“…As expected, the data suggested that KOD Hot Start DNA polymerase is more accurate than the other three DNA polymerases due to a higher fidelity. These results are in line with previous studies [8, 9, 16], which have revealed that usage of high fidelity DNA polymerases decreases the number of errors introduced in synthetic genes during PCR amplification. Moreover, the PCA-DTF procedure appears to be very efficient with KOD Hot Start DNA polymerase due to the rapid elongation rates presented by this DNA polymerase; completion of the gene synthesis protocol is achieved in less than 40 min.Fig.…”
Section: Resultssupporting
confidence: 93%
“…Recently, methods based on a two-step approach were reported for the production of long DNA sequences. Examples of these technologies are the PCR-based thermodynamically balanced inside-out technology (TBIO) [6], the two-step total gene synthesis method [7] that combines both dual asymmetrical PCR (DA-PCR) and overlap-extension (OE-PCR), the PCR-based two-step DNA synthesis (PTDS) [8] and PCR-based accurate synthesis (PAS) [9]. …”
Section: Introductionmentioning
confidence: 99%
“…Lately, improvements in PCR-based gene synthesis methods, as exemplified by the development of the improved PCR synthesis (IPS) and the simplified gene synthesis (SGS) protocols [8, 9], have been described and incorporate significant simplifications over earlier strategies. SGS uses oligonucleotides of 40 nucleotides (nt) in length and 18–20 nt of overlap region, which are assembled in a unique PCR-assembly reaction leading to the direct construction of the full-length DNA molecule.…”
BackgroundGene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression.ResultsHere, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins.ConclusionsAn automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0316-3) contains supplementary material, which is available to authorized users.
“…As expected, the data suggested that KOD Hot Start DNA polymerase is more accurate than the other three DNA polymerases due to a higher fidelity. These results are in line with previous studies [8, 9, 16], which have revealed that usage of high fidelity DNA polymerases decreases the number of errors introduced in synthetic genes during PCR amplification. Moreover, the PCA-DTF procedure appears to be very efficient with KOD Hot Start DNA polymerase due to the rapid elongation rates presented by this DNA polymerase; completion of the gene synthesis protocol is achieved in less than 40 min.Fig.…”
Section: Resultssupporting
confidence: 93%
“…Recently, methods based on a two-step approach were reported for the production of long DNA sequences. Examples of these technologies are the PCR-based thermodynamically balanced inside-out technology (TBIO) [6], the two-step total gene synthesis method [7] that combines both dual asymmetrical PCR (DA-PCR) and overlap-extension (OE-PCR), the PCR-based two-step DNA synthesis (PTDS) [8] and PCR-based accurate synthesis (PAS) [9]. …”
Section: Introductionmentioning
confidence: 99%
“…Lately, improvements in PCR-based gene synthesis methods, as exemplified by the development of the improved PCR synthesis (IPS) and the simplified gene synthesis (SGS) protocols [8, 9], have been described and incorporate significant simplifications over earlier strategies. SGS uses oligonucleotides of 40 nucleotides (nt) in length and 18–20 nt of overlap region, which are assembled in a unique PCR-assembly reaction leading to the direct construction of the full-length DNA molecule.…”
BackgroundGene synthesis is becoming an important tool in many fields of recombinant DNA technology, including recombinant protein production. De novo gene synthesis is quickly replacing the classical cloning and mutagenesis procedures and allows generating nucleic acids for which no template is available. In addition, when coupled with efficient gene design algorithms that optimize codon usage, it leads to high levels of recombinant protein expression.ResultsHere, we describe the development of an optimized gene synthesis platform that was applied to the large scale production of small genes encoding venom peptides. This improved gene synthesis method uses a PCR-based protocol to assemble synthetic DNA from pools of overlapping oligonucleotides and was developed to synthesise multiples genes simultaneously. This technology incorporates an accurate, automated and cost effective ligation independent cloning step to directly integrate the synthetic genes into an effective Escherichia coli expression vector. The robustness of this technology to generate large libraries of dozens to thousands of synthetic nucleic acids was demonstrated through the parallel and simultaneous synthesis of 96 genes encoding animal toxins.ConclusionsAn automated platform was developed for the large-scale synthesis of small genes encoding eukaryotic toxins. Large scale recombinant expression of synthetic genes encoding eukaryotic toxins will allow exploring the extraordinary potency and pharmacological diversity of animal venoms, an increasingly valuable but unexplored source of lead molecules for drug discovery.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0316-3) contains supplementary material, which is available to authorized users.
“…Initiation codon ATG and termination codon TAA were respectively added to 5′and 3′of the lip gene. In order to expressing A. Niger lipase gene in P. pastoris at high levels, the lipA gene was optimized to utilize the frequently used codons in P. pastoris for each amino acid with the help of DNAworks (inservice software) [4] . Totally 26 primers (Pl-P26) which overlapped each other was designed with EcoRI on P1 and NotI on P26.…”
Section: B Codon Optimization Design and Synthesis Of Primersmentioning
Aspergillus niger lipases are important biocatalysts widely used in industries for food processing and pharmaceutical preparation. High-level expression recombinants can lead to cost effective lipase large scale production. Total gene synthesis is an efficient method to enhance the expression level of the gene. According to the mature peptide sequence of A. niger lipase and codons of preference in Pichia pastoris, 26 primers with 20bp overlaps at both 57 and 37 ends between adjacent o1igonucleotides were designed and synthesized. Fragments lipAl(300bp), lipA2(237bp), lipA3(234bp) and lipA4(210bp) were separately synthesized by assembly PCR, and then they were used as the template to get the full length gene. The synthetic gene was cloned into pPIC9K secretory expression vector. The expression efficiency was verified with analysis of mRNA secondary structure using RNA Structure 4.5.
“…In this study, assembly PCR was applied to generate a synthetic gene, a simple and cost effective method that relies on constructing a single synthetic gene encoding multi-immunodominant epitope peptides as a new strategy for developing effective and standard diagnostic markers [23]. Assembly PCR is a flexible technique that can be used for producing long DNA sequence from single-stranded oligos or a mixture of single and double-stranded DNA [20].…”
BackgroundSerological investigation remains the primary approach to achieve satisfactory results in Toxoplasma gondii identification. However, the accuracy of the native antigen used in the current diagnostic kits has proven to be insufficient as well as difficult to standardize, so significant efforts have been made to find alternative reagents as capture antigens. Consequently, multi-epitope peptides are promising diagnostic markers, with the potential for improving the accuracy of diagnostic kits. In this study, we described a simple, inexpensive and improved strategy to acquire such diagnostic markers. The study was aimed at producing novel synthetic protein consisting of multiple immunodominant epitopes of several T. gondii antigens.FindingsTo accomplish our goals, a single synthetic gene of approximately 456 bp, which encodes potential epitopes of T. gondii antigens, was successfully constructed using gene assembly PCR. The constructed gene was cloned into a pET32a expression vector and transformed into BL21 E. coli. The entire protein was successfully expressed and purified. Subsequently, the preliminary diagnostic performance of expressed protein was evaluated by developing IgG enzyme-linked immunosorbent assay (ELISA) and Western blot analysis using human sera. The results showed 100 % sensitivity and specificity.ConclusionA purified protein expressing multi-immunodominant epitopes of T. gondii was generated. Further studies are required to evaluate the immunogenicity in animal models and to verify the immuno-reactivity of USM.TOXO1 as a diagnostic antigen.
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