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At least 1,027 records · Page 57Linked to original sources
PubMed1997

A high throughput screening for rarely transcribed differentially expressed genes.

A novel method combining elements of suppression subtractive hybridization with high throughput differential screening permits the efficient and rapid cloning of rarely transcribed differentially expressed genes. The experimental strategy virtually excludes the possibility of isolating false positive clones. The potential of the method is demonstrated by the isolation of 625 differentially expressed cDNAs from the metastatic adenocarcinoma cell line Bsp73-ASML when subtracted from its non-metastatic counterpart Bsp73-1AS. Northern analysis of 72 randomly selected clones demonstrated that 68 were differentially expressed with respect to Bsp73-ASML, indicating a true positive rate of 94%. Additionally, a large proportion of these clones represented rare transcripts as determined by the exposure time required to detect a signal. Sequence data indicated that of the 625 clones obtained, 92 clones scored perfect or near perfect matches with already known genes. Two hundred and eighty one clones scored between 60 and 95% homology to known human and mouse genes, whereas 252 clones scored no match with any sequences in the public databases. The method we describe is ideally suited whenever subtle changes in gene expression profiles need to be determined.

Adenocarcinoma↗
PubMed2005

Crystal growth in X-ray-transparent plastic tubing: an alternative for high-throughput applications.

A modified capillary-growth method is described that has substantial advantages for standard and high-throughput protein crystal growth. Protein-containing drops are injected into vapor-permeable flexible X-ray-transparent polyester tubing. The protein concentration in the drop increases over time by water transport through the tubing wall at a rate controlled by the wall thickness and ambient relative humidity. Unlike in conventional vapor-diffusion growth, the evaporation rate from the drop is constant over a longer time period, providing more suitable conditions for nucleation, and can be controlled by varying the tubing thickness and surrounding humidity. In situ X-ray diffraction can be performed at room temperature or, by flash-cooling, at low temperatures. Compared with glass capillaries or thick-wall plastic tubing, sealing and handling the tubing and extracting crystals are much easier.

Crystallization↗
PubMed2001

A versatile high throughput screen for dioxygenase activity using solid-phase digital imaging.

We have developed a solid-phase, high throughput (10,000 clones/day) screen for dioxygenase activity. The cis-dihydrodiol product of dioxygenase bioconversion is converted to a phenol by acidification or to a catechol by reaction with cis-dihydrodiol dehydrogenase. Gibbs reagent reacts quickly with these oxygenated aromatics to yield colored products that are quantifiable using a microplate reader or by digital imaging and image analysis. The method is reproducible and quantitative at product concentrations of only 30 microM, with essentially no background from media components. This method is an effective general screen for aromatic oxidation and should be a useful tool for the discovery and directed evolution of oxygenases.

Automation↗
PubMed2002

Automated high throughput screening for serine kinase inhibitors using a LEADseeker scintillation proximity assay in the 1536-well format.

High-throughput screening in the 1536-well format has been largely restricted to solution-based and cell-based screens. In this article, we show the feasibility of a completely automated, robust scintillation proximity assay in the 1536-well format that is suitable to identify inhibitors for a serine/threonine kinase from a compound library. The introduction of [(33)P]phosphate into a biotinylated peptide substrate mirrors the activity of the kinase. The peptide is immobilized on streptavidin-coated LEADseeker imaging beads and [(33)P]phosphate incorporation is detected with the LEADseeker imaging system of Amersham Pharmacia Biotech. To improve the liquid handling procedures for imaging bead suspensions in the low microliter range, we developed a novel trough with an integrated stirring function. A comparison of the 1536-well assay to a 384-well assay revealed a comparable assay quality with Z' factors of about 0.7 for the 384-well format and 0.6 for the 1536-well format. In an automated screen of a random compound collection, 94.4% of the inhibitory compounds could be identified with both assay formats. Dose-response curves were performed for a selection of identified kinase inhibitors and revealed similar IC(50) values for both assay formats.

Automation↗
PubMed2002

A neutravidin-based assay for reverse transcriptase suitable for high throughput screening of retroviral activity.

A non-isotopic neutravidin-based reverse transcriptase (RT) assay adapted for high throughput screening of HIV activity is described. Using a 96-well microtitre plate, HIV particles are lysed and the RT enzyme released into a reaction mixture containing poly(A) RNA, biotinylated oligo d(T) and fluorescein-labelled dUTP (FI-dUTP). With poly(A) as a template and oligo d(T) as primer, the viron RT incorporates FI-dUTP into an elongating DNA strand. The resulting product is captured on a neutravidin-coated 96-well plate and the unincorporated nucleotides removed by a series of washing steps. A simple ELISA is subsequently performed using a monoclonal antifluorescein antibody conjugated to alkaline phosphatase. Quantification of RT activity is facilitated by a colorimetric readout. The assay was validated in the context of a diagnostic HIV-1 phenotyping assay. Using supernatants from HIV-1 infected lymphocyte cultures the assay was shown to be as sensitive as a radioactive assay and the RT activity correlated well with levels of cell-associated HIVp24. Importantly, even minor reductions of RT activity by virus variants with reduced fitness could be distinguished.

Avidin↗
PubMedDate not supplied

High-throughput purification of single compounds and libraries.

The need for increasing productivity in medicinal chemistry and associated improvements in automated synthesis technologies for compound library production during the past few years have resulted in a major challenge for compound purification technology and its organization. To meet this challenge, we have recently set up three full-service chromatography units with the aid of in-house engineers, different HPLC suppliers, and several companies specializing in custom laboratory automation technologies. Our goal was to combine high-throughput purification with the high attention to detail which would be afforded by a dedicated purification service. The resulting final purification laboratory can purify up to 1000 compounds/week in amounts ranging from 5 to 300 mg, whereas the two service intermediate purification units take 100 samples per week from 0.3 to 100 g. The technologies consist of normal-phase and reversed-phase chromatography, robotic fraction pooling and reformatting, a bottling system, an automated external solvent supply and removal system, and a customized, high-capacity freeze-dryer. All work processes are linked by an electronic sample registration and tracking system.

Automation↗
PubMed2005

High-throughput cloning for proteomics research.

Ligation-independent cloning (LIC) is a simple, rapid, and efficient method for high-throughput cloning. In this system, linear plasmid vector and insert DNA are treated to generate complementary single-stranded overhangs that anneal during a short incubation. The LIC system is adaptable for use with any vector following an alteration of the vector sequence. This chapter describes the creation of an LIC-compatible vector, with tips on how to make any vector LIC-enabled. It also includes a protocol for generating high-quality linearized vector template for the LIC reaction. Lastly, a step-by-step protocol of the LIC reaction is outlined, with useful tips and tricks for optimization and screening.

Amino Acid Sequence↗
PubMed2004

High throughput screening of methylation status of genes in prostate cancer using an oligonucleotide methylation array.

Recent work using high-throughput microarray technology has discovered altered expression of a large number of genes in prostate cancer. Many of these alterations may be the consequence of changes in methylation status in the CpG islands of promoter or exon 1 regions of these genes. In order to determine the methylation status of a large number of genes and ESTs we combined the principle of match/mismatch hybridization with the technique of whole genome labeling to develop a highly specific oligonucleotide-based methylation microarray. Using this array, we analyzed the methylation status of 105 genes and ESTs in three prostate cancer cell lines. Between 32 and 47% of these genes and ESTs were methylated in these cell lines. By correlating the methylation status of this array with the results of Affymetrix expression arrays of three prostate cancer cell lines, we determined that methylation of genes played a significant role (37%) in down-regulating the expression of certain genes in prostate cancer. We also tested this array on a number of primary prostate tissue samples. Our results indicated that a subset of genes in this microarray (25/105) were methylated in all prostate cancer samples but not in normal prostate, suggesting the potential significance of alterations in the methylation status of certain genes in the development of prostate cancer.

Acetylation↗
PubMed2000

A system for specific, high-throughput genotyping by allele-specific primer extension on microarrays.

This study describes a practical system that allows high-throughput genotyping of single nucleotide polymorphisms (SNPs) and detection of mutations by allele-specific extension on primer arrays. The method relies on the sequence-specific extension of two immobilized allele-specific primers that differ at their 3'-nucleotide defining the alleles, by a reverse transcriptase (RT) enzyme at optimized reaction conditions. We show the potential of this simple one-step procedure performed on spotted primer arrays of low redundancy by generating over 8000 genotypes for 40 mutations or SNPs. The genotypes formed three easily identifiable clusters and all known genotypes were assigned correctly. Higher degrees of multiplexing will be possible with this system as the power of discrimination between genotypes remained unaltered in the presence of over 100 amplicons in a single reaction. The enzyme-assisted reaction provides highly specific allele distinction, evidenced by its ability to detect minority sequence variants present in 5% of a sample at multiple sites. The assay format based on miniaturized reaction chambers at standard 384-well spacing on microscope slides carrying arrays with two primers per SNP for 80 samples results in low consumption of reagents and makes parallel analysis of a large number of samples convenient. In the assay one or two fluorescent nucleotide analogs are used as labels, and thus the genotyping results can be interpreted with presently available array scanners and software. The general accessibility, simple set-up, and the robust procedure of the array-based genotyping system described here will offer an easy way to increase the throughput of SNP typing in any molecular biology laboratory.

Alleles↗
PubMed2025

Using Prime Editing Guide Generator (PEGG) for high-throughput generation of prime editing sensor libraries.

Prime editing enables the generation of nearly any small genetic variant. However, the process of prime editing guide RNA (pegRNA) design is challenging and requires automated computational design tools. We developed Prime Editing Guide Generator (PEGG), a fast, flexible, and user-friendly Python package that enables the rapid generation of pegRNA and pegRNA-sensor libraries. Here, we describe the installation and use of PEGG (https://pegg.readthedocs.io) to rapidly generate custom pegRNA-sensor libraries for use in high-throughput prime editing screens.

Gene Editing↗
PubMed2004

A novel high-throughput pepT1 transporter assay differentiates between substrates and antagonists.

PepT1 is a transporter of proven pharmaceutical utility for enhancing oral absorption. A high-throughput, robust functional assay, capable of distinguishing PepT1 binders from substrates, allowing identification and/or prediction of drug candidate activation was developed. An MDCK epithelial cell line was transfected with rPepT1. The high level of stable rPepT1 expression that was achieved enabled development of a miniaturized PepT1 assay in a 96-well format, which could be scaled to 384 wells. The assay is based on measurement of membrane depolarization resulting from the cotransport of protons and PepT1 substrates. Membrane potential changes are tracked with a voltage-sensitive fluorescent indicator. Control (mock-transfected) cells are used to determine nonspecific membrane potential changes. A variety of fluorescent dyes were tested during initial assay design, including intracellular pH and membrane potential indicators. A membrane potential indicator was chosen because of its superior performance. Upon PepT1 activation with glycylsarcosine, dose-dependent membrane depolarization was observed with an EC50 of 0.49 mM. Maximum depolarization was dependent on the level of PepT1 expression. Testing of 38 known PepT1 substrates, binders, and nonbinders demonstrated that this assay accurately distinguished substrates from binders and from nonbinders. Initial validation of this novel assay indicates that it is sensitive and robust, and can distinguish between transporter substrates and antagonists. This important distinction has been previously achieved only with lower-throughput assays. This assay might also be used to determine substrate potency and establish a high-quality data set for PepT1 SAR modeling.

Animals↗
PubMed2025

There and back again: historical biogeography of neotropical magnolias based on high-throughput sequencing.

BACKGROUND: The Neotropics are considered one of the most biodiverse areas in the world, housing at least one third of all vascular plant species. One of the genera that has diversified in the Neotropics is Magnolia, with about 174 species of three sections (Macrophylla, Magnolia and Talauma) endemic to the Americas. In this work, we study the biogeographic history of the Neotropical Magnolia species using high-throughput sequencing data. Sequences from 39 species (38 from Magnolia and one from the sister genus Liriodendron) were assembled. The dataset contained sequences from 239 nuclear targets and complete chloroplast genomes. Phylogenomic hypotheses and the ancestral distribution range of Magnolia were reconstructed. RESULTS: The results of the calibrated phylogenetic hypotheses and ancestral range construction suggest that the earliest arrival in the Neotropics were the ancestors of section Talauma (38 million years ago), which colonized the Pacific region. This early presence in South America suggests long-distance, overwater dispersal from North America, the presumed origin of the genus Magnolia. The analysis and the extant Talauma distribution indicate a south to north recolonization. The ancestors of the other two Neotropical sections, Magnolia and Macrophylla, migrated around 19 mya from Asia to North America, radiating southward to the Neotropics afterwards, around 11 mya. CONCLUSIONS: Our results suggest that Neotropical magnolias originated from a North American ancestor. The current sections arrived at the region independently influenced by climatic processes such as temperature drops or the Miocene Climatic Optimum. Additionally, geological processes, such as the movement of the South and North American land masses and the emergence of the Panama isthmus, facilitated the migration between continents.

Magnolia↗
PubMed1997

Microplate alamar blue assay versus BACTEC 460 system for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium.

In response to the need for rapid, inexpensive, high-throughput assays for antimycobacterial drug screening, a microplate-based assay which uses Alamar blue reagent for determination of growth was evaluated. MICs of 30 antimicrobial agents against Mycobacterium tuberculosis H37Rv, M. tuberculosis H37Ra, and Mycobacterium avium were determined in the microplate Alamar blue assay (MABA) with both visual and fluorometric readings and compared to MICs determined in the BACTEC 460 system. For all three mycobacterial strains, there was < or = 1 dilution difference between MABA and BACTEC median MICs in four replicate experiments for 25 to 27 of the 30 antimicrobics. Significant differences between MABA and BACTEC MICs were observed with 0, 2, and 5 of 30 antimicrobial agents against H37Rv, H37Ra, and M. avium, respectively. Overall, MICs determined either visually or fluorometrically in MABA were highly correlated with those determined in the BACTEC 460 system, and visual MABA and fluorometric MABA MICs were highly correlated. MICs of rifampin, rifabutin, minocycline, and clarithromycin were consistently lower for H37Ra compared to H37Rv in all assays but were similar for most other drugs. M. tuberculosis H37Ra may be a suitable surrogate for the more virulent H37Rv strain in primary screening of compounds for antituberculosis activity. MABA is sensitive, rapid, inexpensive, and nonradiometric and offers the potential for screening, with or without analytical instrumentation, large numbers of antimicrobial compounds against slow-growing mycobacteria.

Anti-Bacterial Agents↗
PubMed1991

Endothelin-1 receptor binding assay for high throughput chemical screening.

An endothelin-1 (ET-1) receptor-binding assay, in microtiter format, has been developed for use in high throughput chemical or natural product screening. A rat smooth muscle, clonal cell line derived from embryonic thoracic aorta and designated A10 has been shown to consistently express high-affinity ET-1 receptors. A 96-well microtiter filtration plate, with individual PVDF membranes attached to the bottom of each well, was used for separation. In saturation binding assays, using [125I]Tyr13-ET-1, Scatchard analysis was monophasic, indicating a single high-affinity population of receptors with Kd = 0.12 nM with approximately 40,000 receptors per cell. The Ki values for peptides, known to bind at the ET receptor, were as follows: ET-1, 0.14 nM; ET-2, 0.16 nM; sarafotoxin S6b, 0.6 nM; VIC, 0.2 nM; ET-3, 16 nM; and big human ET, greater than 1 microM. ET receptors on A10 cells were stable for at least 2 months when stored at -20 degrees C. The assay is suitable for automation, because it is stable and reproducible. This method gave a 90% reduction in radioactive waste compared to tissue homogenate assays that use glass fiber filtration and cell harvesters.

Animals↗
PubMed2005

High-throughput SNP genotyping by single-tube PCR with Tm-shift primers.

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.

DNA Primers↗
PubMed2005

High-throughput evaluation of antioxidant and pro-oxidant activities of polyphenols with thymidine protection assays.

A recently reported high-throughput screening strategy has been applied to the rapid selection of new water-soluble antioxidants that display strong protective activities. Based on a competitive immunoassay, a triple-screening procedure was used to evaluate the ability of different compounds to protect thymidine under different oxidative stresses. The pro-oxidant effect of norbadione A in the presence of iron was observed, while some pulvinic acid derivatives proved strongly protective during gamma radiolysis, UV irradiation, and Fenton-like oxidation.

4-Butyrolactone↗
PubMed2006

High-throughput kinase assay based on surface plasmon resonance suitable for native protein substrates.

We report a novel in vitro high-throughput (HTP) kinase assay using surface plasmon resonance (SPR). In vitro tyrosine phosphorylation was performed in a microtiter plate, after which the substrate was captured with an antibody on a sensor chip and phosphotyrosine (pTyr) was detected with an anti-pTyr antibody. The capture and pTyr detection steps were performed using a Biacore A100, which is a sensitive and high-performance flow-cell-based SPR biosensor. This system allowed multiple sample processing (1000 samples/day) and high-quality data sampling. We compared the abilities of the HTP-SPR method and a standard radioisotope assay by measuring the phosphorylation of several substrate proteins by the Fyn tyrosine kinase. Similar results were obtained with both methods, suggesting that the HTP-SPR method is reliable. Therefore, the HTP-SPR method described in this study can be a powerful tool for a variety of screening analyses, such as kinase activity screening, kinase substrate profiling, and kinase HTP screening of kinase inhibitors.

Antibodies↗
PubMed2006

A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.).

A robust and high throughput Agrobacterium genetic transformation procedure has been developed for perennial ryegrass (Lolium perenne L.). Embryogenic callus lines were selected and maintained as plants in vitro. Embryogenic calli derived from meristematic regions of the vegetative tillers were co-cultivated with Agrobacterium tumefaciens strain EHA101 carrying the plasmid pCAMBIA 1305.1 in the presence of acetosyringone for 3-4 days. The calli were grown under 94.8 and 151.6 microM hygromycin selection, respectively for two cycles of 2-weeks each, followed by transfer to regeneration medium with 47.4 microM hygromycin. Regenerated plants were rooted and successfully transferred to soil. The transgenic nature of the regenerated plants was confirmed by DNA gel blot analysis and gene expression demonstrated by GUS histochemical assay and/or reverse transcription PCR. After development of the transformation procedure, we used Agrobacterium strain EHA101 carrying a modified binary plasmid pMH bearing genes of interest. In the past 2 years, we have produced more than 1,000 plants with constructs encoding different genes of interest from perennial ryegrass.

Agrobacterium tumefaciens↗