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Targeted mutagenesis of a promoter or gene is essential for attaining new functions in microbial and protein engineering efforts. In the burgeoning field of synthetic biology, heterologous genes are expressed in new host organisms. Similarly, natural or designed proteins are mutagenized at targeted positions and screened for gain-of-function mutations. Here, we describe methods to attain complete randomization or controlled mutations in promoters or genes. Combinatorial libraries of one hundred thousands to tens of millions of variants can be created using commercially synthesized oligonucleotides, simply by performing two rounds of polymerase chain reactions. With a suitably engineered reporter in a whole cell, these libraries can be screened rapidly by performing fluorescence-activated cell sorting (FACS). Within a few rounds of positive and negative sorting based on the response from the reporter, the library can rapidly converge to a few optimal or extremely rare variants with desired phenotypes. Library construction, transformation and sequence verification takes 6-9 days and requires only basic molecular biology lab experience. Screening the library by FACS takes 3-5 days and requires training for the specific cytometer used. Further steps after sorting, including colony picking, sequencing, verification, and characterization of individual clones may take longer, depending on number of clones and required experiments.

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For difficult overlap extension PCR, a Gibson assembly process was inserted between the two PCR rounds to facilitate the formation of complete gene templates at a moderate temperature. That is, after amplifying each DNA fragment, they were preluded by a Gibson assembly process in equal proportion. Then, the assembled mixture was used as a template for the second PCR round. This idea was tested and verified by taking the cloning example of a single and a double site mutation of the retinoblastoma gene. This scheme associates overlap extension PCR with Gibson assembly exquisitely, significantly improving gene amplification efficiency, particularly in the fusion of long genes and multifragments using overlap extension PCR.

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Owing to difficulties in obtaining functional gene sequences, molecular pathogenic mechanisms in Demodex have been understudied. In this study, overlap extension PCR was used to obtain the sequences of cathepsin L (CatL), a pathogenicity-related gene, to provide a foundation for subsequent functional research. Demodex folliculorum and Demodex brevis mites were obtained from the face skin of Chinese individuals, and Demodex canis mites were isolated from the skin lesions of a dog. RNA was extracted and used to synthesise double-stranded cDNA. PCR amplification, cloning, sequencing, and bioinformatics analysis of CatL were performed. CatL gene sequences of 1005, 1008, and 1008 bp were successfully amplified for D. brevis, D. folliculorum, and D. canis, respectively. These sequences showed 99.9 or 100% identity with templates previously obtained by RNA-seq. The Maximum Likelihood (ML) phylogenetic tree showed that D. folliculorum clustered with D. canis first, then with D. brevis, and finally with other Acariformes mite species. The three Demodex species had nine similar motifs to those of Sarcoptes scabies, Dermatophagoides pteronyssinus, and Dermatophagoides farinae, and motifs 10-13 were valuable for identification. CatL proteins of Demodex species were predicted to be approximately 38 kDa, be located in lysosomes, have a signal peptide but no transmembrane region, and have two functional domains, I29 and Pept_C1. However, interspecific differences were observed in secondary and tertiary protein structures. In conclusion, we successfully obtained CatL sequences of three Demodex species by overlap extension PCR, which creates conditions for further pathogenic mechanism studies.

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Ligating two or more DNA fragments is a regular operation for the subcloning and the engineering of vectors. The overlap extension PCR serves as a straightforward method to solve this issue. However, it takes a relatively long time to design the appropriate overlapping primers and the primers for the full-length sequence, and there has not been a professional offline software for such kind of primer design. Here, we propose a Python script to search, calculate and sort thousands of combinations of primers for users according to the predefined parameters. The results of script running and experimental validation show that this script is capable of generating the optimal pairs of primers based on the proper melting temperatures and lengths of the primers, which facilitates gene modification in research.

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Overlap extension PCR is one of the routinely used methods to generate mutagenic genes for the functional and structural study of proteins. However, it is time-consuming to design the overlapping mutagenic primers and gene primers by manual operation. In this chapter, we present a Python script that is able to search all the possible primer combinations according to the preset definitions and calculate the necessary parameters of each primer for the users, which could facilitate the primer design process. Up to 256 pairs of primers can be provided for selection using this script.

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In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

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In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

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As a group of cytokines, interferons are the first line of defense in the antiviral immunity. In this study, Siberian tiger IFN-ß (PtIFN-ß) and IFN-γ (PtIFN-γ) were successfully amplified, and the two were fused (PtIFN-γ) by overlap extension polymerase chain reaction (SOE-PCR). Bioinformatics analysis disclosed that PtIFN-ß and PtIFN-γ have species-specificity and conservation in the course of evolution. After being expressed in prokaryotes, the antiviral activities and physicochemical properties of PtIFN-ß, PtIFN-γ and PtIFNß-γ were analyzed. In Feline kidney cells (F81), PtIFNß-γ showed more active antiviral activity than PtIFN-ß and PtIFN-γ, which has more stable physicochemical properties (acid and alkali resistance, high temperature resistance). In addition, PtIFN-ß, PtIFN-γ and PtIFN-γ activated the JAK-STAT pathway and induced the transcription and expression of interferon-stimulated genes (ISGs). Janus kinase (JAK) 1 inhibitor inhibited ISGs expression induced by PtIFN-ß, PtIFN-γ and PtIFN-γ. Overall, this research clarified that PtIFN-ß, PtIFN-γ and PtIFNß-γ have the ability to inhibit viral replication and send signals through the JAK-STAT pathway. These findings may facilitate further study on the role of PtIFN in the antiviral immune response, and help to develop approaches for the prophylactic and therapeutic of viral diseases based on fusion interferon.

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Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

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PCR is a powerful tool for generating specific fragments of DNA that can be used to create gene variations or tagged expression constructs. Overlap extension PCR is a valuable technique that is commonly used for cloning large complex fragments, making edits to cloned genes or fusing two gene elements together. After difficulties in utilizing this technique following existing methods, we developed an optimized protocol. To accomplish this, three significant changes were made; 1) touchdown PCR cycling parameters were used to eliminate the need for optimizing PCR cycling conditions, 2) the high-fidelity, high-processivity Q5 DNA polymerase was used to improve full-length amplification quality, and 3) a reduced amount of primer in the final PCR amplification step decreased non-specific amplimers. This modified protocol results in consistent generation of gene fusion products, with little to no background and enhanced efficiency of the transgene construction process.

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The use of interferon α-2 in combination with thymosin α-1 shows higher anti-cancer effect in comparison when both are used individually because of their synergistic effects. In this study we produced an important human interferon α-2-thymosin α-1 (IFNα2-Tα1) fusion protein with probable pharmaceutical properties coupled to its high-level expression, characterization, and study of its biological activity. The IFNα2-Tα1 fusion gene was constructed by over-lap extension PCR and expressed in Escherichia coli expression system. The expression of IFNα2-Tα1 fusion protein was optimized to higher level and its maximum expression was obtained in modified terrific broth medium when lactose was used as inducer. The fusion protein was refolded into its native biologically active form with maximum yield of 83.14% followed by purification with ∼98% purity and 69% final yield. A band of purified IFNα2-Tα1 fusion protein equal to ∼23 kDa was observed on 12 % SDS-PAGE gel. The integrity of IFNα2-Tα1 fusion protein was confirmed by western blot analysis and secondary structure was assessed by CD spectroscopy. When IFNα2-Tα1 fusion protein was subjected to its biological activity analysis it was observed that it exhibits both IFNα2 & Tα1 activities as well as significantly higher anticancer activity as compared to IFNα-2 alone.

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Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.

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Overlap extension polymerase chain reaction (PCR) is a powerful technology for DNA assembly. Based on this technology, we synthesized DNA templates, which were transcribed into sgRNA in vitro, and further detected their efficiency of purified sgRNAs with Cas9 nuclease. The sgRNAs synthesized by this approach can effectively cleave the DNA fragments of interest in vitro and in vivo. Compared with the conventional method for generating sgRNA, it does not require construction of recombinant plasmids and design of primers to amplify sgRNA core fragment. Only several short primers with overlapped sequences are needed to assemble a DNA fragment as the template of sgRNA. This modified and simplified method is highly applicable and less time-consuming.

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AIM: To produce synthetic nucleotides of notifiable dengue virus (1-4 types), Japanese encephalitis, yellow fever and Zika flaviviruses. These notifiable flaviviruses, particularly dengue and Zika, are problematic mosquito-borne infections in the Philippines, as well as in those countries with tropical and subtropical climates. METHOD: An algorithmic design formulation of overlap extension - polymerase chain reaction (OE-PCR) was performed to propagate 50-60 oligomer lengths of select notifiable flaviviral RNAs to DNA nucleotides via the two-step process of OE-PCR. RESULT: Algorithmic OE-PCR design formulation efficiently produced 253-256 bp of notifiable flaviviruses. Comparing the newly designed algorithmic OE-PCR with existing executable programs demonstrated it to be efficient and useful in generating accurate sequences of synthetic flaviviral nucleotides. CONCLUSION: The efficiently and accurately produced novel synthetic nucleotides of notifiable dengue virus 1-4, Japanese encephalitis, yellow fever and Zika flaviviruses using OE-PCR is useful in understanding the dynamics of flaviviral species and holds potential for the development of synthetic nucleotide-based immunogens.

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The growth and production of yeast in the industrial fermentation are seriously restrained by heat stress and exacerbated by heat induced oxidative stress. In this study, a novel synthetic biology approach was developed to globally boost the viability and production ability of S. cerevisiae at high temperature through rationally designing and combing heat shock protein (HSP) and superoxide dismutase (SOD) genetic devices to ultimately synergistically alleviate both heat stress and oxidative stress. HSP and SOD from extremophiles were constructed to be different genetic devices and they were preliminary screened by heat resistant experiments and anti-oxidative experiments, respectively. Then in order to customize and further improve thermotolerance of S. cerevisiae, the HSP genetic device and SOD genetic device were rationally combined. The results show the simply assemble of the same function genetic devices to solve heat stress or oxidative stress could not enhance the thermotolerance considerably. Only S. cerevisiae with the combination genetic device (FBA1p-sod-MB4-FBA1p-shsp-HB8) solving both stress showed 250% better thermotolerance than the control and displayed further 55% enhanced cell density compared with the strains with single FBA1p-sod-MB4 or FBA1p-shsp-HB8 at 42 °C. Then the most excellent combination genetic device was introduced into lab S. cerevisiae and industrial S. cerevisiae for ethanol fermentation. The ethanol yields of the two strains were increased by 20.6% and 26.3% compared with the control under high temperature, respectively. These results indicate synergistically defensing both heat stress and oxidative stress is absolutely necessary to enhance the thermotolerance and production of S. cerevisiae.

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Methods to fuse multiple DNA fragments are extremely useful in synthetic biology and protein engineering. Here, we report a gold nanoparticle-mediated simultaneous overlap extension-PCR (AuNP-mediated SOE-PCR) method that enables the fusion of multiple DNA fragments simultaneously with their amplification in a single reaction using typical PCR conditions. Using greater concentrations of rTaq DNA polymerase and AuNPs significantly improves the performance of SOE-PCR especially for the fusion of more than three DNA fragments. We show that up to six lambda DNA fragments can be simultaneously fused by AuNP-mediated SOE-PCR.

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We developed a simple method (simple cloning) for subcloning DNA fragments into any location of a targeted vector without the need of restriction enzyme, ligase, exonuclease, or recombinase in Escherichia coli. This technology can be applied to common E. coli hosts (e.g., DH5α, JM109, TOP10, BL21(DE3)). The protocol includes three steps: (1) generate DNA insert and linear vector backbone by regular high-fidelity PCR, where these two DNA fragments contain 3' and 5' overlapping termini; (2) generate DNA multimers based on these two DNA fragments by using prolonged overlap extension-PCR (POE-PCR) without primers added; and (3) transform POE-PCR product to competent Escherichia coli cells directly, yielding the desired plasmid. Simple cloning provides a new cloning method with great simplicity and flexibility. Furthermore, this new method can be modified for the preparation of a large-size mutant library for directed evolution in E. coli. Using this method, it is very easy to generate a mutant library with a size of more than 10(7) per 50 µL of the POE-PCR product within 1 day.

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Infectious cDNA clones are an important tool to study the molecular and cellular process of RNA virus infection. In vitro and in vivo transcription systems are the two main strategies used in the generation of infectious cDNA clones for RNA viruses. This study describes the first generation of a full-length infectious cDNA clone of Cowpea mild mottle virus (CPMMV), a Carlavirus. The full-length genome was synthesized by Overlap Extension PCR of two overlapping fragments and cloned in a pUC-based vector under control of the SP6 RNA polymerase promoter. After in vitro run-off transcription, the produced RNA was mechanically inoculated into soybean plants cv. CD206. The systemic infection was confirmed by RT-PCR and further sequencing of amplified cDNA fragments. To simplify the transfection process, the complete genome was subcloned into a binary vector under control of the 35S promoter of cauliflower mosaic virus by the Gibson Assembly protocol. The resulting clones were inoculated by particle bombardment onto soybean seedlings and the recovery of the virus was confirmed 2 weeks later by RT-PCR. Our results indicate the constructs of the full-length cDNA of CPMMV are fully infectious in both in vitro and in vivo transcription strategies.

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Objective To construct the DNA vaccine pIRES2-MLAA34-HSP70,and to detect its immune effect.Methods The acute monocytic leukemia associated antigen gene MLAA-34 and heat-shock protein (HSP)70 gene were extracted by using RT-PCR.The specific overlapping primer was designed,and the fusion gene MLAA34-HSP70 was amplified by using SOE-PCR technique.Then the DNA vaccine pIRES2-MLAA34-HSP70 was constructed,and BALB/c mice were immunized with this DNA vaccine.The splenic lymphocyte killing activity was detected by using MTT,levels of IL-2,IL-4 and IFN-γ were also detected by using ELISA.Results The MLAA34-HSP70 gene (2 956 bp) and the DNA vaccine pIRES2-MLAA34-HSP70 was amplified and constructed successfully.The killing efficiency of DNA vaccine pIRES2-MLAA34-HSP70 in U937 cells was significantly higher than that in other experimental groups and control group (P＜0.01),and levels of IL-4,IL-2 and IFN-γin DNA vaccine pIRES2-MLAA34-HSP70 group were significantly higher than those in the other experimental groups and control group (P＜0.01).Conclnsion The DNA vaccine pIRES2-MLAA34-HSP70 is constructed successfully.It is shown that the DNA vaccine induces strong humoral immunity,which could enhance immune responses to tumor cells and specificlly kill MLAA34 positive cells.

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BACKGROUND: Human monoclonal antibodies are important molecules in clinical research. Current Limitations of mAb technologies namely instability of immortalized B-cell line and probability of forming unusual VH-VL pairs in phage-display method led to mAbs technology based on single plasma cell called ``SYMPLEX''. OBJECTIVE: In this method, cognate VH and VL fragments generated from individual antibody genes exactly the same as natural ones. METHODS: PBMCs of whole blood of an immunized candidate was used as a resource of rearranged Ab genes. Then flow-cytometric screening was performed to isolate VH and VL from PBMCs. Various VH and VLκ were amplified by six pairs of primers. Overlap Extension PCR was accomplished to link VH and Vκ regions. ScFv inserted into T-vector and its sequence was determined and eventually analyzed by using blast analysis tools. RESULTS: Electrophoresis results indicated that VH and VL fragments were separately amplified by PCR with a length of about 400bp and linked through OE-PCR. Hence, ScFv, which was approximately 800bp in size, was constructed then sequencing and BLASTn results of the ScFv fragment consequently proved the accuracy. CONCLUSION: Results showed 88% similarity to available sequences in mentioned databank. ScFv was ultimately inserted into expression vector for producing recombinant human anti-tetanus mAb.

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