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PubMed1976

Automated cataloging: the state of the art.

The art of cataloging is in a state of constant dynamic change. The capabilities of automation are causing changes in the tools we have at our disposal, the education and training we need to work with these tools, the caliber of staff we use at various stages of the cataloging process, and the physical form of the end product of the efforts of the catalog department. But perhaps of more importance is the cooperation and sharing between libraries on both the national and international level that become possible as the result of automated systems.

Automation↗
PubMed1996

Isotope or mass encoding of combinatorial libraries.

BACKGROUND: Combinatorial chemistry using solid-phase synthesis is a rapidly developing technology that can result in a significant reduction in the time required to find and optimize lead compounds. The application of this approach to traditional medicinal chemistry has led to the construction of libraries of small organic molecules on resin beads. A major difficulty in developing large combinatorial libraries is the lack of a facile encoding and decoding methodology to identify active compounds. RESULTS: Several encoding schemes are described which use the ability of mass spectrometry to ascertain isotopic distributions. Molecular tags are attached to resin beads in parallel or on the linker used for chemical library synthesis. The tags are encoded via a controlled ratio of a number of stable isotopes on the tagging molecules, and range from a single to a complex isotopic distribution. CONCLUSIONS: A novel coding scheme is described that is useful for the generation of large encoded combinatorial libraries. The code can be cleaved after assay and analyzed by mass spectrometry in an automated fashion. An important element of the combinatorial discovery process is the ability to extract the structure-activity relationship (SAR) information made available by library screening. The speed and sensitivity of the mass-encoding scheme has the potential to determine the full SAR for a given library.

Chemistry, Organic↗
PubMed1992

Cosmid linking clones localized to the long arm of human chromosome 11.

Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.

Chromosomes, Human, Pair 11↗
PubMed2001

Sequence and structure of the mouse gene for RPE65.

PURPOSE: To determine the genomic organization of the mouse gene for the retinal pigment epithelium (RPE) specific protein RPE65. METHODS: A genomic clone containing the entire Rpe65 gene was isolated from a mouse genomic P1 library. Fragments of this clone were subcloned and sequenced by automated fluorescent dideoxy DNA sequencing and analyzed. Direct sequencing of PCR amplification products was used to complete the structure. Primer extension analysis was used to determine the transcription start site. RESULTS: Southern hybridization of restriction digests of mouse genomic DNA reveals a likely single autosomal gene for Rpe65 with no evidence of pseudogenes. Sequence analysis of the mouse P1 clone for Rpe65 and fragments thereof reveals 14 exons distributed over 27 kbp. The transcription start site is located 57 bp upstream of the initiation codon. The protein encoded by the mouse Rpe65 gene is highly conserved when compared with RPE65s from other species. CONCLUSIONS: RPE65 is a highly conserved protein and it appears that the genes for the mouse and human RPE65s, at least, are also conserved in overall structure.

Amino Acid Sequence↗
PubMed2001

Full-length cDNAs: more than just reaching the ends.

The development of functional genomic resources is essential to understand and utilize information generated from genome sequencing projects. Central to the development of this technology is the creation of high-quality cDNA resources and improved technologies for analyzing coding and noncoding mRNA sequences. The isolation and mapping of cDNAs is an entrée to characterizing the information that is of significant biological relevance in the genome of an organism. However, a bottleneck is often encountered when attempting to bring to full-length (or at least full-coding) a number of incomplete cDNAs in parallel, since this involves the nonsystematic, time consuming, and labor-intensive iterative screening of a number of cDNA libraries of variable quality and/or directed strategies to process individual clones (e.g., 5' rapid amplification of cDNA ends). Here, we review the current state of the art in cDNA library generation, as well as present an analysis of the different steps involved in cDNA library generation.

Automation↗
PubMedDate not supplied

Automated high-throughput synthesis of artificial glycopeptides. Small-molecule probes for chemical glycobiology.

A fully automated method for the synthesis of artificial glycopeptides having two (similar or different) carbon-linked glycosyl moieties on a dipeptide scaffold has been developed. By use of this approach that combines the diversity of peptide/pseudopeptide and glycosides, different glycoside moieties can be incorporated onto the peptide/pseudopeptide backbone in a highly controlled manner. The approach utilizes a stepwise reductive amination with glycoside aldehyde derivatives (model 1) or (ii) glycoside reductive amination followed by glycoside amide bond formation (model 2). Further, an automated method has been utilized in the high-throughput library synthesis of 4 x 96 artificial glycopeptides. These libraries were tested as chemical probes/inhibitors of enzyme systems that convert a glucose moiety into rhamnose prior to incorporation of the rhamnose unit and the conversion of UDP-galactopyranose to UDP-galactofuranose via UDP-galactopyranose mutase enzyme during the biosynthesis of the mycobacterium cell wall.

Combinatorial Chemistry Techniques↗
PubMed1997

[An entire rain forest can be screened at pharmaceutical industry's laboratories].

The pharmaceutical industry has long been heavily reliant on natural products, and today more than half of the twenty best-selling pharmaceuticals are derived from natural sources. Hitherto sample collection from fauna and flora has been based on an ethnobotanical approach involving traditional healers and oral histories of indigenous peoples as sources of information on folk uses of plants and organisms. In the future, however, random sampling combined with automated high-throughput screening (HTS) may come to the forefront in drug design, enabling extensive libraries of active compounds to be built up, and rendering local knowledge largely irrelevant. New technological advances in chemistry, molecular biology and data processing are combined in automated systems whereby HTS, based on bioassay-guided fractionation procedures, is used to isolate active compounds, and enabling the chemical structure of isolated compounds to be determined within 24 hours. The combination of the speed of HTS, and a remarkable decrease in the amount of sample required for the isolation and structure determination of natural compounds, has improved our ability to find unique natural products for drug development. Moreover, it has already been demonstrated that, owing to the relatively minute amounts of material required for HTS, this random sampling approach to bioprospecting for the purposes of drug will benefit efforts to conserve such sensitive biosystems as the rain forests and marine ecosystems. Thus, this technology would seem to be more compatible with the needs of modern drug design in the pharmaceutical industry that the ethnobotanical approach.

Drug Industry↗
PubMed2026

Systematic performance evaluation and application validation of an end-to-end NGS workstation.

Next-generation sequencing (NGS) library preparation is a core component of precision genomics, but it is commonly constrained by inefficiency, variability, and low throughput of manual protocols. To address these limitations, we developed and systematically evaluated a fully automated NGS workstations and further validated its performance across representative application scenarios. The automated system reduced total processing time from 8 to 10 to 4–6 h. At the same time, it maintained similar performance in pre-library metric, including DNA yield and fragment size, as well as post-capture sequencing metrics (Q30 > 90%, mapping rates > 95%, on-target rates 85–90%). The duplication rate was reduced to 5–8%, compared with 10–15% for manual methods, indicating increased library complexity. Bioinformatic evaluation of inter-species read mapping showed minimal cross-contamination, with a maximum contamination ratio of 0.0003%, indicating effective sample isolation in the automated workflow. High concordance in variant detection was observed between automated and manual workflows. Overall, this automated workstation provides a standardized and reproducible workflow that supports scalable precision genomics applications.

High-Throughput Nucleotide Sequencing↗
PubMedDate not supplied

Characterization of small combinatorial chemistry libraries by (1)H NMR. Quantitation with a convenient and novel internal standard.

A novel silane standard, 1,4-bis(trimethylsilyl)benzene (BTMSB), is introduced for the generic quantitation of small organic molecules in DMSO-d(6) solution by (1)H NMR. This standard is an easily weighable solid and is stable for at least 1 month in DMSO solution, and its (1)H NMR spectrum contains a strong singlet in a region usually free of signals. With a set of certified standards, concentration determination with about 2% precision and accuracy is verified after solution preparation with fully automated procedures, thus making very effective the characterization of small combinatorial chemistry libraries for identity and purity when combined with other physicochemical or biochemical tests. As an example, for a set of about 400 compounds, results of (1)H NMR characterization are compared to the more customary LC-UV-MS method. NMR and MS data agree for identity on the vast majority of cases (84% positive and 5% negative), whereas the remaining cases (11%) are marked as highly impure only after NMR spectra analysis. Most importantly, determination of concentration rather than that of relative purity appears the right choice for a correct evaluation of biochemical potency.

Chromatography, High Pressure Liquid↗
PubMed1994

Introduction of YACs into intact yeast cells by a procedure which shows low levels of recombinagenicity and co-transformation.

Yeast artificial chromosomes (YACs) enable the cloning and analysis of large segments of genomic DNA and permit the isolation of sequences which are impossible to maintain in Escherichia coli. However, the construction of genome libraries in YAC vectors is beset by a number of technical problems, not least of which is the creation of cloned fragments which are not true representatives of the donor genome. These artefactual clones arise mainly due to intra-fragment rearrangements or inter-fragment chimaera formation, both phenomena resulting from the activity of the host yeast's mitotic recombination system. We demonstrate that this system is significantly stimulated by the spheroplasting step of the standard YAC transformation system. In contrast, the transformation of intact yeast cells by either the lithium method or a new lithium-free protocol is much less recombinagenic. It is not possible to introduce high molecular weight YACs into yeast using the lithium protocol, but we find that such molecules may be introduced into pde2-mutants using the lithium-free approach. Since intact cells are transformed by this method, automation of post-transformation steps in the construction of YAC libraries is facilitated. Moreover, the frequency of cotransformation (and, therefore, chimera formation) is significantly reduced. However, these advantages do incur a penalty. Yields of YAC transformants by this simplified intact cell approach are reduced some 25- to 30-fold compared to those obtained by the spheroplast transformation route. Nevertheless, the considerable advantages of the new system recommend it for a number of applications.

Chromosomes, Artificial, Yeast↗
PubMedDate not supplied

Managing the film library: what to do until the panacea arrives.

Today's imaging departments are complex and often decentralized. Despite such growth, the film library is often overlooked and minimally planned for. The oversight may come from the anticipation of picture archiving and communications systems (PACS), considered by many to be the panacea for film management, but in reality, still only a concept, or at best, in its early stages. Since most departments do not anticipate going filmless for many years and others expect to continue hard copy imaging at a 20 to 30 percent rate, managing the film library remains an important issue. In general, film librarians' daily work remains labor intensive, despite attempts to automate some tasks. Yet when cutbacks are made, this support staff is often cut. One way to deal with service and expansion issues in the film library is to stop and evaluate the situation, assess the decline in the level of service, and determine what issues need improvement. Managers should gather information, analyze the data and listen to all interested parties to be sure they understand the overall problems, often best done by someone from outside the department. Analyzing and evaluating work processes, even observing the design of work areas, can be useful in understanding productivity inside the department. An invaluable way to gather data is to listen to those who carry out the daily tasks and those who use the services. Until their needs are understood by management, further changes will have minimal success. When a list of issues is assembled, managers should establish a team to review and prioritize them. Team members will need to understand departmental long-term plans before making recommendations. While every library's operations are unique, a key problem remains lost films. Policies and procedures for removing films must come from hospital senior administration, while managing film within the department is everyone's job. With the chaos under control, managers will be ready to face the challenge of electronic imaging.

Efficiency, Organizational↗
PubMed1996

Design, synthesis and use of binary encoded synthetic chemical libraries.

With the advent of combinatorial chemistry a new paradigm is evolving in the field of drug discovery. The approach is based on an integration of chemistry, high-throughput screening and automation engineering. The chemistry arm is usually based on solid-phase synthesis technology as the preferred approach to library construction. One of the most powerful of the solid-phase methods is encoded split synthesis, in which the reaction history experience by each polymeric bead is unambiguously recorded. This split-and-pool approach, employing chemically robust tags, was used to construct a 85,000-membered dihydrobenzopyran library.

Benzopyrans↗
PubMed2002

Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates.

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.

Alleles↗
PubMedDate not supplied

Screening of synthetic peptide libraries with radiolabeled acceptor molecules.

A method has been developed for the identification of specific acceptor molecule-binding sequences from a chemically synthesized peptide library. The peptide resin beads, each bead carrying one peptide sequence, are incubated with radiolabeled acceptor molecule and subsequently immobilized in a thin layer of agarose. Resin beads that carry acceptor molecule-specific sequences are identified by autoradiography and subjected to automated gas-phase sequencing. The method was tested by screening a synthetic pentapeptide library with an anti-beta-endorphin monoclonal antibody.

Amino Acid Sequence↗
PubMed1971

New trends in medical libraries in hospitals.

External and internal forces affecting medical library services are examined. Public Law 89-239, the Heart, Cancer and Stroke Amendments of 1965, and Public Law 89-291, the Medical Library Assistance Act of 1965 which was extended by Public Law 91-212 have an impact on medical libraries. The Veterans Administration relationships with primary beneficiaries of these laws are explained. Internally, effort has been expended through automation, networking, and extension of resources by amalgamation of books and audiovisuals to evolve responsive concepts with emphasis on independence through interdependence. The Veterans Administration Library Service plans and accomplishments in these areas are reviewed.

Information Services↗