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PubMed1989

Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.

The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation, and utility of the polymerase chain reaction method for amplifying DNA. We report the cloning and expression of Taq DNA polymerase in Escherichia coli. From a lambda gt11:Taq library we identified a Taq DNA fragment encoding an epitope of Taq DNA polymerase via antibody probing. The fusion protein from the lambda gt11:Taq candidate selected an antibody from an anti-Taq polymerase polyclonal antiserum which reacted with Taq polymerase on Western blots. We used the lambda gt11 clone to identify Taq polymerase clones from a lambda Ch35:Taq library. The complete Taq DNA polymerase gene has 2499 base pairs. From the predicted 832-amino acid sequence of the Taq DNA polymerase gene, Taq DNA polymerase has significant similarity to E. coli DNA polymerase I. We subcloned and expressed appropriate portions of the insert from a lambda Ch35 library candidate to yield thermostable, active, truncated, or full-length forms of the protein in E. coli under control of the lac promoter.

Amino Acid Sequence↗
PubMed2004

GEISHA, a whole-mount in situ hybridization gene expression screen in chicken embryos.

Despite the increasing quality and quantity of genomic sequence that is available to researchers, predicting gene function from sequence information remains a challenge. One method for obtaining rapid insight into potential functional roles of novel genes is through gene expression mapping. We have performed a high throughput whole-mount in situ hybridization (ISH) screen with chick embryos to identify novel, differentially expressed genes. Approximately 1,200 5' expressed sequence tags (ESTs) were generated from cDNA clones of a Hamburger and Hamilton (HH) stage 4-7 (late gastrula) chick embryo endoderm-mesoderm library. After screening to remove ubiquitously expressed cDNAs and internal clustering and after comparison to GenBank sequences, remaining cDNAs (representing both characterized and uncharacterized genes) were screened for expression in HH stage 3-14 embryos by automated high throughput ISH. Of 786 cDNAs for which ISH was successfully performed, approximately 30% showed ubiquitous expression, 40% were negative, and approximately 30% showed a restricted expression pattern. cDNAs were identified that showed restricted expression in every embryonic region, including the primitive streak, somites, developing cardiovascular system and neural tube/neural crest. A relational database was developed to hold all EST sequences, ISH images, and corresponding BLAST report information, and to enable browsing and querying of data. A user interface is freely accessible at http://geisha.biosci.arizona.edu. Results show that high throughput whole-mount ISH provides an effective approach for identifying novel genes that are differentially expressed in the developing chicken embryo.

Animals↗
PubMed1992

Ion-trap detection of volatile organic compounds in alveolar breath.

We describe a method for the collection and microanalysis of the volatile organic compounds in human breath. A transportable apparatus supplies subjects with purified air and samples their alveolar breath; the volatile organic compounds are captured in an adsorptive trap containing activated carbon and molecular sieve. The sample is thermally desorbed from the trap in an automated microprocessor-controlled device, concentrated by two-stage cryofocusing, and assayed by gas chromatography with ion-trap detection. Compounds are identified by reference to a computer-based library of mass spectra with subtraction of the background components present in the inspired air. We used this device to study 10 normal subjects and determined the relative abundance of the volatile organic compounds in their alveolar breath. The breath-collecting apparatus was convenient to operate and was well tolerated by human volunteers.

Air Pollutants↗
PubMed2003

Exploring protein-protein interactions with phage display.

Protein-protein interactions mediate essentially all biological processes. A detailed understanding of these interactions is thus a major goal of modern biological chemistry. In recent years, genome sequencing efforts have revealed tens of thousands of novel genes, but the benefits of genome sequences will only be realized if these data can be translated to the level of protein function. While genome databases offer tremendous opportunities to expand our knowledge of protein-protein interactions, they also present formidable challenges to traditional protein chemistry methods. Indeed, it has become apparent that efficient analysis of proteins on a proteome-wide scale will require the use of rapid combinatorial approaches. In this regard, phage display is an established combinatorial technology that is likely to play an even greater role in the future of biology. This article reviews recent applications of phage display to the analysis of protein-protein interactions. With combinatorial mutagenesis strategies, it is now possible to rapidly map the binding energetics at protein-protein interfaces through statistical analysis of phage-displayed protein libraries. In addition, naïve phage-displayed peptide libraries can be used to obtain small peptide ligands to essentially any protein of interest, and in many cases, these binding peptides act as antagonists or even agonists of natural protein functions. These methods are accelerating the pace of research by enabling the study of complex protein-protein interactions with simple molecular biology methods. With further optimization and automation, it may soon be possible to study hundreds of different proteins in parallel with efforts comparable to those currently expended on the analysis of individual proteins.

Animals↗
PubMedDate not supplied

Synthesis of a library of 2-alkyl-3-alkyloxy-2H-indazole-6-carboxamides.

A library of 200 2-alkyl-3-alkyloxy-2H-indazole-6-carboxamides was synthesized using parallel solution-phase methods. The indazole cyclization reaction was optimized for library production with the best yields resulting from controlled alcohol/water solvent ratios. The key step, a heterocyclization reaction, proceeds by N,N-bond formation and delivers the 2H-indazole scaffold. Automated preparative HPLC was utilized to provide pure compounds on a 10+ mg scale.

Amides↗
PubMed1999

Homogeneous Cell- and Bead-Based Assays for High Throughput Screening Using Fluorometric Microvolume Assay Technology.

High throughput drug screening has become a critical component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds has resulted in a requirement for assays and instrumentation that are amenable to nonradioactive formats and that can be miniaturized. Homogeneous assays that minimize upstream automation of the individual assays are also preferable. Fluorometric microvolume assay technology (FMAT) is a fluorescence-based platform for the development of nonradioactive cell- and bead-based assays for HTS. This technology is plate format-independent, and while it was designed specifically for homogeneous ligand binding and immunological assays, it is amenable to any assay utilizing a fluorescent cell or bead. The instrument fits on a standard laboratory bench and consists of a laser scanner that generates a 1 mm(2) digitized image of a 100-µmm deep section of the bottom of a microwell plate. The instrument is directly compatible with a Zymark Twistertrade mark (Zymark Corp., Hopkinton, MA) for robotic loading of the scanner and unattended operation in HTS mode. Fluorescent cells or beads at the bottom of the well are detected as localized areas of concentrated fluorescence using data processing. Unbound flurophore comprising the background signal is ignored, allowing for the development of a wide variety of homogeneous assays. The use of FMAT for peptide ligand binding assays, immunofluorescence, apoptosis and cytotoxicity, and bead-based immunocapture assays is described here, along with a general overview of the instrument and software.

Journal Article↗
PubMed2002

In vitro production and screening of DNA polymerase eta mutants for catalytic diversity.

Mutant DNA polymerases have become an increasingly important tool in biotechnology. The ability to examine the activity and specific properties of enzymes has a crucial role in the characterization of the enzyme. We have developed several systems for characterizing DNA polymerases that combine random mutagenesis with in vivo selection systems. However in vivo screening systems for specific properties are sometimes unavailable. The ability to quickly screen for polymerase activity has many applications, including the identification of compounds that can inhibit polymerase activity, identifying the properties of newly discovered polymerases, and engineering new biological properties into existing polymerases. These applications can both expand the knowledge of the basic science of polymerases and can further industrial efforts to identify new drugs that specifically target polymerase activity. Here we present a high-throughput in vitro assay to select for active polymerases. We show the applicability of this assay by measuring the level of activity for a set of in vitro synthesized polymerase mutants and by screening for the incorporation of a fluorescent nucleotide analog by DNA polymerases.

Automation↗
PubMed1995

The impact of IAIMS on the work of information experts. Integrated Advanced Information Management Systems.

Integrated Advanced Information Management Systems (IAIMS) programs differ but have certain characteristics in common. Technological and organizational integration are universal goals. As integration takes place, what happens to those implementing the vision? A survey of 125 staff members, or information experts, involved in information or informatics at an IAIMS-funded institution was conducted during the last year of the implementation phase. The purpose was to measure the impact of IAIMS on the jobs of those in the library and related service units, and the computing, telecommunications, and health informatics divisions. The researchers used newly developed scales measuring levels of integration (knowledge of and involvement with other departments), customer orientation (focus on the user), and informatedness (changes in the nature of work beyond automation of former routines). Ninety-four percent of respondents indicated that their jobs had changed a great deal; the changes were similar regardless of division. To further investigate the impact of IAIMS on librarians in particular, a separate skills survey was conducted. The IAIMS librarians indicated that technology and training skills are especially needed in the new, integrated environment.

Analysis of Variance↗
PubMed2002

Use of semi-permeable membrane devices and solid-phase extraction for the wide-range screening of microcontaminants in surface water by GC-AED/MS.

An automated GC-MS-based screening method was developed for over 400 industrial, agrochemical and household chemicals. Extracted ion chromatograms were used and the method was aimed at creating a minimum number of false positives. The compound polarity range usually associated with solid-phase extraction was extended to include very apolar, bioaccumulative, compounds by using the complementary semi-permeable membrane device technique. Real-life samples were taken at four locations in the main Dutch river systems and one in an agricultural area. Some 150 compounds were detected in the low-ng/l to low-microg/l range. Next to the target compounds, several brominated and chlorinated non-target compounds were detected by means of GC with atomic emission detection and tentatively identified using mass spectral library searching.

Automation↗
PubMed2000

Solid-phase extraction for combinatorial libraries.

Solid-phase extraction (SPE) has during the last three years emerged as a convenient method for the purification of compound libraries prepared by solution synthesis. The widespread use of SPE in combinatorial chemistry can be explained by straightforward SPE method development facilitated by the availability of numerous commercial SPE resins. High-speed automated SPE is readily accomplished by taking advantage of commercial laboratory robot systems. The present review summarizes and discusses advancements made in the use of different SPE resins and molecule tagging techniques for optimization of ion-exchange, reversed-phase, normal-phase and fluorous-phase SPE in combinatorial chemistry.

Chromatography, Liquid↗
PubMed2001

IBC's Drug Discovery Technology - Europe 2001. 23-26 April 2001, Stuttgart, Germany.

Specific sectors within the pharmaceutical industry are rapidly changing in response to technological advances. Genomics, high-throughput automated chemistry, high-throughput screening (HTS), ADME/Tox screening and informatics, provide new opportunities, but also create new bottlenecks. In addition, the selection and validation of biological targets, the proper design of compound libraries, data and knowledge management, and as the last and crucial step, the proof of therapeutic relevance by clinical trials, generates an enormous financial load on biotechnology and pharmaceutical companies. In the future, drug development costs might be reduced due to an ongoing pressure for shorter development cycles of new drugs. IBC's Drug Discovery Technology Europe 2001 conference addressed many aspects relevant to drug discovery technologies, and, along with its US partner conference, provides an annual meeting place for researchers and company executives to interact and exchange ideas.

Journal Article↗
PubMed2002

[CT/MRI-based software for 3-D brachytherapy planning system].

The paper presents a 3D Brachytherapy Planning System based on CT/MRI images. Unlike other percutaneous radiotherapy planning systems, it provides doctors with an interactive source layout function, including accurate and quick dosage distribution calculation and dynamical 2D/3D display. A special template library and an integrated planning evaluation/alert subsystem are incorporated in the system. The system successfully has many functions such as data acquisition, registration, segmentation, image processing, 3D reconstruction and planning report output. Various automated procedures, 3-D graphics and a user-friendly interface GUI enable the system to be a powerful and practical one meeting well the chinical therapeutic demands.

Algorithms↗
PubMed1993

A simple and efficient method for constructing high resolution physical maps.

This paper describes a simple and efficient walking method for constructing high resolution physical maps and discusses its applications to genome analysis. The method is an integration of three strategies: (1) use of a highly redundant library of 3Kb-long subclones; (2) construction of a multidimensional pool from the library; (3) direct application of a PCR (polymerase chain reaction)-based screening technique to the pooled library, with two PCR primers, one from the end of the subcloning vector and the other from the leading edge of the walk. This technique allows not only detection of each overlapping subclone but simultaneous determination of its orientation and the size of its overlap. The end of the subclone with the smallest overlap is sequenced and a primer is designed for the next step in the walk. Iteration of the screening procedure with minimum overlapping subclones results in completion of the high resolution map. Using this method, a 3Kb-resolution map was constructed from an 80Kb region of the bithorax complex of Drosophila melanogaster. The method is general enough to be applicable to DNA from other species, and simple enough to be automated.

Animals↗
PubMed1996

High-quality automated DNA sequencing primed with hexamer strings.

The finishing phase of genome sequencing projects is expensive, in part, because of the cost of de novo synthesis of custom primers and the management burden associated with obtaining and using them for primer walking. One approach to reduce these high costs is the use of a presynthesized library of short oligonucleotides (8-10 bases) rather than long primers. The use of such a library eliminates the need for custom synthesis of oligonucleotides, providing the convenience of priming from any site by combining two to three short oligonucleotides to form a string with the required specificity. The first practical implementation of this strategy presented a robust protocol for using hexamer strings with radioisotopic labelling. Whereas versions of this technique have subsequently been implemented on fluorescent sequencers we felt that there was a need to develop and extensively test a protocol that consistently gave read lengths comparable to dye-terminator sequencing with longer primers. We have developed a new two-cycle fluorescent Sequenase terminator procedure for using hexamer strings. We tested this procedure using a set of 32 different 3 hexamer primer strings, each known to be functional to some degree in radioisotopic sequencing, on single-stranded M13mp18 template and ABI 373 DNA sequencers. The overall success rate of priming with these hexamer primer strings is 97% with the failure of only one string. In this case, the corresponding 18-mer primer also failed to produce usable sequence from M13mp18 template. The average read length from reactions successfully primed with the 31 different hexamer strings was 461 bases with > 99% base-calling accuracy. The current protocol is robust enough to be used in virtually any situation where primer walking on single-stranded templates is used. The success rate and read lengths make it universally applicable to the sequencing of single-stranded templates on automated sequencers. It is also amenable to automation.

Automation↗
PubMed1999

Identification of Epichloë endophytes in planta by a microsatellite-based PCR fingerprinting assay with automated analysis.

Epichloë endophytes are a group of filamentous fungi that include both sexual (Epichloë) and asexual (Neotyphodium) species. As a group they are genetically diverse and form both antagonistic and mutualistic associations with temperate grasses. We report here on the development of a microsatellite-based PCR system for fingerprinting this group of fungi with template isolated from either culture or infected plant material. M13mp19 partial genomic libraries were constructed for size-fractionated genomic DNA from two endophyte strains. These libraries were screened with a mixture of DIG-labeled dinucleotide and trinucleotide repeat probes. Positive clones were sequenced, and nine unique microsatellite loci were identified. An additional microsatellite was serendipitously identified in the 3' untranscribed region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase gene from N. lolii Lp19. Primers were designed for each locus and a panel of endophytes, from different taxonomic groupings, was screened to determine the degree of polymorphism. On the basis of these results a multiplex assay was developed for strain identification with fluorescently labeled primers for five of these loci. Using this system the size of the products amplified can be precisely determined by automated analysis, and an allele profile for each strain can be readily generated. The assay was shown to resolve endophyte groupings to the level of known isozyme phenotype groupings. In a blind test the assay was used successfully to identify a set of endophytes in planta. A reference database of allele sizes has been established for the panel of endophytes examined, and this will be expanded as new strains are analyzed.

Alleles↗
PubMed2005

Small-molecule screening and profiling by using automated microscopy.

Automated fluorescence microscopy provides a powerful tool for analyzing the physiological state of single cells with high throughput and high information content. Here I discuss two types of experiments in which this technology was used to discover and characterize bioactive small molecules. In phenotypic-screening experiments, the goal is to find "hits" with specific effects on cells by screening large libraries of small molecules. An example is screening for small molecules that perturb mitosis by novel mechanisms. In cytological-profiling experiments, the goal is to characterize the bioactivity of a limited number of small molecules in considerable depth, and thus understand their mechanism and toxicities at the cellular level. I discuss an example in which 100 small molecules with known bioactivity were profiled by using multiple fluorescent probes, and clustered into mechanistic classes by automated statistical analysis.

Algorithms↗
PubMed1999

Construction and analysis of arrayed cDNA libraries.

For any attempt to understand the biology of an organism the incorporation of a cDNA-based approach is unavoidable, because it is a major approach to studying gene function. The complete sequence of the genome alone is not sufficient to understand any organism; its gene regulation, expression, splice variation, posttranslational modifications, and protein-protein interactions all need to be addressed. Because the majority of vertebrate genes have probably been identified as ESTs the next stage of the Human Genome Project is attributing functional information to these sequences. In most cases hybridization-based approaches on arrayed pieces of DNA represent the most efficient way to study the expression level and splicing of a gene in a given tissue. Similar technology, now being applied at the protein level using protein expression libraries, high-density protein membranes, and antibody screening, should allow studies of protein localization and modifications. Coupled to these approaches is the use of technologies, which although lacking the highly parallel nature of hybridization, can potentially characterize large numbers of samples individually and with high accuracy. Automated gel-based DNA sequencing is an example of such a technique; protein sequencing and mass fingerprinting are further examples. In the case of mass spectroscopic analysis, the speed and sensitivity are vastly superior to that of gel-based approaches; however, the preparation of samples is more tedious. Our laboratory is developing a system to characterize DNA samples by mass spectrometry, allowing more rapid genotyping than is currently possible using gel-based technologies ([symbol: see text]. Gut, [symbol: see text]. Berlin and H. Lehrach, personal communication, 1998). Such technology would make information on gene polymorphisms widely accessible. Data generated using all of these techniques at the DNA and protein level will be connected by both protein expression libraries and database comparisons; finally, two hybrid library screens will identify many of the protein-protein interactions, linking genes together. In this way we will start to understand the interplay between genes on a global scale, both at the level of molecular interaction and the biological processes they regulate.

Alternative Splicing↗