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Journal of Pharmaceutical and... Jun 2010We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to determine DNA sizes between 500 and 5000 bp with good quantification...
We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to determine DNA sizes between 500 and 5000 bp with good quantification (DNA concentration, <8 ng/l) within 5 min, for the analysis of DNA ligation. On analysis of an electropherogram of a ligation mixture of the pTAC1-T vector and a 789 bp PCR-amplified DNA fragment, the presence of recombinant DNA was easily detected by comparison with an electropherogram obtained without ligase. On analysis of a ligation mixture of pUC19/Eco RI without alkaline phosphatase treatment and a 667 bp Eco RI-digested fragment of foreign DNA, several peaks observed in the electropherogram corresponded to the formation of monomeric and polymeric insert DNAs, self-ligated vector DNA, and recombinant DNA. On the other hand, several peaks were also observed in the electropherogram of the ligation mixture of pUC19/Eco RI with alkaline phosphatase treatment and the 667 bp Eco RI-digested fragment of foreign DNA, the fluorescence intensity corresponding to recombinant DNA apparently being increased. These results indicate the potential of microchip electrophoresis for the analysis of DNA ligation, it offering high resolution in a short time.
Topics: Bacteriophage T4; Base Sequence; Buffers; Cloning, Molecular; DNA Restriction Enzymes; DNA, Circular; DNA, Recombinant; DNA, Viral; Electrophoresis, Microchip; Feasibility Studies; Microfluidic Analytical Techniques; Molecular Sequence Data; Reference Standards
PubMed: 20196235
DOI: 10.1016/j.jpba.2009.12.023 -
Protein Engineering, Design & Selection... Oct 2012DNA ligation is essential to many molecular biology manipulations, but this reaction is often carried out by following generic guidelines or by trial and error....
DNA ligation is essential to many molecular biology manipulations, but this reaction is often carried out by following generic guidelines or by trial and error. Maximizing the desired ligation product is especially important in DNA library construction for directed evolution experiments since library diversity is directly affected by ligation efficiency. Here, we suggest that display vectors that rely on Type IIP restriction sites for cloning should be redesigned to utilize Type IIS restriction sites instead because ligation yield is significantly improved: we observed up to 15- and 2.6-fold increases in desired products for circular and linear ligation reactions, respectively. To guide ligation optimization more rationally, we developed an easily parameterized thermodynamic model that predicts product distributions based on input DNA concentrations and free energies of the ligation events. We applied this model to study ligation reactions using a ribosome display vector redesigned with Type IIS restriction sites (pRDV2). We computationally predicted and experimentally validated the relative abundance of various products in three-piece linear ligations as well as the extent of transformation from vector-insert circular ligations. Based on our results, we provide general insights into ligation and we outline guidelines for optimizing this reaction for both in vivo and in vitro display methodologies.
Topics: Bacteriophage T4; Base Sequence; Cloning, Molecular; DNA; DNA Ligases; Deoxyribonucleases, Type II Site-Specific; Directed Molecular Evolution; Gene Library; Genetic Vectors; Models, Biological; Models, Chemical; Thermodynamics
PubMed: 22543356
DOI: 10.1093/protein/gzs019 -
Chemical Communications (Cambridge,... May 2018Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The...
Current gene synthesis methods are driven by enzymatic reactions. Here we report the one-pot synthesis of a chemically-ligated gene from 14 oligonucleotides. The chemical ligation benefits from the highly efficient click chemistry approach templated by DNA nanostructures, and produces modified DNA that is compatible with polymerase enzymes.
Topics: Alkynes; Azides; Click Chemistry; DNA; Green Fluorescent Proteins; Mutation; Nanostructures; Oligodeoxyribonucleotides; Polymerase Chain Reaction; Protein Engineering; Triazoles
PubMed: 29662975
DOI: 10.1039/c8cc00738a -
Sheng Wu Gong Cheng Xue Bao = Chinese... May 2024To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly...
To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into . After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.
Topics: Escherichia coli; Fructose-Bisphosphatase; Homologous Recombination; Plasmids; Genetic Vectors; DNA; Mutation; Mutagenesis, Site-Directed; Cloning, Molecular
PubMed: 38783816
DOI: 10.13345/j.cjb.230793 -
Proceedings of the National Academy of... Mar 1998A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from Escherichia coli to selectively pair...
A method is described that allows the sequence-specific ligation of DNA. The method is based on the ability of RecA protein from Escherichia coli to selectively pair oligonucleotides to their homologous sequences at the ends of fragments of duplex DNA. These three-stranded complexes were protected from the action of DNA polymerase. When treated with DNA polymerase, unprotected duplex fragments were converted to fragments with blunt ends, whereas protected fragments retained their cohesive ends. By using conditions that greatly favored ligation of cohesive ends, a second DNA fragment could be selectively ligated to a previously protected fragment of DNA. When this second DNA was a vector, selected fragments were preferentially cloned. The method had sufficient power to be used for the isolation of single-copy genes directly from yeast or human genomic DNA, and potentially could allow the isolation of much longer fragments with greater fidelity than obtainable by using PCR.
Topics: Bacteriophage lambda; Base Sequence; Binding Sites; Cloning, Molecular; DNA; DNA, Complementary; DNA, Fungal; DNA, Viral; Escherichia coli; Fibroblast Growth Factor 3; Fibroblast Growth Factors; Genetic Vectors; Humans; Oligodeoxyribonucleotides; Proto-Oncogene Proteins; Rec A Recombinases; Saccharomyces cerevisiae
PubMed: 9482854
DOI: 10.1073/pnas.95.5.2152 -
Journal of Pharmaceutical and... Feb 2018The use of DNA barcodes for species identification is a common laboratory practice. However, PCR amplification of full-length DNA barcode in processed material is...
The use of DNA barcodes for species identification is a common laboratory practice. However, PCR amplification of full-length DNA barcode in processed material is difficult because of severe DNA fragmentation. In this study, an adaptor ligation-mediated PCR protocol was derived to amplify sets of target DNA fragments isolated from two CCMG products. The specially designed adaptor with asymmetric strands and terminal modification avoids amplification of non-target DNA sequences. DNA extracted from Angelica sinensis and Panax notoginseng CCMG were ligated with the adaptors and amplified by an adaptor primer and a single universal barcode primer to obtain partial ITS2 sequence. Results showed that various length of DNA fragments within the ITS2 region were amplified and could be used to identify the concerned species. The adaptor ligation-mediated PCR is therefore a promising universal method for species identification in highly processed herbal products.
Topics: Angelica sinensis; DNA Barcoding, Taxonomic; DNA Primers; DNA, Intergenic; DNA, Plant; Drugs, Chinese Herbal; Medicine, Chinese Traditional; Panax notoginseng; Polymerase Chain Reaction; Sequence Analysis, DNA
PubMed: 29175746
DOI: 10.1016/j.jpba.2017.11.048 -
DNA Research : An International Journal... Dec 2013Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can...
Ligation, the joining of DNA fragments, is a fundamental procedure in molecular cloning and is indispensable to the production of genetically modified organisms that can be used for basic research, the applied biosciences, or both. Given that many genes cooperate in various pathways, incorporating multiple gene cassettes in tandem in a transgenic DNA construct for the purpose of genetic modification is often necessary when generating organisms that produce multiple foreign gene products. Here, we describe a novel method, designated PRESSO (precise sequential DNA ligation on a solid substrate), for the tandem ligation of multiple DNA fragments. We amplified donor DNA fragments with non-palindromic ends, and ligated the fragment to acceptor DNA fragments on solid beads. After the final donor DNA fragments, which included vector sequences, were joined to the construct that contained the array of fragments, the ligation product (the construct) was thereby released from the beads via digestion with a rare-cut meganuclease; the freed linear construct was circularized via an intra-molecular ligation. PRESSO allowed us to rapidly and efficiently join multiple genes in an optimized order and orientation. This method can overcome many technical challenges in functional genomics during the post-sequencing generation.
Topics: Arabidopsis; Cloning, Molecular; DNA; DNA, Plant; Genetic Vectors; Plasmids
PubMed: 23897972
DOI: 10.1093/dnares/dst032 -
Journal of the American Chemical Society Mar 2004Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA...
Cloning DNA typically involves the joining of target DNAs with vector constructs by enzymatic ligation. A commonly used enzyme for this reaction is bacteriophage T4 DNA ligase, which requires ATP as the energy source to catalyze the otherwise unfavorable formation of a phosphodiester bond. Using in vitro selection, we have isolated a DNA sequence that catalyzes the ligation of DNA in the absence of protein enzymes. We have used the action of two catalytic DNAs, an ATP-dependent self-adenylating deoxyribozyme (AppDNA) and a self-ligating deoxyribozyme, to create a ligation system that covalently joins oligonucleotides via the formation of a 3',5'-phosphodiester linkage. The two-step process is conducted in separate reaction vessels wherein the products of deoxyribozyme adenylation are purified before their use as substrates for deoxyribozyme ligation. The final ligation step of the deoxyribozyme-catalyzed sequence of reactions mimics the final step of the T4 DNA ligase reaction. The initial rate constant (k(obs)) of the optimized deoxyribozyme ligase was found to be 1 x 10(-)(4) min(-)(1). Under these conditions, the ligase deoxyribozyme promotes DNA ligation at least 10(5)-fold faster than that generated by a simple DNA template. The self-ligating deoxyribozyme has also been reconfigured to generate a trans-acting construct that joins separate DNA oligonucleotides of defined sequence. However, the sequence requirements of the AppDNA and that of the 3' terminus of the deoxyribozyme ligase limit the range of sequences that can be ligated.
Topics: Base Sequence; DNA; DNA Ligases; DNA, Catalytic; Kinetics; Molecular Sequence Data; Nucleic Acid Conformation
PubMed: 15025472
DOI: 10.1021/ja039713i -
Current Protocols in Molecular Biology May 2001DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair...
DNA ligases catalyze the formation of phosphodiester bonds between juxtaposed 5' phosphate and a 3'-hydroxyl terminus in duplex DNA. This activity can repair single-stranded nicks in duplex DNA and join duplex DNA restriction fragments having either blunt ends or homologous cohesive ends. Two ligases are used for nucleic acid research and their reaction conditions and applications are described in this unit: E. coli ligase and T4 ligase. These enzymes differ in two important properties. One is the source of energy: T4 ligase uses ATP, while E. coli ligase uses NAD. Another important difference is their ability to ligate blunt ends; under normal reaction conditions, only T4 DNA ligase will ligate blunt ends.
Topics: Catalysis; DNA; DNA Ligases; Escherichia coli
PubMed: 18265223
DOI: 10.1002/0471142727.mb0314s08 -
Biosensors & Bioelectronics Mar 2013We report a simple and versatile strategy for the construction of DNA-templated nanoparticle (NP) assemblies using click DNA ligation. Diels-Alder cycloaddition reaction...
We report a simple and versatile strategy for the construction of DNA-templated nanoparticle (NP) assemblies using click DNA ligation. Diels-Alder cycloaddition reaction of maleic acid and sorbic acid is used for click DNA ligation. Sorbic acid-prelabelled DNA nanowires with micron-scale length were synthesized and acted as templates for the construction of NP assemblies. Au NPs were orderly assembled onto the DNA nanowires using the click DNA ligation to form DNA-templated Au NP chains. Furthermore, DNA-templated CdTe quantum dot (QD) chains were synthesized using the method and characterized by confocal fluorescence microscopy (CFM). The DNA-templated CdTe QD chains were functionalized with cell-binding aptamer as the recognition element. The aptamer-functionalized CdTe QD chains, acting as a novel linear-shaped cellular probe, could be used for cancer cell imaging. Target cells could be rapidly and easily distinguished from a mixture of multiple cells using the cellular probe. The click DNA ligation method offers a convenient and efficient way to synthesize DNA-templated NP assemblies, and may become a powerful tool in other DNA-based studies.
Topics: Click Chemistry; DNA; Materials Testing; Molecular Imprinting; Quantum Dots; Surface Properties
PubMed: 23122756
DOI: 10.1016/j.bios.2012.09.026