Browse
Submit Data
Databases
API
Help

Dataset Information

23 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

High-fidelity PCR enzyme with DNA-binding domain facilitates de novo gene synthesis.

ABSTRACT: Nowadays enzymatic synthesis of genes is the most powerful tool for fast resolution of the various tasks in the field of basic and applied biological research. PCR-based gene assembly from overlapping oligonucleotides has become a widely used strategy. However, all the methods described in the literature are not perfect and need an extra processing step. In this study we are verifying Phusion high-fidelity polymerase as a tool to reduce nucleotide mismatches in de novo gene synthesis, thus facilitating subsequent cloning. To test the efficiency of the polymerase, we selected Fel d 4 gene, which is a 581 bp DNA sequence encoding the lipocalin allergen protein, one of the major cat allergens. The approach described here, therefore, would be useful in DNA sequences creation.

SUBMITTER: Dolgova AS

PROVIDER: S-EPMC5453909 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

High-fidelity PCR enzyme with DNA-binding domain facilitates de novo gene synthesis.

Dolgova A S AS Stukolova O A OA

3 Biotech 20170601 2

Nowadays enzymatic synthesis of genes is the most powerful tool for fast resolution of the various tasks in the field of basic and applied biological research. PCR-based gene assembly from overlapping oligonucleotides has become a widely used strategy. However, all the methods described in the literature are not perfect and need an extra processing step. In this study we are verifying Phusion high-fidelity polymerase as a tool to reduce nucleotide mismatches in de novo gene synthesis, thus facil ...[more]

PMID: 28573398

Similar Datasets

De novo DNA synthesis using single molecule PCR.

Project description:The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.

| S-EPMC2553596 | biostudies-literature

De novo synthesis and development of an RNA enzyme.

Project description:Arbitrary manipulation of molecular recognition at the atomic level has many applications. However, systematic design and de novo synthesis of an artificial enzyme based on such manipulation has been a long-standing challenge in the field of chemistry and biotechnology. In this report, we developed an artificial RNA ligase by implementing a synthetic strategy that fuses a series of 3D molecular modelings based on naturally occurring RNA-RNA recognition motifs with a small-scale combinatorial synthesis of a modular catalytic unit. The resulting ligase produces a 3'-5' linkage in a template-directed manner for any combinations of two nucleotides at the reaction site. The reaction rate is 10(6)-fold over that of the uncatalyzed reaction with a yield higher than those of previously reported ligase ribozymes. The strategy may be applicable to the synthesis and development of a variety of nonnatural functional RNAs with defined 3D structures.

| S-EPMC518828 | biostudies-literature

Protein-mediated error correction for de novo DNA synthesis.

Project description:The availability of inexpensive, on demand synthetic DNA has enabled numerous powerful applications in biotechnology, in turn driving considerable present interest in the de novo synthesis of increasingly longer DNA constructs. The synthesis of DNA from oligonucleotides into products even as large as small viral genomes has been accomplished. Despite such achievements, the costs and time required to generate such long constructs has, to date, precluded gene-length (and longer) DNA synthesis from being an everyday research tool in the same manner as PCR and DNA sequencing. A critical barrier to low-cost, high-throughput de novo DNA synthesis is the frequency at which errors pervade the final product. Here, we employ a DNA mismatch-binding protein, MutS (from Thermus aquaticus) to remove failure products from synthetic genes. This method reduced errors by >15-fold relative to conventional gene synthesis techniques, yielding DNA with one error per 10 000 base pairs. The approach is general, scalable and can be iterated multiple times for greater fidelity. Reductions in both costs and time required are demonstrated for the synthesis of a 2.5 kb gene.

| S-EPMC534640 | biostudies-literature

De Novo Design of an Androgen Receptor DNA Binding Domain-Targeted peptide PROTAC for Prostate Cancer Therapy.

Project description:Androgen receptor splice variant-7 (AR-V7), one of the major driving factors, is the most attractive drug target in castration-resistant prostate cancer (CRPC). Currently, no available drugs efficiently target AR-V7 in clinical practice. The DNA binding domain (DBD) is indispensable for the transcriptional activity of AR full length and AR splice variants, including AR-V7. Based on the homodimerization structure of the AR DBD, a novel peptide-based proteolysis-targeting chimera (PROTAC) drug is designed to induce AR and AR-V7 degradation in a DBD and MDM2-dependent manner, without showing any activity on other hormone receptors. To overcome the short half-life and poor cell penetrability of peptide PROTAC drugs, an ultrasmall gold (Au)-peptide complex platform to deliver the AR DBD PROTAC in vivo is developed. The obtained Au-AR pep-PROTAC effectively degrades AR and AR-V7 in prostate cancer cell lines, particularly in CWR22Rv1 cells with DC50 values 48.8 and 79.2 nM, respectively. Au-AR pep-PROTAC results in suppression of AR levels and induces tumor regression in both enzalutamide sensitive and resistant prostate cancer animal models. Further optimization of the Au-AR pep-PROTAC can ultimately lead to a new therapy for AR-V7-positive CRPC.

| S-EPMC9534960 | biostudies-literature

High-fidelity de novo synthesis of pathways using microchip-synthesized oligonucleotides and general molecular biology equipment.

Project description:Engineering and evaluation of synthetic routes for generating valuable compounds require accurate and cost-effective de novo synthesis of genetic pathways. Here, we present an economical and streamlined de novo DNA synthesis approach for engineering a synthetic pathway with microchip-synthesized oligonucleotides (oligo). The process integrates entire oligo pool amplification, error-removal, and assembly of long DNA molecules. We utilized this method to construct a functional lycopene biosynthetic pathway (11.9?kb encoding 10 genes) in Escherichia coli using a highly error-prone microchip-synthesized oligo pool (479 oligos) without pre-purification, and the error-frequency was reduced from 14.25/kb to 0.53/kb. This low-equipment-dependent and cost-effective method can be widely applied for rapid synthesis of biosynthetic pathways in general molecular biology laboratories.

| S-EPMC5522410 | biostudies-literature

SRA-domain proteins required for DRM2-mediated de novo DNA methylation.

Project description:De novo DNA methylation and the maintenance of DNA methylation in asymmetrical sequence contexts is catalyzed by homologous proteins in plants (DRM2) and animals (DNMT3a/b). In plants, targeting of DRM2 depends on small interfering RNAs (siRNAs), although the molecular details are still unclear. Here, we show that two SRA-domain proteins (SUVH9 and SUVH2) are also essential for DRM2-mediated de novo and maintenance DNA methylation in Arabidopsis thaliana. At some loci, SUVH9 and SUVH2 act redundantly, while at other loci only SUVH2 is required, and this locus specificity correlates with the differing DNA-binding affinity of the SRA domains within SUVH9 and SUVH2. Specifically, SUVH9 preferentially binds methylated asymmetric sites, while SUVH2 preferentially binds methylated CG sites. The suvh9 and suvh2 mutations do not eliminate siRNAs, suggesting a role for SUVH9 and SUVH2 late in the RNA-directed DNA methylation pathway. With these new results, it is clear that SRA-domain proteins are involved in each of the three pathways leading to DNA methylation in Arabidopsis.

| S-EPMC2582956 | biostudies-literature

De novo RNA synthesis catalyzed by the Zika Virus RNA polymerase domain.

Project description:Mosquito- and tick-borne pathogens including Chikungunya, Dengue, Japanese encephalitis, West Nile, Yellow fever and Zika virus, represent a new economic and public health challenge. In the absence of effective vaccines and specific therapies, only supportive regimens are administrated for most of these infections. Thus, the development of a targeted therapy is mandatory to stop the rapid progression of these pathogens and preoccupant associated burdens such as Guillain-Barre syndrome, microcephaly. For this, it is essential to develop biochemical tools to help study and target key viral enzymes involved in replication such as helicase complexes, methyl-transferases and RNA-dependent RNA polymerases. Here, we show that a highly purified ZIKV polymerase domain is active in vitro. Importantly, we show that this isolated domain is capable of de novo synthesis of the viral genome and efficient elongation without terminal nucleotide transferase activity. Altogether, this isolated polymerase domain will be a precious tool to screen and optimize specific nucleoside and non-nucleoside inhibitors to fight against Zika infections.

| S-EPMC5457451 | biostudies-literature

De novo enzyme design using Rosetta3.

Project description:The Rosetta de novo enzyme design protocol has been used to design enzyme catalysts for a variety of chemical reactions, and in principle can be applied to any arbitrary chemical reaction of interest. The process has four stages: 1) choice of a catalytic mechanism and corresponding minimal model active site, 2) identification of sites in a set of scaffold proteins where this minimal active site can be realized, 3) optimization of the identities of the surrounding residues for stabilizing interactions with the transition state and primary catalytic residues, and 4) evaluation and ranking the resulting designed sequences. Stages two through four of this process can be carried out with the Rosetta package, while stage one needs to be done externally. Here, we demonstrate how to carry out the Rosetta enzyme design protocol from start to end in detail using for illustration the triosephosphate isomerase reaction.

| S-EPMC3095599 | biostudies-literature

Serine hydroxymethyltransferase anchors de novo thymidylate synthesis pathway to nuclear lamina for DNA synthesis.

Project description:The de novo thymidylate biosynthetic pathway in mammalian cells translocates to the nucleus for DNA replication and repair and consists of the enzymes serine hydroxymethyltransferase 1 and 2? (SHMT1 and SHMT2?), thymidylate synthase, and dihydrofolate reductase. In this study, we demonstrate that this pathway forms a multienzyme complex that is associated with the nuclear lamina. SHMT1 or SHMT2? is required for co-localization of dihydrofolate reductase, SHMT, and thymidylate synthase to the nuclear lamina, indicating that SHMT serves as scaffold protein that is essential for complex formation. The metabolic complex is enriched at sites of DNA replication initiation and associated with proliferating cell nuclear antigen and other components of the DNA replication machinery. These data provide a mechanism for previous studies demonstrating that SHMT expression is rate-limiting for de novo thymidylate synthesis and indicate that de novo thymidylate biosynthesis occurs at replication forks.

| S-EPMC3293584 | biostudies-other

The de novo design and synthesis of yeast chromosome XIII facilitates investigations on aging

Project description:In the era of synthetic biology, design, construction, and utilization of synthetic chromosomes with unique features provide a new strategy to study complex cellular processes such as aging. Herein, we successfully constructed the 884 Kb synXIII of Saccharomyces cerevisiae to investigate replicative aging using this synthetic strains. We verified that up-regulation of a rRNA-related transcriptional factor, RRN9, positively influenced replicative lifespan. Using SCRaMbLE system that enables inducible whole-genome rearrangement on synXIII, we obtained 20 SCRaMbLEd synXIII strains with extended lifespan. Transcriptome analysis revealed the expression of genes involved in global protein synthesis is up-regulated in longer-lived strains. We established causal links between genotypic change and the long-lived phenotype via reconstruction of some key structural variations observed in post-SCRaMbLE strains and further demonstrated combinatorial effects of multiple aging regulators on lifespan extension. Our findings underscored the potential of synthetic yeasts in unveiling the function of aging-related genes.

2024-10-30 | PXD057330 |

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data