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
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
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]
Screening of accurate clones for gene synthesis in yeast.
J Biosci Bioeng. 2014 Sep 5;
Authors: Yarimizu T, Nakamura M, Hoshida H, Akada R
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]
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…
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
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]
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…
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
De novo synt…
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
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]
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
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]
Structure-function analysis of Drosophila notch using genomic rescue transgenes.
Methods Mol Biol. 2014;1187:29-46
Authors: Leonardi J, Jafar-Nejad H
One of the evolutionarily conserved pos…