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Nucleic Acids Research Aug 2004We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency....
We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. This method should be useful to facilitate the preparation of high-quality site saturation libraries.
Topics: DNA Primers; Directed Molecular Evolution; Electrophoresis, Agar Gel; Gene Library; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Engineering
PubMed: 15304544
DOI: 10.1093/nar/gnh110 -
BMC Biotechnology Dec 2008Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more...
BACKGROUND
Mutagenesis plays an essential role in molecular biology and biochemistry. It has also been used in enzymology and protein science to generate proteins which are more tractable for biophysical techniques. The ability to quickly and specifically mutate a residue(s) in protein is important for mechanistic and functional studies. Although many site-directed mutagenesis methods have been developed, a simple, quick and multi-applicable method is still desirable.
RESULTS
We have developed a site-directed plasmid mutagenesis protocol that preserved the simple one step procedure of the QuikChange site-directed mutagenesis but enhanced its efficiency and extended its capability for multi-site mutagenesis. This modified protocol used a new primer design that promoted primer-template annealing by eliminating primer dimerization and also permitted the newly synthesized DNA to be used as the template in subsequent amplification cycles. These two factors we believe are the main reasons for the enhanced amplification efficiency and for its applications in multi-site mutagenesis.
CONCLUSION
Our modified protocol significantly increased the efficiency of single mutation and also allowed facile large single insertions, deletions/truncations and multiple mutations in a single experiment, an option incompatible with the standard QuikChange. Furthermore the new protocol required significantly less parental DNA which facilitated the DpnI digestion after the PCR amplification and enhanced the overall efficiency and reliability. Using our protocol, we generated single site, multiple single-site mutations and a combined insertion/deletion mutations. The results demonstrated that this new protocol imposed no additional reagent costs (beyond basic QuikChange) but increased the overall success rates.
Topics: DNA Primers; Mutagenesis, Insertional; Mutagenesis, Site-Directed; Plasmids; Polymerase Chain Reaction; Sequence Deletion
PubMed: 19055817
DOI: 10.1186/1472-6750-8-91 -
Nucleic Acids Research Feb 2019The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone...
The assembly of DNA fragments with homologous arms is becoming popular in routine cloning. For an in vitro assembly reaction, a DNA polymerase is often used either alone for its 3'-5' exonuclease activity or together with a 5'-3' exonuclease for its DNA polymerase activity. Here, we present a 'T5 exonuclease DNA assembly' (TEDA) method that only uses a 5'-3' exonuclease. DNA fragments with short homologous ends were treated by T5 exonuclease and then transformed into Escherichia coli to produce clone colonies. The cloning efficiency was similar to that of the commercial In-Fusion method employing a proprietary DNA polymerase, but higher than that of the Gibson method utilizing T5 exonuclease, Phusion DNA polymerase, and DNA ligase. It also assembled multiple DNA fragments and did simultaneous site-directed mutagenesis at multiple sites. The reaction mixture was simple, and each reaction used 0.04 U of T5 exonuclease that cost 0.25 US cents. The simplicity, cost effectiveness, and cloning efficiency should promote its routine use, especially for labs with a budget constraint. TEDA may trigger further development of DNA assembly methods that employ single exonucleases.
Topics: Cloning, Molecular; Escherichia coli; Exodeoxyribonucleases; Genetic Vectors; Mutagenesis, Site-Directed; Polyethylene Glycols; Tromethamine
PubMed: 30462336
DOI: 10.1093/nar/gky1169 -
ACS Synthetic Biology Jul 2020Genetic engineering of natural product biosynthetic gene clusters represents an attractive approach to access new and complex bioactive small molecules. However, due to...
Genetic engineering of natural product biosynthetic gene clusters represents an attractive approach to access new and complex bioactive small molecules. However, due to the large number and size of some genes involved in specialized metabolism, notably those encoding modular polyketide synthase and nonribosomal peptide synthetase megaproteins, it remains difficult to introduce precise genetic mutations to probe domain activity or alter chemical product formation. Here, we report the development and validation of a robust method combining oligonucleotide recombineering and CRISPR/Cas9 targeting for rapid site-directed mutagenesis of cloned pathways, which can be directly transferred to a heterologous host for expression. We rapidly generated 12 point mutations and identified several important determinants of successful mutagenesis, including the protospacer/PAM sequence and presence of regions of local homology. Our approach may be broadly applicable for researchers interested in probing natural product biosynthesis or performing pathway engineering.
Topics: Base Sequence; CRISPR-Cas Systems; Escherichia coli; Multigene Family; Mutagenesis, Site-Directed; Oligonucleotides; Peptide Synthases; Plasmids; Point Mutation; Polyketide Synthases; Recombination, Genetic; Saccharomyces cerevisiae
PubMed: 32584552
DOI: 10.1021/acssynbio.0c00265 -
Biomolecules Feb 2023The high-degree polymerization of isomaltooligosaccharide (IMO) not only effectively promotes the growth and reproduction of Bifidobacterium in the human body but also...
The high-degree polymerization of isomaltooligosaccharide (IMO) not only effectively promotes the growth and reproduction of Bifidobacterium in the human body but also renders it resistant to rapid degradation by gastric acid and can stimulate insulin secretion. In this study, we chose the engineered strain expressed dextranase (PsDex1711) as the research model and used the AutoDock vina molecular docking technique to dock IMO4, IMO5, and IMO6 with it to obtain mutation sites, and then studied the potential effect of key amino acids in this enzyme on its hydrolysate composition and enzymatic properties by site-directed mutagenesis method. It was found that the yield of IMO4 increased significantly to 62.32% by the mutant enzyme H373A. Saturation mutation depicted that the yield of IMO4 increased to 69.81% by the mutant enzyme H373R, and its neighboring site S374R IMO4 yield was augmented to 64.31%. Analysis of the enzymatic properties of the mutant enzyme revealed that the optimum temperature of H373R decreased from 30 °C to 20 °C, and more than 70% of the enzyme activity was maintained under alkaline conditions. The double-site saturation mutation results showed that the mutant enzyme H373R/N445Y IMO4 yield increased to 68.57%. The results suggest that the 373 sites with basic non-polar amino acids, such as arginine and histidine, affect the catalytic properties of the enzyme. The findings provide an important theoretical basis for the future marketable production of IMO4 and analysis of the structure of dextranase.
Topics: Humans; Dextranase; Molecular Docking Simulation; Polymerization; Amino Acids; Mutagenesis, Site-Directed
PubMed: 36830669
DOI: 10.3390/biom13020300 -
BioTechniques Nov 2022Despite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed...
Despite the development of various methods and commercial kits, site-directed mutagenesis of large plasmids remains a challenge in many laboratories. A site-directed mutagenesis method was developed for large plasmids by directly transforming two overlapping PCR fragments into . This method successfully generated mutations for plasmids of 8.3 kb and 11.0 kb with high efficiencies. The method only requires Q5 DNA polymerase and , which greatly reduces costs. The procedure is simple, including PCR reaction, treatment and transformation. This simple, efficient and economical site-directed mutagenesis method for large plasmids is likely to be widely applied in the future.
Topics: Plasmids; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Escherichia coli; DNA-Directed DNA Polymerase
PubMed: 36398840
DOI: 10.2144/btn-2022-0085 -
Scientific Reports Aug 2023Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary...
Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic β-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and K and k values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.
Topics: Paenibacillus; Bacillus; Mutagenesis, Site-Directed; Binding Sites
PubMed: 37591902
DOI: 10.1038/s41598-023-40072-1 -
Protein Science : a Publication of the... Jul 2015It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to... (Review)
Review
It is well recognized that protein product development is far more challenging than that for small-molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long-term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site-specific mutagenesis, and preparation of nontraditional types of dosage forms-semiaqueous solutions, nonfreeze-dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.
Topics: Animals; Chemistry, Pharmaceutical; Drug Stability; Excipients; Humans; Mutagenesis, Site-Directed; Protein Stability; Proteins
PubMed: 25858529
DOI: 10.1002/pro.2684 -
Acta Biochimica Et Biophysica Sinica Dec 2017The crystal structure of mature Polygonatum cyrtonema lectin (PCL) showed three similar carbohydrate-binding sites (CBS I, CBS II, and CBS III). The Gln58 and Asp60...
The crystal structure of mature Polygonatum cyrtonema lectin (PCL) showed three similar carbohydrate-binding sites (CBS I, CBS II, and CBS III). The Gln58 and Asp60 residues of CBS II are substituted with His58 and Asn60. To establish the relationship between the key amino acid residues and structure or activity of PCL, we constructed four recombinant mutants in CBS I, CBS II, and CBS III. The experimental results indicate that CBS I, CBS III and the disulfide bond play vital roles in the binding with mannose. Furthermore, molecular dynamics simulations and binding free energy calculation illustrate that CBS I has a direct and strong relationship with the activity of PCL. CBS II does not play a critical role in the model for mannose binding by PCL. Although CBS III does not enhance the activity, it helps to maintain the activity and 3D structure. These results suggest that the carbohydrate-binding site of PCL may be in a hydrophilic environment, and Asn and Tyr are the key amino acids involved in its binding with sugar, but Gln and Asp are not necessary to maintain its activity.
Topics: Binding Sites; Hemagglutination; Mannose; Molecular Dynamics Simulation; Mutagenesis, Site-Directed; Plant Lectins; Polygonatum; Structure-Activity Relationship
PubMed: 29121159
DOI: 10.1093/abbs/gmx116 -
Genetics and Molecular Research : GMR May 2015Site-directed mutagenesis is an essential technique for investigating the mechanisms of gene regulation on a molecular level, as well as for exploring post-translational...
Site-directed mutagenesis is an essential technique for investigating the mechanisms of gene regulation on a molecular level, as well as for exploring post-translational modifications and functional structure at the protein level. Polymerase chain reaction combining in vitro synthesis of oligonucleotide primers allows for site-directed mutation to be performed with ease. However, site-directed mutagenesis is difficult when larger plasmids are involved. Here, we present a novel method for generating large gene site-directed mutagenesis products based on a cut-and-paste-based cloning strategy. This method uses 4 primers, incorporating relevant mutations and restriction enzyme site sequences, to generate 2 DNA fragments by polymerase chain reaction. The fragments are then ligated into TA cloning vectors. Large genes containing mutations of interest were obtained by cutting and then pasting, and then inserting one fragment into another T-vector. We demonstrated the practicality of this method by creating a G59S mutation within the p150(Glued)-encoding gene.
Topics: Base Sequence; Cloning, Molecular; DNA; DNA Primers; Genetic Vectors; HeLa Cells; Humans; Mutagenesis, Site-Directed; Mutation; Protein Kinases
PubMed: 26125756
DOI: 10.4238/2015.May.25.10