Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Library Automation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 559 records · Page 31Linked to original sources
PubMed2001

Green fluorescent protein-based halide indicators with improved chloride and iodide affinities.

The green fluorescent protein YFP-H148Q is sensitive to halides by a mechanism involving halide binding and a shift in pK(a). However, a limitation of YFP-H148Q is its low halide sensitivity, with K(d)>100 mM for Cl(-). Indicators with improved sensitivities are needed for cell transport studies, particularly in drug discovery by high-throughput screening, and for measurement of Cl(-) concentration in subcellular organelles. YFP-H148Q libraries were generated in which pairs of residues in the vicinity of the halide binding site were randomly mutated. An automated procedure was developed to screen bacterial colonies for improved halide sensitivity. Analysis of 1536 clones revealed improved anion sensitivities with K(d) down to 2 mM for I(-) (I152L), 40 mM for Cl(-) (V163S), and 10 mM for NO(3)(-) (I152L). The anion-sensitive mechanism of these indicators was established and their utility in cells was demonstrated using transfected cells expressing the cystic fibrosis transmembrane conductance regulator chloride channel.

3T3 Cells↗
PubMedDate not supplied

An integrated instrument control and informatics system for combinatorial materials research.

The use of high-throughput synthesis and characterization techniques is increasingly prevalent in materials science research. We describe the London University Search Instrument, a research apparatus designed for the high-throughput synthesis and characterization of thick-film sample libraries of ceramic compounds. The instrument is constructed largely from commodity components, which pose particular engineering challenges for achieving the automated operation required for efficient high-throughput experimentation. This paper describes the architecture and implementation of the software system that provides integrated instrument control and data management functions.

Journal Article↗
PubMed2003

Automatic discovery and classification of bioinformatics Web sources.

MOTIVATION: The World Wide Web provides an incredible resource to genomics researchers in the form of query access to distributed data sources--e.g. BLAST sequence homology search interfaces. The number of these autonomous sources and their rate of change outpaces the speed at which they can be manually classified, meaning that the available data is not being utilized to its full potential. Manually maintaining a wrapper library will not scale to accommodate the growth of genomics data sources on the Web, challenging us to produce an automated system that can find, classify and wrap new sources without constant human intervention. Previous research has not addressed the problem of automatically locating, classifying and integrating classes of bioinformatics data sources. RESULTS: This paper presents an overview of a system for finding classes of bioinformatics data sources and integrating them behind a unified interface. We describe our approach for automatic classification of new Web sources into relevance categories that eliminates the human effort required to maintain a current repository of sources. Our approach is based on a meta-data description of classes of interesting sources that describes the important features of an entire class of services without tying that description to any particular Web source. We examine the features of this format in the context of BLAST sources to show how it relates to Web sources that are being described. We then show how a description can be used to determine if an arbitrary Web source is an instance of the described service. To validate the effectiveness of this approach, we have constructed a prototype that correctly classifies approximately two-thirds of the BLAST sources we tested. We conclude with a discussion of these results, the factors that affect correct automatic classification and areas for future study.

Algorithms↗
PubMed1991

Complementary DNA sequencing: expressed sequence tags and human genome project.

Automated partial DNA sequencing was conducted on more than 600 randomly selected human brain complementary DNA (cDNA) clones to generate expressed sequence tags (ESTs). ESTs have applications in the discovery of new human genes, mapping of the human genome, and identification of coding regions in genomic sequences. Of the sequences generated, 337 represent new genes, including 48 with significant similarity to genes from other organisms, such as a yeast RNA polymerase II subunit; Drosophila kinesin, Notch, and Enhancer of split; and a murine tyrosine kinase receptor. Forty-six ESTs were mapped to chromosomes after amplification by the polymerase chain reaction. This fast approach to cDNA characterization will facilitate the tagging of most human genes in a few years at a fraction of the cost of complete genomic sequencing, provide new genetic markers, and serve as a resource in diverse biological research fields.

Amino Acid Sequence↗
PubMed1999

Evaluation of accuracy and repeatability of identification of food-borne pathogens by automated bacterial identification systems.

The performances of five automated microbial identification systems, relative to that of a reference identification system, for their ability to accurately and repeatedly identify six common food-borne pathogens were assessed. The systems assessed were the MicroLog system (Biolog Inc., Hayward, Calif.), the Microbial Identification System (MIS; MIDI Inc., Newark, Del.), the VITEK system (bioMérieux Vitek, Hazelwood, Mo.), the MicroScan WalkAway 40 system (Dade-MicroScan International, West Sacramento, Calif.), and the Replianalyzer system (Oxoid Inc., Nepean, Ontario, Canada). The sensitivities and specificities of these systems for the identification of food-borne isolates of Bacillus cereus, Campylobacter jejuni, Listeria monocytogenes, Staphylococcus aureus, Salmonella spp., and verotoxigenic Escherichia coli were determined with 40 reference positive isolates and 40 reference negative isolates for each pathogen. The sensitivities of these systems for the identification of these pathogens ranged from 42.5 to 100%, and the specificities of these systems for the identification of these pathogens ranged from 32.5 to 100%. Some of the systems had difficulty correctly identifying the reference isolates when the results were compared to those from the reference identification tests. The sensitivity of MIS for the identification of S. aureus, B. cereus, E. coli, and C. jejuni, for example, ranged from 47.5 to 72. 5%. The sensitivity of the Microlog system for the identification of E. coli was 72.5%, and the sensitivity of the VITEK system for the identification of B. cereus was 42.5%. The specificities of four of the five systems for the identification of all of the species tested with the available databases were greater than or equal to 97.5%; the exception was MIS for the identification of C. jejuni, which displayed a specificity of 32.5% when it was tested with reference negative isolates including Campylobacter coli and other Campylobacter species. All systems had >80% sensitivities for the identification of Salmonella species and Listeria species at the genus level. The repeatability of these systems for the identification of test isolates ranged from 30 to 100%. Not all systems included all six pathogens in their databases; thus, some species could not be tested with all systems. The choice of automated microbial identification system for the identification of a food-borne pathogen would depend on the availability of identification libraries within the systems and the performance of the systems for the identification of the pathogen.

Bacillus cereus↗
PubMed1996

Automated DNA sequencing requiring no DNA template purification.

A critical component in automated fluorescent DNA sequencing is good quality of DNA template. In an effort to reduce the dependence of sequencing success on DNA template quality, the effect of heat-soaked PCR on automated sequencing reactions has been examined. We have found that the heat-soaked PCR protocol considerably improves the overall quality of sequence data and significantly reduces the dependence on the quality of DNA templates. The improvement is corroborated by our ability to obtain over 500 bp of readable sequence per reaction using DNA from E. coli lysates as template obviating DNA purification.

Automation↗
PubMed2002

The application of multi-component reactions in drug discovery.

Multi-component reactions (MCRs) enable the facile, automated and high throughput generation of small organic molecules. MCRs have been used to create diversity oriented and biased combinatorial libraries, to accomplish the synthesis of highly complex natural products as well as for the large-scale production of drug candidates. This provides medicinal chemists with a powerful tool to create novel chemical diversity, matching the space of biological targets with relevant chemistry. The discovery of novel MCRs has become an increasingly active area of research, yielding novel chemical scaffolds for drug discovery efforts.

Animals↗
PubMed2006

[Meta-analysis of diagnostic capability of frequency-doubling technology (FDT) for primary glaucoma].

OBJECTIVE: To evaluate the overall diagnostic capabilities of frequency-doubling technology (FDT) in patients of primary glaucoma, with standard automated perimetry (SAP) and/or optic disc appearance as the gold standard. METHODS: A comprehensive electric search in MEDLINE, EMBASE, Cochrane Library, BIOSIS, Previews, HMIC, IPA, OVID, CNKI, CBMdisc, VIP information, CMCC, CCPD, SSreader and 21dmedia and a manual search in related textbooks, journals, congress articles and their references were performed to identify relevant English and Chinese language articles. Criteria for adaptability were established according to validity criteria for diagnostic research published by the Cochrane Methods Group on Screening and Diagnostic Tests. Quality of the included articles was assessed and relevant materials were extracted for studying. Statistical analysis was performed with Meta Test version 0.6 software. Heterogeneity of the included articles was tested, which was used to select appropriate effect model to calculate pooled weighted sensitivity and specificity. Summary Receiver Operating Characteristic (SROC) curve was established and the area under the curve (AUC) was calculated. Finally, sensitivity analysis was performed. RESULTS: Fifteen English articles (21 studies) of 206 retrieved articles were included in the present study, with a total of 3172 patients. The reported sensitivity of FDT ranged from 0.51 to 1.00, and specificity from 0.58 to 1.00. The pooled weighted sensitivity and specificity for FDT with 95% confidence intervals (95% CI) after correction for standard error were 0.86 (0.80 - 0.90), 0.87 (0.81 - 0.91), respectively. The AUC of SROC was 93.01%. Sensitivity analysis demonstrated no disproportionate influences of individual study. CONCLUSIONS: The included articles are of good quality and FDT can be a highly efficient diagnostic test for primary glaucoma based on Meta-analysis. However, a high quality perspective study is still required for further analysis.

Aged↗
PubMed1999

Antigen sequence- and library-based mapping of linear and discontinuous protein-protein-interaction sites by spot synthesis.

The knowledge (antigen-derived peptide scans)- and library (de novo)-based mapping of linear and discontinuous antibody epitopes as well as protein-protein contact sites in general by spot synthesis now is a well established technique. Due to its automation, this technique also promises great potential for applications in functional genomics. It should help to elucidate the complex network of interacting protein molecules involved in signal transduction events (Adam-klages et al. 1996; Hoffmüller et al. 1999). Although little chemistry is involved in the preparation of peptide scans or libraries and the synthesis procedure is relatively simple, the laboratories of immunologists or molecular biologists are often not equipped to perform spot synthesis. In this case scans or libraries can be purchased from commercial suppliers.

Antigen-Antibody Reactions↗
PubMed1998

Molecular characterization of the mouse gene encoding cellular retinaldehyde-binding protein.

PURPOSE: To clone and characterize the mouse gene encoding cellular retinaldehyde-binding protein (CRALBP). CRALBP appears to modulate enzymatic generation and processing of 11-cis-retinol and regeneration of visual pigment in the vertebrate visual cycle. Mutations in human CRALBP segregate with autosomal recessive retinitis pigmentosa. METHODS: A genomic clone encompassing the 5' end of the CRALBP gene through exon 6 was isolated from a mouse 129/Sv genomic DNA library. Exons 7 and 8 were PCR amplified from mouse eye cDNA and 129/SvJ genomic DNA. The gene structure was determined by automated DNA sequence analysis. RESULTS: The sequence of 6855 nucleotides was determined, including all 8 exons, 3 introns plus 3932 and 629 bases from the 5'- and 3'-flanking regions, respectively. The lengths of introns 3-6 were determined by PCR amplification. Northern analysis identifies a approximately 2.1 kb transcript in mouse eye; Southern analysis supports a single copy gene. CONCLUSIONS: The mouse CRALBP gene is similar to the human gene; the coding sequence is approximately 87% identical, the non-coding sequence approximately 65% identical. In contrast to the human gene, the mouse gene contains a consensus TATA box. One of two photoreceptor consensus elements important for CRALBP expression in human retinal pigment epithelium is also present in the mouse gene. Additional conserved and species-specific consensus sequences are identified. The mouse CRALBP genomic clones and structure provide valuable tools for developing an in vivo model to study protein function and gene regulation.

Animals↗
PubMed2004

Enzyme-like proteins from an unselected library of designed amino acid sequences.

Combinatorial libraries of de novo amino acid sequences can provide a rich source of diversity for the discovery of novel proteins with interesting and important activities. However, since arbitrary sequences rarely fold into well ordered protein-like structures, randomly generated libraries will yield functional proteins only very rarely. To enhance the likelihood of finding functional de novo proteins, we use binary patterning of polar and non-polar amino acids to design focused libraries of sequences that are predisposed to fold into ordered structures. Proteins isolated from binary patterned libraries have been shown previously to fold into well ordered and native-like three-dimensional structures. To probe the potential of such libraries to also yield proteins with enzyme-like activity, we measured the esterase activity of S-824, a de novo binary patterned protein whose alpha-helical three-dimensional structure was reported recently. Protein S-824 displayed a rate enhancement (k(cat)/k(uncat)) of 8700. The observed activity is similar to, or better than, that observed for several esterases designed previously using rational design or automated computational methods. Moreover, the observed activity rivals those of the first catalytic antibodies. To assess whether the activity of S-824 is representative of other proteins in binary patterned libraries, we measured the esterase activity of six additional proteins from two libraries. These libraries were 'naïve' in that they were neither designed to bind substrate, nor subjected to high throughput screens for activity. All six of the additional proteins displayed esterase activity significantly above background. These findings demonstrate that novel proteins with enzyme-like properties are surprisingly common in focused libraries designed by binary patterning. Moreover, the activity of these unselected proteins provides a reference state for the levels of activity that have been obtained by selection and/or computational design.

Amino Acid Sequence↗
PubMed2001

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Animals↗
PubMed2003

Approaches towards the automated interpretation and prediction of electrospray tandem mass spectra of non-peptidic combinatorial compounds.

Combinatorial chemistry is widely used within the pharmaceutical industry as a means of rapid identification of potential drugs. With the growth of combinatorial libraries, mass spectrometry (MS) became the key analytical technique because of its speed of analysis, sensitivity, accuracy and ability to be coupled with other analytical techniques. In the majority of cases, electrospray mass spectrometry (ES-MS) has become the default ionisation technique. However, due to the absence of fragment ions in the resulting spectra, tandem mass spectrometry (MS/MS) is required to provide structural information for the identification of an unknown analyte. This work discusses the first steps of an investigation into the fragmentation pathways taking place in electrospray tandem mass spectrometry. The ultimate goal for this project is to set general fragmentation rules for non-peptidic, pharmaceutical, combinatorial compounds. As an aid, an artificial intelligence (AI) software package is used to facilitate interpretation of the spectra. This initial study has focused on determining the fragmentation rules for some classes of compound types that fit the remit as outlined above. Based on studies carried out on several combinatorial libraries of these compounds, it was established that different classes of drug molecules follow unique fragmentation pathways. In addition to these general observations, the specific ionisation processes and the fragmentation pathways involved in the electrospray mass spectra of these systems were explored. The ultimate goal will be to incorporate our findings into the computer program and allow identification of an unknown, non-peptidic compound following insertion of its ES-MS/MS spectrum into the AI package. The work herein demonstrates the potential benefit of such an approach in addressing the issue of high-throughput, automated MS/MS data interpretation.

Automation↗
PubMed1994

Solid-phase cloning to create sublibraries suitable for DNA sequencing.

A solid-phase method is described to create subclones, suitable for DNA sequencing, from lambda or cosmid libraries. The purified target DNA is sonicated and two linkers, with one oligonucleotide biotinylated in the 5'-end, are ligated to the ends of the fragments produced by sonication. After size separation, the fragments are immobilised onto a solid support and the non-biotinylated strand of each immobilised fragment is eluted. In this way, a library of single-stranded fragments is obtained. All fragments contain 'universal' flanking sequences of 22 bases introduced by the linker ligation. These flanking sequences can subsequently be used for solid-phase cloning into a single-stranded vector containing the complementary sequences. Thus, cloning can be achieved without the use of ligase or restriction enzymes. The resulting subclones are used for direct solid-phase sequencing and the immobilised strand can be used to selectively remove homologous DNA from the library of single-stranded fragments. Thus, a sublibrary of non-sequenced fragments can be created. Here, we show that a library of clones, suitable for direct solid-phase sequencing, can be obtained starting with lambda DNA. The efficiency of selective hybridisation of homologous and non-homologous fragments was investigated. The possibility of using this approach for automated cloning strategies for large-scale genomic and cDNA sequencing is discussed.

Base Sequence↗
PubMed2001

Folded-back solution structure of monomeric factor H of human complement by synchrotron X-ray and neutron scattering, analytical ultracentrifugation and constrained molecular modelling.

Factor H (FH) is a regulatory cofactor for the protease factor I in the breakdown of C3b in the complement system of immune defence, and binds to heparin and other polyanionic substrates. FH is composed of 20 short consensus/complement repeat (SCR) domains, for which the overall arrangement in solution is unknown. As previous studies had shown that FH can form monomeric or dimeric structures, X-ray and neutron scattering was accordingly performed with FH in the concentration range between 0.7 and 14 mg ml(-1). The radius of gyration of FH was determined to be 11.1-11.3 nm by both methods, and the radii of gyration of the cross-section were 4.4 nm and 1.7 nm. The distance distribution function P(r) showed that the overall length of FH was 38 nm. The neutron data showed that FH was monomeric with a molecular mass of 165,000(+/-17,000) Da. Analytical ultracentrifugation data confirmed this, where sedimentation equilibrium curve fits gave a mean molecular mass of 155,000(+/-3,000) Da. Sedimentation velocity experiments using the g*(s) derivative method showed that FH was monodisperse and had a sedimentation coefficient of 5.3(+/-0.1) S. In order to construct a full model of FH for scattering curve and sedimentation coefficient fits, homology models were constructed for 17 of the 20 SCR domains using knowledge of the NMR structures for FH SCR-5, SCR-15 and SCR-16, and vaccinia coat protein SCR-3 and SCR-4. Molecular dynamics simulations were used to generate a large conformational library for each of the 19 SCR-SCR linker peptides. Peptides from these libraries were combined with the 20 SCR structures in order to generate stereochemically complete models for the FH structure. Using an automated constrained fit procedure, the analysis of 16,752 possible FH models showed that only those models in which the 20 SCR domains were bent back upon themselves were able to account for the scattering and sedimentation data. The best-fit models showed that FH had an overall length of 38 nm and is flexible. This length is significantly less than a predicted length of 73 nm if the 20 SCR structures had been arranged in an extended arrangement. This outcome is attributed to several long linker sequences. These bent-back domain structures may correspond to conformational flexibility in FH and enable the multiple FH binding sites for C3 and heparin to come into close proximity.

Amino Acid Sequence↗
PubMed1994

The 5'-sequence of the murine Hox-b3 (Hox-2.7) gene and its intron contain multiple transcription-regulatory elements.

We sought to clone and characterize the murine Hox-b3 gene. In Xenopus embryos, the homologous gene has been shown to be responsive to retinoic acid, an agent which has profound effects on tissue growth and development. By plaque hybridization, using a partial, murine Hox-b3 cDNA as a probe, we screened a genomic library and isolated a series of overlapping clones. Restriction fragments from positive clones were sequenced by the dideoxy method on an automated DNA sequencer. We report the genomic sequence of the murine Hox-b3 gene. The sequence has been submitted to the GenBank database (accession number U02278). Our sequence extends from the P1 promoter through the coding sequence of the gene to the 3'-untranslated region. In common with other homeobox genes, there is an intron between the conserved hexapeptide and the homeobox. It is 866 bp long and has 3'- and 5'-splice sites very similar to the consensus, a long polypyrimidine tract and a potential branch point near the 3'-splice site. We have analyzed the sequence 5' to the initiation codon and the intron for putative control elements, and have identified a series of putative transcription factor binding sites in the P1 promoter and intron, including two for the retinoid X receptor-beta. Their possible significance is discussed. The sequences we have identified may be responsible for the observed pattern of expression of the gene. This sequence and the clones from which it is derived will enable a molecular dissection of the P1 promoter region.

Amino Acid Sequence↗
PubMed2006

A rapid assay for dihydropteroate synthase activity suitable for identification of inhibitors.

The enzymes 6-hydroxymethylpterin pyrophosphokinase (HPPK) and dihydropteroate synthase (DHPS) catalyze sequential steps in folate biosynthesis. They are present in microorganisms but absent in mammals and therefore are especially suitable targets for antimicrobials. Sulfa drugs (sulfonamides and sulfones) currently are used as antimicrobials targeting DHPS, although resistance to these drugs is increasing. The most widely used assay that measures activity of these enzymes, to assess new inhibitors in vitro, is not amenable to automation. This article describes a simple, coupled, enzymatic spectrophotometric assay where the product of the DHPS reaction, dihydropteroate, is reduced to tetrahydropteroate by excess dihydrofolate reductase (DHFR) using the cofactor NADPH. The oxidation of NADPH is monitored at 340 nm. The activity of both HPPK and DHPS can be measured in this assay, and it has been used to measure kinetic parameters of wild-type and sulfa drug-resistant DHPS enzymes to demonstrate the utility of the assay. It is a sensitive and reproducible assay that can be readily automated and used in multiwell plates. This NADPH-coupled microplate photometric assay could be used for rapid screening of chemical libraries for novel inhibitors of folate biosynthesis as the first step in developing new antimicrobial drugs targeting the folate biosynthetic pathway.

Dapsone↗