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At least 325 records · Page 18Linked to original sources
PubMed2003

A generic high-throughput screening assay for kinases: protein kinase a as an example.

A generic high-throughput screening assay based on the scintillation proximity assay technology has been developed for protein kinases. In this assay, the biotinylated (33)P-peptide product is captured onto polylysine Ysi bead via avidin. The scintillation signal measuring the product formation increases linearly with avidin concentration due to effective capture of the product on the bead surface via strong coulombic interactions. This novel assay has been optimized and validated in 384-well microplates. In a pilot screen, a signal-to-noise ratio of 5- to 9-fold and a Z' factor ranging from 0.6 to 0.8 were observed, demonstrating the suitability of this assay for high-throughput screening of random chemical libraries for kinase inhibitors.

Adenosine Triphosphate↗
PubMed2006

Development of a Drosophila seizure model for in vivo high-throughput drug screening.

An important application of model organisms in neurological research has been to identify and characterise therapeutic approaches for epilepsy, a recurrent seizure disorder that affects > 1% of the human population. Proconvulsant-treated rodent models have been widely used for antiepileptic drug discovery and development, but are not suitable for high-throughput screening. To generate a genetically tractable model that would be suitable for large-scale, high-throughput screening for antiepileptic drug candidates, we characterized a Drosophila chemical treatment model using the GABA(A) receptor antagonist picrotoxin. This proconvulsant, delivered to Drosophila larvae via simple feeding methods suitable for automated screening, generated robust generalised seizures with lethality occurring at doses between 0.3 and 0.5 mg/mL. Electrophysiological analysis of CNS motor neuron output in picrotoxin-treated larvae revealed generalised seizures within minutes of drug exposure. At subthreshold doses for seizure induction, picrotoxin produced an increased frequency of motor neuron action potential bursting, indicating that CNS GABAergic transmission regulates patterned activity. Mutants in the Drosophila Rdl GABA(A) receptor are resistant to picrotoxin, confirming that seizure induction occurs via a conserved GABA(A) receptor pathway. To validate the usefulness of this model for in vivo drug screening, we identified several classes of neuroactive antiepileptic compounds in a pilot screen, including phenytoin and nifedipine, which can rescue the seizures and lethal neurotoxicity induced by picrotoxin. The well-defined actions of picrotoxin in Drosophila and the ease with which compounds can be assayed for antiseizure activity makes this genetically tractable model attractive for high-throughput in vivo screens to identify novel anticonvulsants and seizure susceptibility loci.

Amino Acid Sequence↗
PubMed2003

Capillary electrophoresis-based single strand DNA conformation analysis in high-throughput mutation screening.

The generation of the draft human genome sequence has created new possibilities for diagnosis, prevention, and treatment of human disease. One consequence of these new possibilities is an increasing need for methods and technology that can be used for high-throughput screening for mutations in large DNA sample materials. In recent years, a number of mutation screening methods have emerged that are based on the analysis of sequence-dependent changes in the conformation of single- and double-stranded DNA using capillary electrophoresis. Common features of these methods are high sensitivity and reproducibility as well as the possibility for automation and massive parallelization. Thus, at present they are among the most attractive technologies for high-throughput mutation screening. This review describes the recent advances in capillary electrophoresis-based single strand conformation polymorphism (CE-SSCP) for detection of unknown mutations, and assesses its practical usability for high-throughput mutation screening based on the available literature. In addition, future prospects are outlined in light of the recent advances in microchip-based capillary electrophoresis.

DNA Mutational Analysis↗
PubMed2006

[A universal high-throughput novel method of constructing the vectors].

In this paper, a simple universal high-throughput method of constructing the vectors was developed. It could add proper adapter when the PCR primers were designed, and the purpose fragments were cloned by PCR, and the various complementary sticky ends were created by T4 DNA polymerase's 3' -exodeoxyribonuclease activity. If all these fragments were put together with DNA ligase, they would recombinate in an orientation. If they had been transformated, the tansformants would be identified. Let's take the Oryza sativa single-cross homologous recombination chloroplast expression vector pRSMGA which was constructed with seven fragments as an example, if the vector pRSMGA was constructed in using the method what had mentioned, only twice recombination and transformation would be done. Scores of experiments had proved that it is a simple universal high-throughput novel method to construct the complicated vectors, which has not appeared in the periodical.

Genetic Vectors↗
PubMed2005

Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection.

BACKGROUND: As we enter the post-genome sequencing era and begin to sift through the enormous amount of genetic information now available, the need for technologies that allow rapid, cost-effective, high-throughput detection of specific nucleic acid sequences becomes apparent. Multiplexing technologies, which allow for simultaneous detection of multiple nucleic acid sequences in a single reaction, can greatly reduce the time, cost and labor associated with single reaction detection technologies. METHODS: The Luminex xMAP system is a multiplexed microsphere-based suspension array platform capable of analyzing and reporting up to 100 different reactions in a single reaction vessel. This technology provides a new platform for high-throughput nucleic acid detection and is being utilized with increasing frequency. Here we review specific applications of xMAP technology for nucleic acid detection in the areas of single nucleotide polymorphism (SNP) genotyping, genetic disease screening, gene expression profiling, HLA DNA typing and microbial detection. CONCLUSIONS: These studies demonstrate the speed, efficiency and utility of xMAP technology for simultaneous, rapid, sensitive and specific nucleic acid detection, and its capability to meet the current and future requirements of the molecular laboratory for high-throughput nucleic acid detection.

Animals↗
PubMed2000

Automated high-throughput liquid-liquid extraction for initial purification of combinatorial libraries.

An automated high-throughput liquid-liquid extraction (LLE) methodology has been developed and utilized for the initial purification of the combinatorial library samples containing unreacted amines and other water-soluble byproducts or impurities. Various extraction solvents were evaluated along with different extraction devices. The LLE method was automated using 96-well-format plates and a robotic liquid-handling workstation. In the optimized LLE method, crude combinatorial library samples were dissolved in a water-immiscible organic solvent, butyl acetate, and added to each well in a 96-well-format plate packed with an inert support material coated with hydrochloric acid. Separation occurs based on the partitioning of the compounds between two liquid phases. Product recovery, purity, and amine removal efficiency were determined by HPLC with and without precolumn derivatization. The automated method was successfully applied to the cleanup of some representative combinatorial library samples with greater than 98% amine removal and an average product purity of 90%. The application of the automated high-throughput LLE method should greatly reduce the labor, time, and cost associated with the purification of combinatorial libraries.

Amines↗
PubMed2001

PCR candidate region mismatch scanning: adaptation to quantitative, high-throughput genotyping.

Linkage and association analyses were performed to identify loci affecting disease susceptibility by scoring previously characterized sequence variations such as microsatellites and single nucleotide polymorphisms. Lack of markers in regions of interest, as well as difficulty in adapting various methods to high-throughput settings, often limits the effectiveness of the analyses. We have adapted the Escherichia coli mismatch detection system, employing the factors MutS, MutL and MutH, for use in PCR-based, automated, high-throughput genotyping and mutation detection of genomic DNA. Optimal sensitivity and signal-to-noise ratios were obtained in a straightforward fashion because the detection reaction proved to be principally dependent upon monovalent cation concentration and MutL concentration. Quantitative relationships of the optimal values of these parameters with length of the DNA test fragment were demonstrated, in support of the translocation model for the mechanism of action of these enzymes, rather than the molecular switch model. Thus, rapid, sequence-independent optimization was possible for each new genomic target region. Other factors potentially limiting the flexibility of mismatch scanning, such as positioning of dam recognition sites within the target fragment, have also been investigated. We developed several strategies, which can be easily adapted to automation, for limiting the analysis to intersample heteroduplexes. Thus, the principal barriers to the use of this methodology, which we have designated PCR candidate region mismatch scanning, in cost-effective, high-throughput settings have been removed.

Adenosine Triphosphatases↗
PubMed2005

High-throughput sequencing: a failure mode analysis.

BACKGROUND: Basic manufacturing principles are becoming increasingly important in high-throughput sequencing facilities where there is a constant drive to increase quality, increase efficiency, and decrease operating costs. While high-throughput centres report failure rates typically on the order of 10%, the causes of sporadic sequencing failures are seldom analyzed in detail and have not, in the past, been formally reported. RESULTS: Here we report the results of a failure mode analysis of our production sequencing facility based on detailed evaluation of 9,216 ESTs generated from two cDNA libraries. Two categories of failures are described; process-related failures (failures due to equipment or sample handling) and template-related failures (failures that are revealed by close inspection of electropherograms and are likely due to properties of the template DNA sequence itself). CONCLUSIONS: Preventative action based on a detailed understanding of failure modes is likely to improve the performance of other production sequencing pipelines.

Automation↗
PubMed2002

Adapting chromophore-assisted laser inactivation for high throughput functional proteomics.

Recent advances in genomics and proteomics have generated a change in emphasis from hypothesis-based to discovery-based investigations. Genomic and proteomic studies based on differential expression microarrays or comparative proteomics often provide many potential candidates for functionally important roles in normal and diseased cells. High throughput technologies to address protein and gene function in situ are still necessary to exploit these emerging advances in gene and protein discovery in order to validate these identified targets. The pharmaceutical industry is particularly interested in target validation, and has identified it as the critical early step in drug discovery. An especially powerful approach to target validation is a direct protein knockdown strategy called chromophore-assisted laser inactivation (CALI) which is a means of testing the role of specific proteins in particular cellular processes. Recent developments in CALI allow for its high throughput application to address many proteins in tandem. Thus, CALI may have applications for high throughput hypothesis testing, target validation or proteome-wide screening.

Drug Industry↗
PubMed2004

Stationary phases with special structural properties for high-throughput separation techniques: preparation, characterization and applications.

Stationary phases with specific structural properties for high-throughput liquid chromatographic (LC) techniques are described. Special attention was paid to phases with special structural properties, mainly containing internal functional group (e.g. amide). Such materials are generally called "embedded phases". There are phases created in amidation process of aminopropylated silica gel, especially phases based on biological compounds, like phospholipids and cholesterol, which are called immobilized artificial membranes (IAM's). The synthesis and applications of polar embedded amide LC stationary phases were also reviewed. Methods of characterization of synthesized packing materials were presented, with general focusing on spectroscopic measurements like (13C and 29Si CP/MAS NMR and FT-IR), elemental and thermal analysis as well as chromatographic quantitative structure-retention relationships (QSRR) and extended chemometric tests. The potential applications of various dedicated stationary phases in a high-throughput LC screening procedures were also presented.

Absorption↗
PubMed2006

Development and application of a high-throughput assay for glmS riboswitch activators.

Riboswitches are newly-discovered gene control elements that are promising targets for antibacterial drug development. To facilitate the rapid discovery and development of riboswitch-targeted compounds, modern drug discovery techniques such as structure-based design and high-throughput screening will need to be applied. One promising riboswitch drug target is the glmS riboswitch, which upon binding glucosamine-6-phosphate (GlcN6P) functions as a ribozyme and catalyzes self-cleavage. Herein we report the development of a high-throughput assay for glmS ribozyme cleavage that relies on fluorescence resonance energy transfer (FRET). This assay can be used to screen for compounds that bind to and activate glmS ribozyme cleavage. To validate the screen, we demonstrate that the assay can identify the active compounds from a library of GlcN6P analogs whose affinities for ribozyme were determined by commonly used electrophoretic methods with radiolabeled RNA. Furthermore, the primary screen of a library of 960 compounds previously approved for use in humans identified five active compounds, one of which is a GlcN6P analog known to stimulate ribozyme activity. These results demonstrate that modern high-throughput screening techniques can be applied to the discovery of riboswitch-targeted drug compounds.

Bacillus subtilis↗
PubMed2002

High-throughput analytical techniques for reaction optimization.

This review describes recent developments in approaches to high-throughput reaction optimization, as well as the associated analytical techniques. The studies discussed include the use of UV-visible, IR-thermographic and mass spectrometric methods for application in catalyst development, process optimization and materials science. Other methods of potential use in a high-throughput format are also discussed.

Combinatorial Chemistry Techniques↗
PubMed2001

Development of a high throughput equilibrium dialysis method.

The identification of large numbers of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with drug-like properties early during the lead prioritization and development processes. One of the major steps in lead prioritization is an assessment of compound binding to plasma proteins, because it affects both the pharmacokinetics and pharmacodynamics of the compound in vivo. Equilibrium dialysis is the preferred method to determine the free drug fraction, because it is less susceptible to experimental artifacts. However, even low-volume standard equilibrium dialysis is currently not amenable to the HTS format. Those considerations dictate the development of a high throughput equilibrium dialysis device, without compromising the analytical quality of the data. The present paper demonstrates successful development of a 96-well format equilibrium dialysis plate. Plasma protein binding of three drugs, propranolol, paroxetine, and losartan, with low, intermediate, and high binding properties, respectively, were chosen for assay validation. The data indicate that the apparent free fraction obtained by this method correlates with the published values determined by the traditional equilibrium dialysis techniques.

Antidepressive Agents↗
PubMed2003

A homogeneous 384-well high-throughput binding assay for a TNF receptor using alphascreen technology.

To take advantage of the growing knowledge of cellular signaling pathways, modern-day drug discovery faces an increasing challenge to develop assays to screen for compounds that modulate protein-protein interactions. One bottleneck in achieving this goal is a lack of suitable and robust assay technologies amenable to a high-throughput format. In this report, we describe how we utilized Alphascreen trade mark technology to develop a high-throughput assay to monitor ligand binding to a member of the tumor necrosis factor receptor superfamily. We expressed a fusion protein consisting of the extracellular domain of the OX40 receptor with the constant domains of human IgG. In the presence of OX40 ligand, we determined a binding affinity constant consistent with reported values and optimized the protocol to develop a simple, homogeneous, and sensitive binding assay in a 384-well format. Finally, we assessed if this system could identify small peptides capable of inhibiting the OX40 receptor and ligand interaction. The results showed that the assay was able to detect such peptides and could be used to launch a high-throughput screening campaign for small molecules able to prevent OX40 receptor activation.

Biochemistry↗
PubMed2002

Enantioselective hydrogenation of 3-alkylidenelactams: high-throughput screening provides a surprising solution.

High-throughput screening of 256 potential catalysts (8 metal precursors x 32 phosphine ligands) has identified [(BDPP)Ir(COD)]BF4 as a catalyst for the enantioselective hydrogenation of 3-alkylidenelactams. This result is surprising given the highly flexible backbone of the BDPP ligand and the ineffectiveness of this catalyst in other applications. The asymmetric hydrogenation appears fairly general for five- and six-membered lactams and in one case has been scaled up to a 20 kg level.

Alkadienes↗
PubMed2005

High-throughput RNAi screening in vitro: from cell lines to primary cells.

Small interfering RNAs (siRNAs) are being used to induce sequence-specific gene silencing in cultured cells to study mammalian gene function. Libraries of siRNAs targeting entire human gene classes can be used to identify genes with specific cellular functions. Here we describe high-throughput siRNA delivery methods to facilitate siRNA library screening experiments with both immortalized and primary cells. We adapted chemical reverse transfection for immortalized adherent cell lines in a 96-well format. The method is fast, robust, and exceptionally effective for many cell types. For primary cells and immortalized cells that are recalcitrant to lipofection-based methods, we developed electropermeabilization (electroporation) conditions that facilitate siRNA delivery to a broad range of cell types, including primary human T-cells, hMSC, NHA, NDHF-Neo, HUVEC, DI TNC1, RPTEC, PC12, and K562 cells. To enable high-throughput electropermeabilization of primary cells, we developed a novel 96-well electroporation device that provides highly efficient and reproducible delivery of siRNAs. The combination of high-throughput chemical reverse transfection and electroporation makes it possible to deliver libraries of siRNAs to virtually any cell type, enabling gene function analysis and discovery on a genome scale.

Animals↗
PubMed2000

Catalytic antibodies that hydrolyze (-)-cocaine obtained by a high-throughput procedure.

Antibodies to a 2beta-carboxamido-2beta-phosphonate transition-state analog of (-)-cocaine benzoate ester hydrolysis were elicited in mice. A large number of hybridoma cell lines were propagated, and the catalytic activity of culture fluid was determined with a high-throughput photometric assay using cocaine benzoyl thioester as substrate. Binding avidity of the hybridoma supernatants to the phosphonate hapten was also determined. The initial rate constants for cocaine benzoyl thioester hydrolysis and binding avidity for a large number of hybridoma supernatants elicited to the phosphonate hapten did not always correlate. The lack of correlation of substrate hydrolysis with the binding affinity of 70 catalytic antibodies was also observed for (-)-cocaine hydrolysis using derivatization and HPLC analysis of methyl ecgonine as meta-nitrococaine. The k(cat) values for cocaine benzoyl thioester hydrolysis by monoclonal antibodies 3, 5, and 12 were 38, 4.2, and 0. 6 min(-1), respectively. For monoclonal antibody 5, the selectivity ratios (K(i) value divided by the K(m) value for the hydrolysis of cocaine benzoyl thioester) with ecgonine benzoyl ester, ecgonine methyl ester, norcocaine, and ecgonine were 101, 25, 9.4, and 4, respectively. Three active esterolytic monoclonal antibodies identified with the high-throughput assay procedure were examined in detail for their ability to hydrolyze (-)-cocaine. The k(cat) values for the hydrolysis of (-)-cocaine with monoclonal antibodies 3, 5, and 12 were 6.6, 0.4, and 0.1 min(-1), respectively. Hydrolysis of (-)-cocaine by monoclonal antibody 3 approached the k(cat) value for that of human butyrylcholinesterase. Cocaine esterolytic catalytic antibodies that approach or exceed the catalytic efficiency of human butyrylcholinesterase may represent a new pharmacological intervention approach to the treatment of cocaine abuse, and the high-throughput process described here represents an advance in the effort to develop clinically useful antibodies.

Animals↗
PubMed2006

High throughput microsomal stability assay for insoluble compounds.

High throughput metabolic stability assays are widely implemented in drug discovery to guide structural modification, predict in vivo performance, develop structure-metabolic stability relationships, and triage compounds for in vivo animal studies. However, these methods are often developed and validated using commercial drugs. Many drug discovery compounds differ from commercial drugs, with many having high lipophilicity, high molecular weight and low solubility. The impact of very low solubility on metabolic stability assay results was explored. Two metabolic stability assays, the 'aqueous dilution method' and the 'cosolvent method, were compared. For commercial drugs and most discovery compounds having reasonable drug-like properties, the two methods gave comparable results. For highly lipophilic, insoluble drug discovery compounds, the 'aqueous dilution method' gave artificially higher stability results. The cosolvent method performs compound dilutions in solutions with higher organic solvent content and adds solutions directly to microsomes to assist with solubilization, minimize precipitation and reduce non-specific binding to plastics. This method is more applicable in drug discovery where compounds of a wide range of solubility are studied.

Acetonitriles↗