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Gene Synthesis

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Independent Generation and Reactivity of Uridin-2′-yl Radical.

J Org Chem. 2014 Oct 17;

Authors: Paul R, Greenberg MM

Abstract
The uridin-2′-yl radical (1) has been proposed as an intermediate during RNA oxidation. However, its reactivity has not been thoroughly studied due to the complex conditions under which it is typically generated. The uridin-2′-yl radical was independently generated from a benzyl ketone (2a) via Norrish type I photocleavage upon irradiation at λmax = 350 nm. Dioxygen and β-mercaptoethanol are unable to compete with loss of uracil from 1 in phosphate buffer. Thiol trapping competes with uracil fragmentation in less polar solvent conditions. This is ascribed mostly to a reduction in the rate constant for uracil elimination in the less polar solvent. Hydrogen atom transfer to 1 from β-mercaptoethanol occurs exclusively from the α-face to produce arabinouridine. Mass balances range from 72 to 95%. Furthermore, the synthesis of 2a is amenable to formation of the requisite phosphoramidite for solid-phase oligonucleotide synthesis. This and the fidelity with which the urdin-2′-yl radical is generated from 2a suggest that this precursor should be useful for studying the radical’s reactivity in synthetic oligonucleotides.

PMID: 25325847 [PubMed - as supplied by publisher]

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5-Nitroindole oligonucleotides with alkynyl side chains: universal base pairing, triple bond hydration and properties of pyrene “click” adducts.
Org Biomol Chem. 2014 Sep 19;
Authors: Ingale SA, Leo…

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Characterizing aptamer small molecule interactions with backscattering interferometry.
Analyst. 2014 Sep 17;
Authors: Kammer MN, Olmsted IR, Kussrow AK, Morris MJ, Jackson GW, Bornhop DJ
Abs…

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Synthesis of branched DNA using oxidatively cleavable tritylsulfenyl as a hydroxy protecting group.

Curr Protoc Nucleic Acid Chem. 2014;58:2.18.1-2.18.19

Authors: Seio K, Kanamori T, Sekine M

Abstract
The application of oxidatively cleavable tritylsulfenyl (TrS) group to the synthesis of branched DNA is described. The TrS protecting group can be removed by treatment with 1 M aqueous iodine, while it is stable toward an oxaziridine-type oxidant. At the same time, the sulfur-oxygen linkage showed sufficient stability under the acidic and basic conditions used in oligonucleotide synthesis. These properties of the TrS group enabled the synthesis of branched DNA using a branched phosphoramidite in which the two hydroxy groups are protected by a 4,4′-dimethoxytrityl (DMTr) group or a TrS group. In this unit, we describe an example of the synthesis of a three-way branched DNA using a branched phosphoramidite. Curr. Protoc. Nucleic Acid Chem. 58:2.18.1-2.18.19. © 2014 by John Wiley & Sons, Inc.

PMID: 25199636 [PubMed - in process]

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admin on September 10th, 2014
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Screening of accurate clones for gene synthesis in yeast.

J Biosci Bioeng. 2014 Sep 5;

Authors: Yarimizu T, Nakamura M, Hoshida H, Akada R

Abstract
Methods for error-less gene synthesis are desired because synthesized genes often contain mutations. By cloning PCR-assembled oligonucleotide fragments fused to a selection marker in yeast, we developed a novel method to screen accurate clones in gene synthesis. As a model case, the 555-bp luciferase gene from Gaussia princeps (GLuc) was synthesized to contain yeast-optimized codons (called yGLuc hereafter). After standard PCR-mediated oligonucleotide assembly, many clones showed no luciferase activity. Of these clones, most contained randomly located nucleotide deletions that produced frameshifts and resulted in premature termination. To exclude clones with premature termination, the synthesized yGLuc gene was cloned in-frame to fuse with the URA3 coding sequence, which served as a selection marker in the yeast Kluyveromyces marxianus. Ura(+) transformation selection was expected to eliminate clones with frameshift errors. The results showed that in-frame marker selection increased the frequency of active yGLuc gene to 79%. We used this strategy to synthesize the 1812-bp gene from Rhizopus oryzae that encodes glucoamylase. Five out of seven Ura(+) clones exhibited amylase activity. Of the functional clones, one contained the correct sequence, and four contained sequences with nucleotide changes, suggesting that in-frame selection frequently produced functional mutants. The K. marxianus non-homologous end joining mediated cloning method for gene synthesis will be useful for synthetic biological studies.

PMID: 25201012 [PubMed - as supplied by publisher]

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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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