow cytometry allows high-throughput drug screening and signaling profiling.

Flow cytometry allows high-content, multiparameter analysis of single cells, making it a promising tool for drug discovery and profiling of intracellular signaling. To add high-throughput capacity to flow cytometry, we developed a cell-based multiplexing technique called fluorescent cell barcoding (FCB). In FCB, each sample is labeled with a different signature, or barcode, of fluorescence intensity and emission wavelengths, and mixed with other samples before antibody staining and analysis by flow cytometry. Using three FCB fluorophores, we were able to barcode and combine entire 96-well plates, reducing antibody consumption 100-fold and acquisition time to 5-15 min per plate. Using FCB and phospho-specific flow cytometry, we screened a small-molecule library for inhibitors of T cell-receptor and cytokine signaling, simultaneously determining compound efficacy and selectivity. We also analyzed IFN-gamma signaling in multiple cell types from primary mouse splenocytes, revealing differences in sensitivity and kinetics between B cells, CD4+ and CD4- T cells and CD11b-hi cells.

Animals↗
PubMed2006

CART variance stabilization and regularization for high-throughput genomic data.

MOTIVATION: mRNA expression data obtained from high-throughput DNA microarrays exhibit strong departures from homogeneity of variances. Often a complex relationship between mean expression value and variance is seen. Variance stabilization of such data is crucial for many types of statistical analyses, while regularization of variances (pooling of information) can greatly improve overall accuracy of test statistics. RESULTS: A Classification and Regression Tree (CART) procedure is introduced for variance stabilization as well as regularization. The CART procedure adaptively clusters genes by variances. Using both local and cluster wide information leads to improved estimation of population variances which improves test statistics. Whereas making use of cluster wide information allows for variance stabilization of data. AVAILABILITY: Sufficient details for our CART procedure are given so that the interested reader can program the method for themselves. The algorithm is also accessible within the Java software package BAMarray(TM), which is freely available to non-commercial users at www.bamarray.com. CONTACT: hemant.ishwaran@gmail.com.

Artificial Intelligence↗
PubMed2004

Large-scale screening of Arabidopsis circadian clock mutants by a high-throughput real-time bioluminescence monitoring system.

Using a high-throughput real-time bioluminescence monitoring system, we screened large numbers of Arabidopsis thaliana mutants for extensively altered circadian rhythms. We constructed reporter genes by fusing a promoter of an Arabidopsis flowering-time gene - either GIGANTEA (GI) or FLOWERING LOCUS T (FT) - to a modified firefly luciferase gene (LUC(+)), and we transferred the fusion gene (P(GI)::LUC(+) or P(FT)::LUC(+)) into the Arabidopsis genome. After mutagenesis with ethyl methanesulfonate, 50 000 M(2) seedlings carrying the P(GI)::LUC(+) and 50 000 carrying P(FT)::LUC(+) were screened their bioluminescence rhythms. We isolated six arrhythmic (AR) mutants and 29 other mutants that showed more than 3 h difference in the period length or phase of rhythms compared with the wild-type strains. The shortest period length was 16 h, the longest 27 h. Five of the six AR mutants carrying P(GI)::LUC(+) showed arrhythmia in bioluminescence rhythms in both constant light and constant dark. These five AR mutants also showed arrhythmia in leaf movement rhythms in constant light. Genetic analysis revealed that each of the five AR mutants carried a recessive mutation in a nuclear gene and the mutations belonged to three complementation groups, and at least one of which was mapped on a novel locus. Our results suggest that the three loci identified here may contain central clock or clock-related genes, at least one of which may be a novel.

Arabidopsis↗
PubMed2006

NMR-based quality control approach for the identification of false positives and false negatives in high throughput screening.

The quality of the data generated in a high throughput screening (HTS) run is fundamental for selecting bona fide inhibitors and for ensuring the capture of the full richness of inhibitors present in a chemical library. For this purpose a quality control filter based on three one dimensional (1D) proton NMR experiments is proposed. The approach called SPAM (Solubility, Purity and Aggregation of the Molecule) Filter requires the acquisition of a 1D reference spectrum, a WaterLOGSY spectrum and/or a selective longitudinal relaxation filter spectrum for the identified hits dissolved in aqueous solution and in the presence of a water soluble reference molecule. This palette of experiments permits the rapid characterization of the identity, purity, solubility and aggregation state of the active compound. This knowledge is crucial for deriving accurate IC(50) and K(1) values of the inhibitors, for identifying false negatives and for detecting promiscuous inhibitors. Only compounds that pass through the SPAM Filter can be considered as starting points for medicinal chemistry efforts directed toward lead optimization. Examples of this approach in the identification of false positives in a screening run against the enzyme thymidine phosphorylase (TP) and the rescue of a false negative in a screening run against the Ser/Thr kinase AKT1 are presented.

Algorithms↗
PubMed2001

High-throughput genomic and proteomic analysis using microarray technology.

BACKGROUND: High-density microarrays are ideally suited for analyzing thousands of genes against a small number of samples. The next step in the discovery process is to take the resulting genes of interest and rapidly screen them against thousands of patient samples, tissues, or cell lines to further investigate their involvement in disease risk or the response to medication. METHODS: We used a microarray technology platform for both single-nucleotide polymorphisms (SNPs) and protein expression. Each microarray contains up to 250 elements that can be customized for each application. Slides contained either a 16- or 96-microarray format (4000-24,000 elements per slide), allowing the corresponding number of samples to be rapidly processed in parallel. RESULTS: Results for SNP genotyping and protein profiling agreed with results of restriction fragment length polymorphism (RFLP) analysis or ELISA, respectively. Genotyping analyses, using the microarray technology, on large sample sets over multiple polymorphisms in the NAT2 gene were in full agreement with traditional methodologies, such as sequencing and RFLP analysis. The multiplexed protein microarray had correlation coefficients of 0.82-0.99 (depending on analyte) compared with ELISAs. CONCLUSIONS: The integrated microarray technology platform is adaptable and versatile, while offering the high-throughput capabilities needed for drug development and discovery applications.

Arylamine N-Acetyltransferase↗
PubMed2002

Multiscale and Bayesian approaches to data analysis in genomics high-throughput screening.

Tremendous amounts of data are produced by high-throughput screening methods currently employed in drug discovery and product development. A typical cDNA microarray or oligonucleotide-based gene chip experiment easily generates over 10,000 data points for each array or chip. The challenge of inferring meaningful information is formidable given the size and number of these datasets. This paper reviews the current status of statistical tools available for gene expression analysis, with emphasis on Bayesian approaches and multiscale wavelet filtering. Fundamental concepts of Bayesian and multiscale modeling are discussed from the perspective of their potential to address important issues related to the analysis of gene expression data, such as the fact that genomic data often have non-Gaussian distributions and feature localization and multiple scales in both frequency and measurement dimension. Recent publications in these areas are reviewed. Wavelet filtering and the advantages of multiscale methods are demonstrated by application to publicly available gene expression data from the National Cancer Institute (NCI). Multiscale methods, including multiscale principal component analysis (MSPCA), are applied to extract gene subsets and to visualize data in multidimensions for comparisons. Similarity in cell lines and gene selection are effectively visualized and quantitatively compared.

Animals↗
PubMed2003

High throughput electrophysiology: new perspectives for ion channel drug discovery.

Proper function of ion channels is crucial for all living cells. Ion channel dysfunction may lead to a number of diseases, so-called channelopathies, and a number of common diseases, including epilepsy, arrhythmia, and type II diabetes, are primarily treated by drugs that modulate ion channels. A cornerstone in current drug discovery is high throughput screening assays which allow examination of the activity of specific ion channels though only to a limited extent. Conventional patch clamp remains the sole technique with sufficiently high time resolution and sensitivity required for precise and direct characterization of ion channel properties. However, patch clamp is a slow, labor-intensive, and thus expensive, technique. New techniques combining the reliability and high information content of patch clamping with the virtues of high throughput philosophy are emerging and predicted to make a number of ion channel targets accessible for drug screening. Specifically, genuine HTS parallel processing techniques based on arrays of planar silicon chips are being developed, but also lower throughput sequential techniques may be of value in compound screening, lead optimization, and safety screening. The introduction of new powerful HTS electrophysiological techniques is predicted to cause a revolution in ion channel drug discovery.

Animals↗
PubMed2002

ARACHNE: a whole-genome shotgun assembler.

We describe a new computer system, called ARACHNE, for assembling genome sequence using paired-end whole-genome shotgun reads. ARACHNE has several key features, including an efficient and sensitive procedure for finding read overlaps, a procedure for scoring overlaps that achieves high accuracy by correcting errors before assembly, read merger based on forward-reverse links, and detection of repeat contigs by forward-reverse link inconsistency. To test ARACHNE, we created simulated reads providing approximately 10-fold coverage of the genomes of H. influenzae, S. cerevisiae, and D. melanogaster, as well as human chromosomes 21 and 22. The assemblies of these simulated reads yielded nearly complete coverage of the respective genomes, with a small number of contigs joined into a smaller number of supercontigs (or scaffolds). For example, analysis of the D. melanogaster genome yielded approximately 98% coverage with an N50 contig length of 324 kb and an N50 supercontig length of 5143 kb. The assembly accuracy was high, although not perfect: small errors occurred at a frequency of roughly 1 per 1 Mb (typically, deletion of approximately 1 kb in size), with a very small number of other misassemblies. The assembly was rapid: the Drosophila assembly required only 21 hours on a single 667 MHz processor and used 8.4 Gb of memory.

Algorithms↗
PubMed2005

Facile, comprehensive, high-throughput genotyping of human genital papillomaviruses using spectrally addressable liquid bead microarrays.

Human papillomavirus (HPV) is the worldwide cause of carcinoma of the uterine cervix, a cancer that is the second most common neoplasm in women, resulting in nearly 250,000 deaths a year. The magnitude of the risk of cancer after HPV infection, however, is virus type-specific. Over 40 HPV types can infect the genital tract. Comprehensive, high-throughput typing assays for HPV, however, are not currently available. Blending multiplex PCR and multiplex hybridization using spectrally addressable liquid bead microarrays we have developed a high-throughput, fast, single-tube-typing assay capable of simultaneously typing 45 HPV. The overall incidence of HPV in 429 women tested using this new assay was 72.2% for those with squamous intraepithelial lesions, 51.5% for those with atypical squamous cells of undetermined significance and 15.4% for women with normal cytology, respectively. This compared well with the incidence of HPV detected by a parallel non-typing generic high-risk assay. The new assay detected a wide spectrum of HPV types and a high incidence of mixed infections. We believe our assay may find widespread applications in areas requiring virus type-specific information, such as in epidemiological studies, cancer screening programs, monitoring therapeutic interventions, and evaluating the efficacy of HPV vaccine trials.

DNA, Viral↗
PubMed2002

Functional genomics: high-throughput mRNA, protein, and metabolite analyses.

A tremendous amount of DNA sequence information is now available to scientists and engineers. These DNA sequences provide the foundation for studying how the genome of an organism is functioning and they are particularly useful for metabolic engineers interested in manipulating plants for the production of chemicals and enzymes. Functional genomics relies on high-throughput techniques for measuring the mRNA (the transcriptome), protein (the proteome), and metabolite (the metabolome) components of plants as well as their organs and tissues. Microarray technologies, recent advances in protein mass spectrometry, and high-throughput metabolite analyses are beginning to provide detailed information on the total mRNA, protein, and metabolite components of plants. This knowledge will allow scientists to monitor changes in proteins and metabolites in plants. Ultimately, it may allow them to discover new metabolic pathways and to model metabolic and regulatory networks in plants.

Base Sequence↗
PubMed2004

Analytical validation of the tag-it high-throughput microsphere-based universal array genotyping platform: application to the multiplex detection of a panel of thrombophilia-associated single-nucleotide polymorphisms.

BACKGROUND: We have developed a novel, microsphere-based universal array platform referred to as the Tag-It platform. This platform is suitable for high-throughput clinical genotyping applications and was used for multiplex analysis of a panel of thrombophilia-associated single-nucleotide polymorphisms (SNPs). METHODS: Genomic DNA from 132 patients was amplified by multiplex PCR using 6 primer sets, followed by multiplex allele-specific primer extension using 12 universally tagged genotyping primers. The products were then sorted on the Tag-It array and detected by use of the Luminex xMAP system. Genotypes were also determined by sequencing. RESULTS: Empirical validation of the universal array showed that the highest nonspecific signal was 3.7% of the specific signal. Patient genotypes showed 100% concordance with direct DNA sequencing data for 736 SNP determinations. CONCLUSIONS: The Tag-It microsphere-based universal array platform is a highly accurate, multiplexed, high-throughput SNP-detection platform.

Genotype↗
PubMed1995

High-throughput method for determination of apolipoprotein E genotypes with use of restriction digestion analysis by microplate array diagonal gel electrophoresis.

Molecular epidemiological research has identified the association of a common apolipoprotein E (apo E) isoform (E4 as opposed to E3), with risk both of coronary artery disease and of Alzheimer dementia. In addition, the role of apo E genotype (usually E2/E2) in Type III hyperlipidemia is well known. However, both for diagnostic and research purposes, apo E genotyping is cumbersome. The preferred approach is electrophoretic sizing of restriction digestion fragments, enabling simultaneous analysis of the two codons (112 and 158) that represent the six common genotypes (E2/E2; E2/E3; E2/E4; E3/E3; E3/E4; E4/E4). However, the consequent demands of high-yield PCR, high-resolution, high-throughput electrophoresis, and sufficient detection sensitivity have left shortfalls in published protocols. In conjunction with a high-throughput electrophoresis system we described recently, microplate array diagonal gel electrophoresis (MADGE), we have constructed extensively optimized, simplified protocols for DNA isolation from mouthwash samples for PCR setup and high-yield PCR, for restriction digestion, and for subsequent MADGE gel image analysis. The integral system enables one worker to readily undertake apo E genotyping of as many as hundreds of DNA samples per day, without special equipment.

Apolipoproteins E↗
PubMed2006

High-throughput assay of DNA methylation based on methylation-specific primer and SAGE.

Mapping of genomic DNA methylation is a dispensable part of functional genome. We have developed a novel method based on methylation-specific primer and serial analysis of gene expression, called MSP-SAGE, with potential of high-throughput quantification of genomic DNA methylation. We used a 6-mer methylation-specific primer to extend the methylated CpG sequences other than non-methylated CpG sequences. The 17 bp tags contained methylated CpG sequence, which were obtained from extended methylation sequence by digestion of restriction endonuclease, and then the tags were concatenated and cloned for sequencing. We can identify the locations of methylation according to the sequences of tags and quantify the methylation status from the frequency of the tags. MSP-SAGE has a good linearity in a broad methylation range from 5% to 100% with good accuracy and high precision. The proof-of-principle study shows that MSP-SAGE is a reliable high-throughput assay for quantification of DNA methylation.

Alleles↗