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…
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…
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
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]
Synthesis of Cross-Linked DNA Containing Oxidized Abasic Site Analogues.
J Org Chem. 2014 Jun 20;
Authors: Ghosh S, Greenberg MM
DNA interstrand cross-links are an important family of DNA d…
Speeding up Directed Evolution: Combining the Advantages of Solid-Phase Combinatorial Gene Synthesis with Statistically Guided Reduction of Screening Effort.
ACS Synth Biol. 2014 Jun 12;
Error removal in microchip-synthesized DNA using immobilized MutS.
Nucleic Acids Res. 2014 May 14;
Authors: Wan W, Li L, Xu Q, Wang Z, Yao Y, Wang R, Zhang J, Liu H, Gao X, Hong J
The development of economical de novo gene synthesis methods using microchip-synthesized oligonucleotides has been limited by their high error rates. In this study, a low-cost, effective and improved-throughput (up to 32 oligos per run) error-removal method using an immobilized cellulose column containing the mismatch binding protein MutS was produced to generate high-quality DNA from oligos, particularly microchip-synthesized oligonucleotides. Error-containing DNA in the initial material was specifically retained on the MutS-immobilized cellulose column (MICC), and error-depleted DNA in the eluate was collected for downstream gene assembly. Significantly, this method improved a population of synthetic enhanced green fluorescent protein (720 bp) clones from 0.93% to 83.22%, corresponding to a decrease in the error frequency of synthetic gene from 11.44/kb to 0.46/kb. In addition, a parallel multiplex MICC error-removal strategy was also evaluated in assembling 11 genes encoding ∼21 kb of DNA from 893 oligos. The error frequency was reduced by 21.59-fold (from 14.25/kb to 0.66/kb), resulting in a 24.48-fold increase in the percentage of error-free assembled fragments (from 3.23% to 79.07%). Furthermore, the standard MICC error-removal process could be completed within 1.5 h at a cost as low as $0.374 per MICC.
PMID: 24829454 [PubMed - as supplied by publisher]
Efficient Functionalization of Oligonucleotides by New Achiral Nonnucleosidic Monomers.
Org Lett. 2014 May 12;
Authors: Kupryushkin MS, Nekrasov MD, Stetsenko DA, Pyshnyi DV
Purification of a Pd20-TNFα fusion protein that prevents liver metastasis of gastric cancer.
Tumour Biol. 2014 May 2;
Authors: Hu SJ, Jiang RX, Xie HH, Ma AL, Shi HL, Shen H, Hao ZM
The specific binding peptide pd20 of gastric cancer cells with a high potential for liver metastasis was fused with human tumour necrosis factor (TNF) α, and a prokaryotic expression vector was established to express the pd20-TNFα fusion protein. After purification and identification, the preventive effects of the fusion protein on liver metastasis of gastric cancer were observed in mice. The whole gene synthesis method was used for pd20-TNFα fusion gene preparation, and a pd20-TNFα prokaryotic expression vector was constructed. The vector was induced and expressed in Escherichia coli BL21. The expression products were analysed and verified by SDS-PAGE electrophoresis and Western blot analysis. The Ni-NTA column method was used to purify the fusion protein, and the L929 cytotoxicity method was used to detect biological activity. Flow cytometry apoptosis experiments and invasion assays were performed to observe the effects of the fusion protein on apoptosis and metastasis of gastric cancer cells with high potential for liver metastasis. Thirty nude mice with liver metastasis of gastric cancer were established and then randomly divided into three groups of ten mice each. The Pd20-TNFα recombinant protein (1.2 × 10(6) U/kg day) or standard TNFα (1.2 × 10(6) U/kg day) saline was administered via tail vein injection for 7 consecutive days. The pathological changes in various organs of nude mice were observed 4 weeks later. The size of the gastric cancer, the incidence of liver metastasis and the number of liver metastases were measured and calculated. We successfully constructed a Pd20-TNFα recombinant plasmid and prepared the fusion protein. Detection of the pd20-TNFα protein by immunofluorescence showed a very strong expression in liver tissue, suggesting a targeting of the fusion protein to the liver. The L929 cytotoxicity assays showed that the pd20-TNFα fusion purified protein had a significant lethal effect on L929 cells, with a killing activity of up to 7.6 × 10(6) IU/ml. The apoptosis experiments showed that as the concentration of the fusion protein increased, the early gastric cancer cell apoptosis also increased, with the early apoptosis rate increasing from 5.99 % to 9.04 %. Cell invasion experiments showed that the purified pd20-TNFα fusion protein significantly inhibited the in vitro invasion of XGC9811-L cells, with the penetrating cells being significantly decreased compared with the control group per unit time (P < 0.01). Vector experiments showed that the pd20-TNFα recombinant protein group had significantly reduced cancer lesions and liver metastasis in nude mice compared with the control group. We successfully purified a pd20-TNFα fusion protein and confirmed that it had significant biological activity promoting early gastric cancer cell apoptosis, thereby inhibiting gastric cancer cell invasion.
PMID: 24789430 [PubMed - as supplied by publisher]