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Processing of cDNA and genomic kilobase-size clones for massive screening, mapping and sequencing by hybridization.
Efficient procedures for managing a large number of M13 or plasmid clones have been developed. In addition to picking, clones are directly arrayed in multiwell plates by dispensing diluted transformation mixtures. Metal pin arrays are used for fast inoculations of preparative plates filled by medium or by PCR mixture. Growth of M13 clones in multiwell plates is optimized to obtain a consistently high yield, and a PCR protocol is defined for reliable amplification of several thousand M13 or plasmid inserts per day in BioOvens. Over 80,000 cDNA inserts have been amplified. The phages or amplified inserts are spotted on nylon filters using an array of pins having a flat bottom, 0.3 mm in diameter. The procedures are suitable for an automated processing of hundreds of thousands of short clones from representative cDNA and genomic libraries. Hybridization of arrayed clones with oligonucleotide and complex probes can simplify the search for new genes and accelerate large-scale sequencing.
Development of an improved automated gas-chromatographic chiral analysis system: application to non-natural amino acids and natural protein hydrolysates.
In the use of combinatorial chemistry as a novel strategy for drug discovery, the chirality assessment of building blocks used for library construction is particularly important in the evaluation of biological actions of generated libraries. The procedure for chiral analysis of lead compounds in screening may be of a high priority, particularly in the case of the protection of intellectual rights. Previously, an automated amino acid analysis system using enantiomer labeling was developed. The system incorporates a reactor, which allows automated esterification and acylation of amino acids, and is connected to an on-line gas chromatographic system. A capillary column coated with a chiral phase is employed for the separation of the enantiomers. This particular system is improved with a newly constructed "high-throughput auto-derivatizer" in combination with a new GC-system. The resulting data can be processed by newly constructed software. The analyses of amino acid derivatives or hydrolysates of proteins and peptides are carried out routinely within ca. 45 min, including derivatization. Using this system several non-natural amino acids were tested with respect to the stereoisomeric configuration. In addition, acid hydrolysates of food proteins and tissues obtained by autopsy were analyzed as an application to proteome research.
Combinatorial approaches as a component of high-throughput experimentation (HTE) in catalysis research.
We consider the application of high-throughput experimentation (HTE), including combinatorial methods, to catalyst discovery and early-phase optimization. While combinatorial- and parallel-testing methods have an already substantial history in catalysis, recent work by several groups promises significant efficiency gains. In molecular catalysis, progress is noted in library design, library synthesis by pooled, parallel, and discrete formats, catalyst testing and reaction optimization; the prime constraint for organometallic catalysts is the limited scope of synthesis procedures for non-peptide-based ligand libraries. Routes described for the synthesis of heterogeneous catalysts include hydrothermal synthesis, arraying of solution precursors, automated impregnation and precipitation, and arraying of solid precursors. The key challenge in applying HTE to heterogeneous catalysis is testing; we distinguish here between Stage 1, or "discovery" testing and Stage 2, optimization testing, and describe techniques with potential in each case. Recent examples from the literature and our own work are used to illustrate these principles and the prospects for HTE applied to catalysis.
Automated DNA sequencing of the human HPRT locus.
The complete sequence of 57 kb of the human HPRT locus has been determined using automated fluorescent DNA sequencing. The strategy employed increasingly directed sequencing methods: A randomly generated M13 library was sequenced to generate contiguous overlapping sets of sequences (contigs). M13 clones at the ends of these contigs were further sequenced using M13 (universal and reverse) and custom oligonucleotide primers to order the contigs and to complete the sequencing project. The human HPRT sequence includes 1676 bp 5' and 15,238 bp 3' to exons 1 and 9, respectively. The sequence contains 49 representatives of the Alu repeat, along with several other types of repetitive sequences. The Alu sequences exhibit a biased orientation, with those sequences in the first half of the locus oriented in the minus direction relative to transcription of the gene (3'----5' = 77%, P less than 0.005) and those sequences in the latter half of the locus oriented randomly (5'----3' = 67%, P less than 0.5). The development and performance of the sequencing strategy and the features of the human HPRT gene are presented.
A microcomputer-based, net-lending interlibrary loan system.
A microcomputer-based, net-lending interlibrary loan system was developed at Lane Medical Library, Stanford University. The system, designed to generate the monthly billing invoices and all necessary statistical reports, has reduced the time required for logging-in procedures and compilation of monthly, quarterly, and annual statistics. User menus, help screens, and choice fields were developed explicitly for library staff who have little or no computer experience. The program was written using the DataEase database management software running on IBM PC, XT, AT, or compatible with a minimum of 512K RAM. Described are features of this automated interlibrary loan management system and its use in a net-lending interlibrary loan department. It focuses on data entry in the "Library Directory" and "ILL Log Sheet," details of billing invoices, and statistical reports, and flexibility in modifying tax rates, borrowing fees, and other parameters.
Infrared analysis of urinary stones: a trial of automated identification.
A Search algorithm included in the Opus software of Bruker (Germany) was evaluated for analysis of urinary stones. Three reference libraries containing respectively 85 (single components), 1,059 (binary mixtures) and 4,565 (ternary mixture) digitized spectra were created and used to identify unknown spectra (n=320), applying the automatic procedure. Identification of the major component was correct in 83% of cases but the percentage of identification significantly decreased for the second and the third components. In cases of identification of the two first components, quantitative assessment was correct within tolerance limits +/- 15%. The computer results are judged unsatisfactory with regard to pathology because computer-aided identification is not sufficiently sensitive and specific to differentiate species with similar spectral pattern, even for the identification of main component, and also to detect minor components. It can be of assistance to guide spectral analysis, but it cannot replace human identification.
From A to Z: automated catalogue to Web OPAC and Z39.50.
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Investigating the genetic control of stem cell behavior.
The precise control of hematopoietic stem cell fate decisions involves molecules that are differentially expressed in the stem cell versus its non-stem progeny. The construction of representative and high quality cDNA libraries containing stem cell-specific sequences is a first step in elucidating stem cell control mechanisms. Automated bioinformatics in conjunction with high-throughput random sequencing and high-density parallel array hybridization studies make it possible to dissect stem cell molecular pathways and networks. It is the differential analysis of these interacting pathways which will provide the greatest insights into the biological differences underlying stem cell behavior.
A new approach to understanding T cell development: the isolation and characterization of immature CD4-, CD8-, CD3- T cell cDNAs by subtraction cloning.
During T cell development in the mammalian thymus, immature T cells are observed that lack the cell surface markers CD4, CD8, and CD3. A subtracted cDNA library was constructed to isolate cDNAs that are specific for these immature T cells. Tissue-specific expression of 97 individual cDNAs were examined using different cell types by Northern blot analysis, and six cDNAs were analyzed by reverse transcriptase (RT) polymerase chain reaction (PCR) detection of RNA. Approximately 50% of the clones could not be detected on Northern blots, and 40% of the clones were expressed by at least one other cell-type including monocytes, mature T cells, and B cells. Eight cDNA clones appear to be specific for the CD4-, CD8-, CD3- T cell line, used to construct the library, as determined by Northern blot analysis. In addition, 330 cDNA clones were subjected to partial automated DNA sequence determination. Database searches, with both nucleotide and protein translations, revealed cDNAs that exhibit interesting similarities to human cell-cycle gene 1, platelet-derived growth factor receptor, c-fms oncogene (CSF-1) receptor, and members of the immunoglobulin gene superfamily. This approach of employing subtraction coupled with large scale partial cDNA sequence determination can be useful to identify genes that may be involved in early T cell growth, cellular recognition or differentiation.
PCR buffer optimization with uniform temperature regimen to facilitate automation.
To facilitate PCR(1,2) reactions in large numbers with uniform conditions, the annealing temperature was fixed and the stringency of the reactions was adjusted by optimizing the ion conditions of the reaction. The buffer system is based primarily on Tris (T), ammonium (N), and potassium (K) to adapt assay conditions to different primer pairs. The TNK buffers have permitted successful screening of a 60,000-clone yeast artificial chromosome (YAC) library with more than 200 primer pairs.
Process redesign: making your film library work for you.
When Riverside Methodist Hospitals in Columbus, Ohio, experienced serious problems in its radiology film library, administrators recognized that poor processes were to blame and used quality management techniques to create a new system. A radiology information system was part of Riverside's long-range plan, but an effective manual process was implemented first. Riverside had decided that closing the library to referring physician access would result in more successful operations. An Operational Changes Planning Committee composed of file clerks, technologists, managers, radiologists and referring physicians planned the redesign. The committee identified four goals: Reduce unmet requests. Improve response time. Benchmark operations of comparable organizations. Implement an effective manual process prior to automation. Steps in the redesign process included identifying the problem, applying statistical tools to the situation and understanding customer requirements. Flow charts, benchmarking, data collection within the department and focus groups provided important quantitative information. Creating an improved process included the following actions: Work simplification. Work elimination (especially non-value-added work). Uncovering and removing causes of delay. Automation of repetitive tasks via the RIS. Reduction and elimination of any rework steps. One important change was to close the library to physician access. There were moderate gains in effectiveness as a result of redesign of the manual process. Results have continued to improve, and the redesign process provided greater commitment and cooperation for the more significant improvements that followed the RIS introduction.
Synthesis of highly substituted 5-(trifluoromethyl)ketoimidazoles using a mixed-solid/solution phase motif.
Using a combination of solid phase synthesis for the preparation of N-substituted-N-acylglycines 7 followed by solution-phase ring transformation of trifluoromethylacyl munchnone intermediate 8, a library of 200 trisubstituted-5-trifluoromethylketo (TFMK) imidazoles 9 was prepared. In a sublibrary, bromoacetate resin 4 was treated with 5 amines in parallel to give N-substituted glycines 5 followed by acylation with 12 acid chlorides to provide, upon cleavage from the resin, 60 individual N-substituted-N-acylglycines 7. The glycines 7 were converted to munchnones 8 by treatment with trifluoroacetic anhydride followed by reaction with benzamidine to give trisubstituted-5-TFMK-imidazoles 9. The structural content of the library was analyzed using PlateView of the LCMS results, and individual members were isolated by automated preparative LCMS.
Use of statistical design of experiments in the optimization of amide synthesis utilizing polystyrene-supported N-hydroxybenzotriazole resin.
Two fields that routinely perform reaction optimization studies are chemical development (prior to scale-up) and medicinal or combinatorial chemistry (prior to analogue synthesis or library production). To date, the use of statistical design of experiments (DoE) in conjunction with automated synthesizers has been applied in process research to a greater extent than in the medicinal or combinatorial laboratories. We have applied DoE in conjunction with an automated synthesizer to optimize the synthesis of amides employing resin-bound N-hydroxybenzotriazole (PS-HOBt) active esters as intermediates. This methodology allowed the rapid development of an improved protocol for the parallel synthesis of amides by conversion of carboxylic acids to PS-HOBt esters followed by treatment with appropriate amines. Product isolation involved only simple filtration and evaporation.
An automated sample preparation system for large-scale DNA sequencing.
Recent advances in DNA sequencing technologies, both in the form of high lane-density gels and automated capillary systems, will lead to an increased requirement for sample preparation systems that operate at low cost and high throughput. As part of the development of a fully automated sequencing system, we have developed an automated subsystem capable of producing 10,000 sequence-ready ssDNA templates per day from libraries of M13 plaques at a cost of $0.29 per sample. This Front End has been in high throughput operation since June, 1997 and has produced > 400,000 high-quality DNA templates.
Electronic health record meets digital library: a new environment for achieving an old goal.
Linking the electronic health record to the digital library is a Web-era reformulation of the long-standing informatics goal of seamless integration of automated clinical data and relevant knowledge-based information to support informed decisions. The spread of the Internet, the development of the World Wide Web, and converging format standards for electronic health data and digital publications make effective linking increasingly feasible. Some existing systems link electronic health data and knowledge-based information in limited settings or limited ways. Yet many challenging informatics research problems remain to be solved before flexible and seamless linking becomes a reality and before systems become capable of delivering the specific piece of information needed at the time and place a decision must be made. Connecting the electronic health record to the digital library also requires positive resolution of important policy issues, including health data privacy, government encouragement of high-speed communications, electronic intellectual property rights, and standards for health data and for digital libraries. Both the research problems and the policy issues should be important priorities for the field of medical informatics.
Analyses of compound libraries obtained by high-throughput parallel synthesis: strategy of quality control by high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance techniques.
The growing interest in combinatorial chemistry has led to a new source of compounds from which a large number of leads has emerged over recent years. Parallel synthesis, in particular, allows a quick production of a wide number of individual compounds. A rapid analytical control is needed to determine their quality. A strategy using automated, fast reversed-phase C18 high-performance liquid chromatography with diode-array detection (LC-DAD-MS) followed by atmospheric pressure chemical ionisation mass spectrometry (APCI-MS) and NMR has been developed for their characterisation and purity control. Complementary NMR analyses are done on selected compounds to provide a better structural characterisation of the expected compounds and their potential side-products. Validated libraries are then registered in ISIS databases using automated procedures.
Molecular cloning of a phosphoinositide-specific phospholipase C from catfish olfactory rosettes.
A 2.7 kb clone encoding the partial (about 66%) sequence of a phosphoinositide-specific phospholipase C (PLC) was isolated from a cDNA library constructed from channel catfish (Ictalurus punctatus) olfactory rosettes. The clone, designated 30c7, was completely sequenced by automated DNA sequencing and was found to share significant homology with rat and bovine PLCs of the delta 1 isotype. In situ hybridization showed that 30c7 transcripts were expressed in a small subpopulation of olfactory neurons, as well as in other cell types in the olfactory epithelium. Polymerase chain reaction (PCR) analysis indicated that the enzyme was also expressed in several additional tissues, including brain, gill, heart, liver and skeletal muscle. These results suggest that the PLC encoded by clone 30c7 is expressed in several tissues and therefore may have a role in mediating transduction events in diverse tissues as well as in a small group of olfactory neurons.