21 results about "Exonuclease" patented technology

This invention discloses a method for producing single-stranded DNA using Cas nickase. This method solves the problem of limitations on the enzyme cleavage sites when using nicking endonucleases, and improves the nicking efficiency of plasmids by performing multiple nicking using multiple gRNAs in a single enzyme cleavage system, while also effectively improving the efficiency of subsequent digestion by exonucleases.

Multiplex PCR amplification may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers. Multiplex PCR-based assays may be improved by using a load-balanced set of primers with different proportions of primers in the set to provide balanced amplicon read depth.

Linear DNA with improved resistance against exonucleases and methods for its production. The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) with improved resistance to nuclease digestion. The present invention relates to methods comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adaptor molecules and intermediate adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to generate a linear DNA product (e.g., a closed linear DNA product). The present invention also relates to linear deoxyribonucleic acid (DNA) products (e.g., a closed linear DNA product) with improved resistance to nuclease digestion and uses thereof.

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

A chimeric DNA polymerase includes: a first fragment having at least 80% homology to at least part of an N-end domain of KOD DNA polymerase; a second fragment having at least 80% homology to at least part of an exonucleolytic domain of Pab DNA polymerase; a third fragment having at least 80% homology to at least part of the N-end domain of KOD DNA polymerase; a fourth fragment having at least 80% homology to at least part of a palm domain of Pfu DNA polymerase; a fifth fragment having at least 80% homology to at least part of a finger domain of Pab DNA polymerase; a sixth fragment having at least 80% homology to at least part of the palm domain of Pfu DNA polymerase; and a seventh fragment having at least 80% homology to at least part of a thumb domain of KOD DNA polymerase.

This invention belongs to the fields of microbiology and molecular biology, specifically relating to a homologous recombinase combination in myxobacteria and its application in gene editing. Specifically, this invention provides four pairs of homologous recombinase systems capable of efficiently mediating in vivo recombination in myxobacteria. Each pair includes a protein YqaJ with exonuclease function and a protein RecT with single-strand annealing function. This invention provides a method for myxobacterial gene editing: under the action of homologous recombinase pairs, mutant strains are obtained through positive screening using resistance tags. The resistance tags are then successfully removed through the combined action of the Cre / loxP system and the reverse screening gene galK. This method achieves a gene editing efficiency of up to 100%, has a short cycle time, requires no purification, and can be used for iterative deletion of multiple genes to achieve genome simplification in myxobacteria, or for inserting functional promoters and other elements to activate silent gene clusters. This method is of great significance for the bioexploration and functional genomics research of myxobacteria.

This invention relates to the field of biopharmaceutical technology, and discloses a method for preparing smegglutinin using circRNA and related compositions. The circRNA molecule contains an open reading frame and an internal ribosome entry site sequence. A recombinant plasmid containing a T7 promoter, introns, and coding sequences is constructed and linearized. A linear RNA intermediate is obtained through in vitro transcription; subsequently, it is placed in a circularization buffer for self-splicing and circularization; and purified to obtain the circRNA molecule stock solution. The related pharmaceutical composition contains circRNA molecules, a four-component lipid mixture, and excipients containing carbohydrates and nonionic surfactants. This invention constructs circRNA molecules with closed-loop structures and utilizes the internal ribosome entry site sequence to initiate translation. The lack of ends in the closed-loop structure avoids exonuclease degradation, thus prolonging the protein expression cycle, improving the purity of the stock solution, and reducing the risk of immune responses caused by linear RNA impurities.

The application provides a method for rapidly screening aptamers based on exonuclease degradation rate, a reaction system with added aptamers and targets, and a control system with only added aptamers, after adding exonuclease, part of samples are taken out every certain time and added into a quenching solution containing a fluorescent dye, the fluorescent intensity is detected and the relative fluorescent intensity of the reaction system and the control system is compared, and the binding capacity of the aptamers and the targets is indirectly represented. The application can qualitatively detect the affinity of multiple groups of aptamers in a high-throughput and large-batch manner, greatly shortens the screening period, reduces the cost, and is beneficial to subsequent quantitative detection.

This invention belongs to the technical field of biomedicine, specifically relating to a lipid nanoparticle delivery system for in vivo CAR-T cell generation, its preparation method, and its applications. The delivery system of this invention first utilizes a high-affinity CD3ε targeting peptide to enable lipid nanoparticles (LNPs) to precisely target and efficiently transfect T cells. Secondly, it employs circular RNA (circRNA) as the CAR encoding vector; its closed circular structure resists exonuclease degradation. Finally, the aforementioned peptides and circRNA-encapsulated LNPs are integrated into a targeted delivery system using microfluidic preparation technology. The results of the embodiments demonstrate that the delivery system of this invention possesses advantages such as extremely short treatment cycles, simplified preparation processes, highly efficient targeted delivery, durable CAR expression, and high safety.

The invention provides a DNA (deoxyribonucleic acid) cloning method, particularly provides heat-resistant exonuclease and a method for seamlessly cloning DNA by using the heat-resistant exonuclease at a relatively high temperature, and finds that the method disclosed by the invention can realize efficient and rapid cloning for the first time.

The application discloses a non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metal beta-lactamase and a screening method thereof. The screening method uses dNaMTP and dTPT3TP, or dCNMOTP and dTPT3TP as non-natural base nucleoside triphosphate substrates, uses OneTaq DNA polymerase to amplify a DNA library containing non-natural bases, and uses lamda exonuclease to degrade 5' end phosphorylated DNA chains in PCR products, so as to obtain two ssDNA libraries. The non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metal beta-lactamase is screened based on the libraries, and three non-natural base DNA aptamers with a length of 70 nt are obtained. The results of a biological membrane layer interference analysis show that the three non-natural base DNA aptamers have good binding affinity to Bacillus cereus 5 / B / 6 metal beta-lactamase.

This invention relates to a disaccharide exoglycosidase β-agarase with both galactosidase and β-galactosidase activities and its applications, belonging to the field of biotechnology. This invention discloses for the first time a method for developing a β-agarase from *Pseudomonas alterniflora* (…). Pseudoalteromonas An enzyme, ORF0276, and its encoding gene, possessing both galactosidase and disaccharide exonuclease activities, were obtained from the genome of strain Q02 (sp.). This enzyme exhibits strong thermostability, high pH tolerance, and stable physicochemical properties, thus possessing potential for industrial applications. When degrading agar polysaccharide substrates, it can act on the β-1,4 glycosidic bonds of the sugar chains to produce a product primarily composed of neo-agarbiose, which can be used for the industrial preparation of neo-agarbiose (NA2). This research provides a molecular template and theoretical reference for the discovery of such multi-functional tool enzymes.

This invention discloses an immunomodulatory composition and its preparation method based on sheep liver protein hydrolysate, belonging to the field of health food. The composition, by weight, comprises 30-60 parts sheep liver protein hydrolysate, 10-25 parts wolfberry polysaccharide, and 10-20 parts astragalus polysaccharide, preferably containing 5-15 parts yeast β-glucan and 2-8 parts vitamin C. The proportion of oligopeptides <1000Da in the hydrolysate is not less than 85%. This invention employs a gentle deodorizing pretreatment with yeast and ginger extract, combined with a continuous, integrated process of two-stage enzymatic hydrolysis using endonucleases and exonucleases. This deodorizes while promoting protein conformational development, improving hydrolysis efficiency and achieving a balance between high oligopeptide yield and excellent sensory quality. Verification using the King's Law shows that the components exhibit significant synergistic effects, superior to single components in enhancing immunity, anti-oxidation, and auxiliary liver protection. It is highly safe and can be applied in health food and special medical food fields.

This invention discloses a method for constructing a dual-omics library based on single-cell whole-genome methylation and transcriptomics, belonging to the field of sequencing technology. The method includes: lysing single cells in a sorting tube pre-filled with lysis buffer; applying a nucleophilic coating to the inner wall of the sorting tube to allow cell nuclei to adsorb and obtain a supernatant containing mRNA; reverse transcribing the supernatant to obtain cDNA and constructing a transcriptome library; lysing the cell nucleus to release DNA and converting it to bisulfite; ligating the converted DNA with a molecularly tagged adapter to obtain methylation intermediates; treating with exonucleases; merging intermediates with different molecular tags; purifying with magnetic beads, single-stranding, ligating the other end with a sequencing adapter, and amplifying and purifying to obtain a methylated library. This invention achieves simultaneous separation of RNA and DNA in the same tube for library construction, reducing separation and transfer steps, minimizing RNA degradation and nucleic acid loss, and improving the throughput of methylation library construction and the consistency of dual-omics data.

The application discloses a method for regulating catalytic behavior of 3'->5' exonuclease based on deoxyribose configuration adjustment and application thereof, and belongs to the technical field of nucleic acid chemistry, biological catalytic regulation and molecular engineering. In view of the problems of poor position accuracy and unpredictable effect of the existing nucleic acid enzyme regulation method, a hydrazine compound containing an NH-NH2 functional group is used to modify and reduce an AP site in DNA, so that the deoxyribose of the AP site is changed from a closed ring configuration to a stable open chain configuration. The DNA after the AP site is modified is incubated with a nucleic acid enzyme with 3'->5' exonuclease activity, so that the nucleic acid enzyme is specifically catalyzed to stop at a nucleotide downstream of the modified AP site, and the catalytic behavior of the nucleic acid enzyme is accurately and controllably regulated with single nucleotide resolution. The application does not need to rely on steric hindrance or enzyme molecular structure modification, the regulation effect is stable and predictable, and the application can be widely applied to high-resolution DNA damage positioning, controllable DNA assembly and DNA oxidative damage level detection.

The application discloses an exonuclease fusing an NTPase domain and application thereof, and belongs to the technical field of bioengineering. The exonuclease is a Ppl protein, which comprises a PHP domain at the N terminal and an NTPase domain at the C terminal, wherein the PHP domain has 3'-5' ssDNA exonuclease activity. The NTPase domain of the application regulates the exonuclease activity of the PHP domain through conformational change. Under the condition of high NTP concentration, the NTPase domain inhibits the activity of the PHP domain; when the NTP concentration decreases, the inhibition is released, and the exonuclease activity is significantly activated. The activated PHP domain shows specific cleavage activity to DNA substrates with 3'-hydroxyl overhang. The unique 'NTP concentration sensing and enzyme activity regulation' characteristics make it a core module for developing biological sensors, gene circuits and controllable nucleic acid tools.

The application relates to the technical field of medicines, in particular to application of a VER155008 compound in preparation of a coronavirus-dependent RNA polymerase antagonist. Through construction of a reliable screening model of SARS-CoV-2 RdRp inhibitors, the application tests the inhibiting effect of a plurality of nucleoside analogs on SARS-CoV-2 RdRp, finds that the VER155008 compound has obvious inhibiting effect on SARS-CoV-2 RdRp, the VER155008 compound has SARS-CoV-2 exonuclease nsp14 resistance, and can effectively inhibit replication of coronaviruses HCoV-OC43 and HCoV-NL63. Therefore, the application provides key data for research and development of a novel anti-new coronavirus lead compound, and provides a theoretical basis for research and development of an effective coronavirus treatment drug.

Multiplex PCR amplification may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers. Multiplex PCR-based assays may be improved by using a load-balanced set of primers with different proportions of primers in the set to provide balanced amplicon read depth.

This invention discloses a time-temperature indicator for determining whether frozen food has undergone room temperature thawing, its preparation method, and its application. The time-temperature indicator of this invention comprises: a single-stranded DNA forming a stem-loop structure, with fluorescent and quenching groups modified at both ends respectively; an exonuclease that specifically cleaves linear single-stranded DNA; and a dispersing solvent; the single-stranded DNA forming the stem-loop structure has no dangling sequences at its 5' and 3' ends, and the fluorescent and quenching groups are close to each other, thus not producing fluorescence; in the dispersing solvent, the melting temperature T of the single-stranded DNA stem-loop is... m The temperature range is 0℃–25℃. The time-temperature indicator of this invention achieves irreversible fluorescence changes from freezing to room temperature by modifying a stem-loop structure of a single-stranded DNA sequence with fluorescent and quenching groups, as well as by the cleavage of linear single-stranded DNA by exonucleases. This allows for accurate determination of whether a frozen product has been thawed at room temperature.

The disclosed invention relates to a linear double stranded DNA product with enhanced exonuclease resistance due to the presence of protected nucleotides, preferably phosphorothioated nucleotides. The linear double stranded DNA comprises: a sense and antisense strand, at least two protected nucleotides at internal positions in the antisense strand, a single cassette comprising a coding sequence, and wherein the antisense strand has a protected nucleotide upstream and downstream of the cassette. By flanking the cassette, the protected nucleotides protect the cassette from exonuclease degradation. In preferred embodiments, the cassette comprises a promoter, and the sense strand also comprises protected nucleotides upstream and downstream of the cassette. A method of manufacturing the DNA product is disclosed, comprising rolling circle amplification (RCA) of a template in the presence of phosphorothioated nucleotides thereby inserting said nucleotides into the product in a sequence and concentration dependent manner, and restriction digesting the RCA product such that each fragment comprises a single cassette. The DNA products of the invention are transfected into cells, resulting in expression of recombinant GFP, and also used for in vitro transcription (IVT) reactions.

The application discloses a DNA detection kit based on a CRISPR / Cas9 system and exonuclease lambda Exo and application thereof, and belongs to the technical field of molecular biology and gene detection, wherein the kit comprises Cas9, Spacer RNA, Scaffold RNA, lambda Exo, pDNA and a single-stranded nucleic acid reporter molecule; in detection, the pDNA guides the lambda Exo to open the double strand at a specific site of a dsDNA target to form an R-Loop structure, and then the assembled SCas9 RNP recognizes the target sequence and activates the trans-cleavage activity to cut the reporter molecule to generate a signal. The strategy activates the trans-cleavage activity of the Cas9, and simultaneously eliminates the inherent cis-cleavage activity, thereby not only eliminating the dependence of the traditional CRISPR system on a PAM sequence, but also enabling the nucleic acid detection to be performed in the same tube as an isothermal amplification system, so that the false positive risk is reduced.