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Gene Synthesis

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Systematic targeted gene deletion using the gene-synthesis method in fission yeast.
J Microbiol Methods. 2014 Aug 19;
Authors: Nam M, Lee SJ, Han S, Kim D, Lee M, Kang EJ, Park HO, Lee AR, Lee S, Ki…

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Optimization and scale-up of oligonucleotide synthesis in packed bed reactors using computational fluid dynamics modeling.

Biotechnol Prog. 2014 Jul 31;

Authors: Wolfrum C, Josten A, Götz P

Abstract
A computational fluid dynamics (CFD) model for the analysis of oligonucleotide synthesis in packed bed reactors was developed and used to optimize the scale up of the process. The model includes reaction kinetics data obtained under well defined conditions comparable to the situation in the packed bed. The model was validated in terms of flow conditions and reaction kinetics by comparison with experimental data. Experimental validation and the following model parameter studies by simulation were performed on the basis of a column with 0.3 g oligonucleotide capacity. The scale-up studies based on CFD modelling were calculated on a 440 g scale (oligonucleotide capacity). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 2014.

PMID: 25105730 [PubMed - as supplied by publisher]

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SpeedyGenes: an improved gene synthesis method for the efficient production of error-corrected, synthetic protein libraries for directed evolution.
Protein Eng Des Sel. 2014 Aug 9;
Authors: Currin A…

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Gene Assembly from Chip-Synthesized Oligonucleotides.
Curr Protoc Chem Biol. 2012 Mar 1;2012
Authors: Eroshenko N, Kosuri S, Marblestone AH, Conway N, Church GM
Abstract
De novo synt…

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Preparing compound heterozygous reference material using gene synthesis technology: a model of thrombophilic mutations.

Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub. 2014 Jul 18;

Authors: Beranek M, Drastikova M, Dulicek P, Palicka V

Abstract
AIMS: The aim of our study is to present a novel approach for preparing a compound heterozygous reference material (hetRM) using gene synthesis technology with inverted insertion of wild-type and mutant fragments into a single cloning vector. Factor II (G20210A) and Factor V (G1691A Leiden) gene mutations were used as an experimental model.
METHODS: During the gene synthesis, DNA fragments were aligned in the following order: G1691 FV wild-type forward strain, G20210 FII wild-type forward strain, 1691A FV mutant reverse strain, 20210A FII mutant reverse strain. The complete chain was inserted into a pIDT SMART cloning vector and amplified in an E. coli competent strain. For assessing hetRM characteristics and commutability, we used real-time PCR with subsequent melting curve analysis, real-time PCR with hydrolysis probes, allele-specific amplification, reverse hybridization, and dideoxynucleotide DNA sequencing.
RESULT: All five methods yielded concordant results of DNA analysis of the hetRM. Differences in real-time PCR cycle threshold values after six-months of storage at -80 °C were not statistically significant from those obtained from freshly prepared hetRM aliquots, which is a good indication of their stability.
CONCLUSION: By applying the procedures of gene synthesis and cloning technology, we prepared and verified a model genetic reference material for FII G20210A and FV G1691A testing with a compound heterozygous genotype. The hetRM was stable, commutable, and available in large quantities and in a wide concentration range.

PMID: 25059235 [PubMed - as supplied by publisher]

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The Maize TFome – Development of a transcription factor open reading frame collection for functional genomics.

Plant J. 2014 Jul 23;

Authors: Burdo B, Gray J, Goetting-Minesky MP, Wittler B, Hunt M, Li T, Velliquette D, Thomas J, Gentzel I, Brito MD, Mejía-Guerra MK, Connolly LN, Qaisi D, Li W, Casas MI, Doseff AI, Grotewold E

Abstract
Establishing the architecture of the gene regulatory networks (GRNs) responsible for the control of transcription of all genes in an organism is a natural development that follows genome sequence elucidation. GRN reconstruction requires the availability of a series of molecular tools and resources that so far have been limited to a few model organisms. One such resource consists of collections of transcription factor (TF) open reading frames (ORFs) cloned into vectors that facilitate easy expression in plants or microorganisms. In this study, we describe the development of a publicly available maize TF ORF collection (TFome) of 2,034 clones corresponding to 2,017 unique gene models in recombination-ready vectors that make possible the facile mobilization of the TF sequences into a number of different expression vectors. The collection also includes several hundred co-regulators (CoREG), which we classified into well-defined families, and for which propose here a standard nomenclature, as we have previously done for TFs. We describe the strategies employed to overcome the limitations associated with cloning ORFs from a genome that remains incompletely annotated, with a partial full-length cDNA set available, and with many TF/CoREG genes lacking experimental support. This required, in many instances, combining genome-wide expression data with gene synthesis approaches. The strategies developed will be valuable for developing similar resources for other agriculturally important plants. Information on all the clones generated is available through the GRASSIUS knowledgebase (http://grassius.org/). This article is protected by copyright. All rights reserved.

PMID: 25053252 [PubMed - as supplied by publisher]

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Structure-function analysis of Drosophila notch using genomic rescue transgenes.
Methods Mol Biol. 2014;1187:29-46
Authors: Leonardi J, Jafar-Nejad H
Abstract
One of the evolutionarily conserved pos…

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Formation of the N(2)-acetyl-2,6-diaminopurine oligonucleotide impurity caused by acetyl capping.
Bioorg Med Chem Lett. 2014 Jun 18;
Authors: Rodriguez AA, Cedillo I, Mowery BP, Gaus HJ, Krishnamoo…

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Phylogenomic Guided Identification of Industrially Relevant GH1 β-Glucosidases Through DNA Synthesis and Nanostructure-Initiator Mass Spectrometry.

ACS Chem Biol. 2014 Jul 1;

Authors: Heins RA, Cheng X, Nath S, Deng K, Bowen BP, Chivian DC, Datta S, Friedland GD, D’Haeseleer P, Wu D, Tran-Gyamfi M, Scullin CS, Singh S, Shi W, Hamilton MG, Bendall ML, Sczyrba A, Thompson J, Feldman T, Guenther JM, Gladden JM, Cheng JF, Adams PD, Rubin EM, Simmons BA, Sale KL, Northen TR, Deutsch S

Abstract
Harnessing the biotechnological potential of the large number of proteins available in sequence databases requires scalable methods for functional characterization. Here we propose a workflow to address this challenge by combining phylogenomic guided DNA synthesis with high-throughput Mass Spectrometry, and applied it to the systematic characterization of GH1 β-glucosidases, a family of enzymes necessary for biomass hydrolysis, an important step in the conversion of lignocellulosic feedstocks to fuels and chemicals. We synthesized and expressed 175 GH1s, selected from over 2000 candidate sequences to cover maximum sequence diversity. These enzymes were functionally characterized over a range of temperatures and pHs using Nanostructure-Initiator Mass Spectrometry (NIMS), generating over 10,000 data points. Combined with HPLC-based sugar profiling we observed GH1 enzymes active over a broad temperature range, and towards many different di-saccharides in the β configuration. For some GH1s we also observed activity towards laminarin, a more complex oligosaccharide present as a major component of macroalgae. An area of particular interest was the identification of GH1 enzymes compatible with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), a next-generation biomass pre-treatment technology. We thus searched for GH1 enzymes active at 70 °C and 20% [C2mim][OAc] over the course of a 24-hour saccharification reaction. Using our unbiased approach we identified multiple enzymes of different phylogentic origin with such activities. Our approach of characterizing sequence diversity through targeted gene synthesis coupled to high-throughput screening technologies is a broadly applicable paradigm for a wide range of biological problems.

PMID: 24984213 [PubMed - as supplied by publisher]

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Synthesis of Cross-Linked DNA Containing Oxidized Abasic Site Analogues.
J Org Chem. 2014 Jun 20;
Authors: Ghosh S, Greenberg MM
Abstract
DNA interstrand cross-links are an important family of DNA d…

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