Biotechniques

A Simple Way to Treat PCR Products Prior to Sequencing Using ExoSAP-IT(R).

Publication Date: 2008 May PMID: 18476841
Authors: Bell, J.
Journal: Biotechniques

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A major step towards efficient sample preparation with bead-beating.

Publication Date: 2008 May PMID: 18476840
Authors: Verollet, R.
Journal: Biotechniques

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De Novo Assembly and Genomic Structural Variation Analysis with Genome Sequencer FLX 3K Long-Tag Paired End Reads.

Publication Date: 2008 May PMID: 18476839
Authors: Jarvie, T. - Harkins, T.
Journal: Biotechniques

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Prestige Antibodiestrade mark-Monospecific Antibodies Designed for Immunohistochemical Analysis.

Publication Date: 2008 May PMID: 18476838
Authors: Gunneras, S. - Agaton, C. - Djerbi, S. - Hansson, M.
Journal: Biotechniques

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High Content Analysis with AxioVision ASSAYbuildertrade mark: Applications in Pharmaceutical Biology.

Publication Date: 2008 May PMID: 18476837
Authors: Kraus, B. - Wolff, H.
Journal: Biotechniques

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Protein detection enhanced by 3DNA dendrimer signal amplification.

Publication Date: 2008 May PMID: 18476836
Authors: Mora, J. - Zielinski, T. - Nelson, B. - Getts, R.
Journal: Biotechniques

DNA dendrimers, conjugated with both anti-biotin antibodies and up to 350 labeling entities, were designed and adapted to protein microarray and enzyme-linked immunosorbent assay (ELISA) to improve the limits of protein detection with no additional steps or equipment. Application of conjugated dendrimers to standard ELISA cytokine detection resulted in up to threefold improvement of the limits of detection with no significant increase in the inter- and intra-assay coefficient of variation (CV) compared to streptavidin horseradish peroxidase (SA-HRP) detection. The adaptation of conjugated dendrimers to protein microarray cytokine detection resulted in up to 10-fold improvement of the limits of detection, but assay conditions would have to be optimized to decrease the intra- and inter-assay %CVs.

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Simplicity, function, and legibility in an enhanced ambigraphic nucleic acid notation.

Publication Date: 2008 May PMID: 18476835
Authors: Rozak, D. - Rozak, A.
Journal: Biotechniques

We previously showed that an ambigraphic nucleic acid notation, based on symmetrical lowercase Roman characters, permits users to complement DNA by physically rotating the sequence text 180 degrees . This article describes an enhanced ambigraphic notation, which uses concept-related symbol design, rather than the arbitrary set of symbols that constitute the Roman alphabet, to logically encode the four DNA bases and 11 ambiguity characters. As ambigrams, the symbols continue to permit the rapid derivation of complementary sequences and visualization of palindromic DNA. In addition, the new AmbiScript notation uses legibility principles to support the identification of sequence polymorphism and improves writing efficiency by requiring fewer strokes per character than the International Union of Pure and Applied Chemistry (IUPAC) notation.

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A quantitative real-time PCR method for absolute telomere length.

Publication Date: 2008 May PMID: 18476834
Authors: O'Callaghan, N. - Dhillon, V. - Thomas, P. - Fenech, M.
Journal: Biotechniques

Telomere shortening is an important risk factor for cancer and accelerated aging. Here we describe the development of a simple and reproducible method to measure absolute telomere length. Based on Cawthon's quantitative real-time PCR (qRT-PCR) assay, our method uses an oligomer standard that can be used to generate absolute telomere length values rather than relative quantification. We demonstrate a strong correlation between this improved method and the "gold standard" of telomere length measurement-terminal restriction fragment analysis (TRF) by Southern hybridization. The capability to generate absolute telomere length values should allow a more direct comparison of results between experiments within and between laboratories.

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Isolation and quantitative estimation of diesel exhaust and carbon black particles ingested by lung epithelial cells and alveolar macrophages in vitro.

Publication Date: 2008 May PMID: 18476833
Authors: Saxena, R. - Ian Gilmour, M. - Hays, M.
Journal: Biotechniques

A new procedure for isolating and estimating ingested carbonaceous diesel exhaust particles (DEP) or carbon black (CB) particles by lung epithelial cells and macrophages is described. Cells were incubated with DEP or CB to examine cell-particle interaction and ingestion. After various incubation periods, the cells were separated from free extracellular DEP or CB particles by Ficoll density gradient centrifugation and dissolved in hot sodium dodecyl sulfate detergent. Insoluble DEP or CB residues were isolated by high-speed centrifugation, and the elemental carbon (EC) concentrations in the pellets were estimated by a thermal-optical-transmittance method (i.e., carbon analysis). From the EC concentration, the amount of ingested DEP or CB could be calculated. The described technique allowed the determination of the kinetics and dose dependence of DEP uptake by LA4 lung epithelial cells and MHS alveolar macrophages. Both cell types ingested DEP to a similar degree; however, the MHS macrophages took up significantly more CB than the epithelial cells. Cytochalasin D, an agent that blocks actin polymerization in the cells, inhibited approximately 80% of DEP uptake by both cell types, indicating that the process was actin-dependent in a manner similar to phagocytosis. This technique can be applied to examine the interactions between cells and particles containing EC and to study the modulation of particle uptake in diseased tissue.

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Purification of inclusion body-forming peptides and proteins in soluble form by fusion to Escherichia coli thermostable proteins.

Publication Date: 2008 May PMID: 18476832
Authors: Thapa, A. - Shahnawaz, M. - Karki, P. - Raj Dahal, G. - Golam Sharoar, M. - Yub Shin, S. - Sup Lee, J. - Cho, B. - Park, I. S.
Journal: Biotechniques

Proteins and peptides expressed in the prokaryotic system often form inclusion bodies. Solubilization and refolding procedures can be used for their recovery, but this process remains difficult. One strategy for improving the solubility of a protein of interest is to fuse it to a highly soluble protein. To select a suitable fusion partner capable of solubilizing the aggregation-prone (inclusion body-forming) proteins and peptides, Escherichia coli thermostable proteins were identified and tested. Among them, trigger factor (TF) protein was selected because of its high expression and stability. Using an expression system based on fusion to TF, selected proteins and peptides that otherwise form inclusion bodies were expressed in soluble state and were purified like other soluble proteins. This system provides a convenient method for production of aggregation-prone proteins and peptides.

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A new technique for selective identification and mapping of enhancers within long genomic sequences.

Publication Date: 2008 May PMID: 18476831
Authors: Chernov, I. - Stukacheva, E. - Akopov, S. - Didych, D. - Nikolaev, L. - Sverdlov, E.
Journal: Biotechniques

We report a new experimental method of direct selection, identification, and mapping of potential enhancer sequences within extended stretches of genomic DNA. The method allows simultaneous cloning of a quantity of sequences instead of tedious screening of the separate ones, thus providing a robust and high-throughput approach to the mapping of enhancers. The selection procedure is based on the ability of such sequences to activate a minimal promoter that drives expression of a selective gene. To this end a mixture of short DNA fragments derived from the segment of interest was cloned in a retroviral vector containing the neomycin phosphotransferase II gene under control of a cytomegalovirus (CMV) minimal promoter. The pool of retroviruses obtained was used to infect HeLa cells and then to select neomycin-resistant colonies containing constructs with enhancer-like sequences. The pool of the genomic fragments was rescued by PCR and cloned, forming a library of the potential enhancers. Fifteen enhancer-like fragments were selected from 1-Mb human genome locus, and enhancer activity of 13 of them was verified in a transient transfection reporter gene assay. The sequences selected were found to be predominantly located near 5' regions of genes or within gene introns.

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TEV protease-mediated cleavage in Drosophila as a tool to analyze protein functions in living organisms.

Publication Date: 2008 May PMID: 18476830
Authors: Harder, B. - Schomburg, A. - Pflanz, R. - Kustner, K. - Gerlach, N. - Schuh, R.
Journal: Biotechniques

Drosophila provides a powerful experimental system to analyze gene functions in a multi-cellular organism. Here we describe an in vivo method that interferes with the integrity of selected proteins through site-specific cleavage in Drosophila. The technique is based on the highly specific seven-amino-acid recognition site of the tobacco etch virus (TEV) protease. We established transgenic fly lines that direct TEV protease expression in various tissues without affecting fly viability. The insertion of the TEV protease recognition site in defined positions of target proteins mediates their sequence-specific cleavage after controlled TEV protease expression in the fly. Thereby, this technique is a powerful tool that allows the in vivo manipulation of selected proteins in a time- and tissue-specific manner.

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Affymetrix Whole-Transcript Human Gene 1.0 ST array is highly concordant with standard 3' expression arrays.

Publication Date: 2008 May PMID: 18476829
Authors: Pradervand, S. - Paillusson, A. - Thomas, J. - Weber, J. - Wirapati, P. - Hagenbuchle, O. - Harshman, K.
Journal: Biotechniques

The recently released Affymetrix Human Gene 1.0 ST array has two major differences compared with standard 3' based arrays: (i) it interrogates the entire mRNA transcript, and (ii) it uses DNA targets. To assess the impact of these differences on array performance, we performed a series of comparative hybridizations between the Human Gene 1.0 ST and the Affymetrix HG-U133 Plus 2.0 and the Illumina HumanRef-8 BeadChip arrays. Additionally, both RNA and DNA targets were hybridized on HG-U133 Plus 2.0 arrays. The results show that the overall reproducibility of the Gene 1.0 ST array is best. When looking only at the high intensity probes, the reproducibility of the Gene 1.0 ST array and the Illumina BeadChip array is equally good. Concordance of array results was assessed using different inter-platform mappings. Agreements are best between the two labeling protocols using HG-U133 Plus 2.0 array. The Gene 1.0 ST array is most concordant with the HG-U133 array hybridized with cDNA targets. This may reflect the impact of the target type. Overall, the high degree of correspondence provides strong evidence for the reliability of the Gene 1.0 ST array.

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pBINPLUS/ARS: an improved plant transformation vector based on pBINPLUS.

Publication Date: 2008 May PMID: 18476828
Authors: Belknap, W. - Rockhold, D. - McCue, K.
Journal: Biotechniques

Binary plant transformation vectors are widely used for introduction of transgenes into plants via Agrobacterium tumefaciens-mediated transformation. We report the construction of a binary plant vector pBINPLUS/ARS based on the pBINPLUS vector. Improvements introduced into pBINPLUS/ARS include the use of nonproprietary (ubiquitin-3 gene of Solanum tuberosum) promoter and terminator sequences for transcription of the NptII selectable marker and introduction of rare 8-bp restriction enzyme sites flanking both the NptII coding sequence (PmeI) and the entire selectable marker gene (FseI). This vector offers all of the advantages of its predecessor pBINPLUS and its helper plasmid pUCAP, which use the proprietary nopaline synthase promoter and terminator, while allowing for facile modification of selectable marker sequences in complex binary vector constructs. pBINPLUS/ARS has been used to introduce transgenes into potato and other crop species and is available to all researchers in academic, government, and industrial laboratories for proof-of-principle and commercial applications.

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Molecular dynamics and forces of a motile cell simultaneously visualized by TIRF and force microscopies.

Publication Date: 2008 May PMID: 18476827
Authors: Iwadate, Y. - Yumura, S.
Journal: Biotechniques

Cells must exert traction forces onto the substratum for continuous migration. Molecular dynamics such as actin polymerization at the front of the cell and myosin II accumulation at the rear should play important roles in the exertion of forces required for migration. Therefore, it is important to reveal the relationships between the traction forces and molecular dynamics. Traction forces can be calculated from the deformation of the elastic substratum under a migrating cell. A transparent and colorless elastic substratum with a high refractive index (1.40) and a low Young's modulus (1.0 kPa) were made from a pair of platinum-catalyzed silicones. We used this substratum to develop a new method for simultaneous recording of molecular dynamics and traction forces under a migrating cell in which total internal refractive fluorescence (TIRF) and force microscopies were combined. This new method allows the detection of the spatiotemporal distribution of traction forces produced by individual filopodia in migrating Dictyostelium cells, as well as simultaneous visualization of these traction forces and the dynamics of filamentous myosin II.

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