230 results about "Endonuclease" patented technology

The invention discloses a kit for detecting miRNAs of multiple extracellular vesicles based on a TtAgo-driven rolling circle amplification technology and application of the kit, a platform combines TtAgo protein and RCA reaction, cooperates with endonuclease activity and DNA polymerase activity of TtAgo, accurately controls reaction kinetic balance between temperature and enzyme activity, not only does not damage the cutting activity of TtAgo, but also can detect miRNAs of multiple extracellular vesicles. The method provided by the invention can be used for detecting multiple extracellular vesicles miRNAs, ensures strong polymerization amplification performance, creates a strong one-step single-tube experiment, greatly simplifies the experimental process, shortens the detection time, realizes ultra-sensitive and specific detection of nucleic acid markers, and can be used for detecting multiple extracellular vesicles miRNAs.

Methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.

The invention belongs to the field of nucleic acid detection and molecular biology, and particularly relates to a method and a kit for eliminating false positive results in nucleic acid amplification reaction. According to the method, after a nucleic acid amplification reaction (taking recombinase polymerase amplification, namely RPA, for example) is completed, a target amplification product is subjected to selective enzyme digestion by utilizing restriction endonuclease, non-target products (such as primer dimers and non-specific amplification products) are not cut, and meanwhile, the non-target products which are not subjected to enzyme digestion are removed by combining solid-phase separation, so that the target amplification product is obtained. Therefore, the false positive result is eliminated. The invention also discloses a kit containing the restriction enzyme. According to the method, the specificity and the signal-to-noise ratio of a nucleic acid amplification reaction, especially RPA, are remarkably improved, the cost is low, operation is easy, the method is compatible with an existing technical platform, the method is suitable for detection scenes such as clinical molecular diagnosis, environmental monitoring and food safety, and the problem of misjudgment caused by false positive signals is effectively avoided.

The invention belongs to the technical field of health-care food, and particularly relates to functional milk assisting in lowering blood pressure and a preparation method thereof.The functional milk comprises, by weight, 99.5% of raw milk and 0.5% of a potassium ion bridged citrus fiber-green tea polyphenol network, raw milk is sequentially subjected to trypsin and proline specific incision enzyme treatment, HPCD-ultrasonic treatment and pulsed magnetic field treatment. The invention provides a systematic solution, which exceeds a simple extraction and addition mode in the prior art, and solves the stability problem of the antihypertensive active component in the processing and storage process.

The invention discloses a high-efficiency wide-targeting-range C-to-G double-enzyme synergistic base editor and application thereof, and belongs to the technical field of gene editing. The editor is a fusion protein comprising a Cas protein having reduced or lost endonuclease activity, a cytosine deaminase (CDA), and a cytosine DNA glycosylase (CDG). In order to solve the problems that an existing C-to-G editor is low in efficiency and limited in targeting range, the C-to-G editor synergistically and efficiently generates a base removal (AP) site on a target DNA through the dual effects of CDA and CDG, so that C-to-G base transversion is promoted. According to the editor, the C-to-G editing efficiency is remarkably improved, an editing window can be effectively expanded or moved, the targeting flexibility is greatly enhanced, meanwhile, high genome specificity is kept, and the editor shows strong application potential in various organisms such as yeast and plants.

The invention relates to the technical field of bioengineering, in particular to a preparation process for extracting antihypertensive peptide from euphausia superba. The process comprises the following steps: adding a ferulic acid / arginine double-grafted pullulan compound in a preparation process, and breaking an emulsification interface by utilizing beta-cyclodextrin adsorption and phospholipase A2 cracking to obtain a soluble pretreatment product; meanwhile, short-time round-trip pH pulse is implemented in the incision enzyme hydrolysis process to activate potential enzyme cutting sites, finally, hydrophobic aggregation peptides are removed through weak acid rapid elution, and excision hydrolysis is performed to obtain a target product with the molecular weight of 0.5-3 kDa, so that dynamic induction and antioxidant steady-state dual control of hydrolysis reaction is realized; the generation proportion of short peptides and the ACE inhibition persistence are improved, and the flavor and the storage stability are remarkably improved. The method is controllable in technological process, high in repeatability and suitable for large-scale preparation of the euphausia superba protein functional peptide and development and application of antihypertensive food.

Provided herein are methods and compositions for truncating and, optionally, joining polynucleotides. The methods bypass inefficient cloning methods and the requirement for expensive enzymes by using in vivo homologous recombination in conjunction with specific endonuclease digestion. The methods may further be used to assemble long fragments of DNA from existing DNA constructs or libraries or to generate novel DNA libraries.

The invention discloses a whole genome methylation detection method based on MeDIP-MSRE, and belongs to the technical field of epigenetics detection. According to the method, methylation immunoprecipitation sequencing and a methylation sensitive restriction enzyme technology are innovatively combined, firstly, a methylation specific antibody is used for conducting immunoprecipitation on sample DNA, and whole genome methylation fragments are enriched; then carrying out enzyme digestion on the enriched product by adopting methylation sensitive restriction enzyme, specifically removing an unmethylated DNA region, and reserving a complete methylation sequence; and finally, constructing a methylation map through high-throughput sequencing. According to the method, traditional hydrosulfite chemical conversion is not needed, DNA damage and base conversion deviation caused by the traditional hydrosulfite chemical conversion are avoided, meanwhile, high sensitivity of MeDIP and high specificity of methylation sensitive restriction endonuclease are fused, and the fidelity, sensitivity and specificity of detection are remarkably improved.

Compositions and methods for detecting structural variations (SVs) in target genes or for genetic mapping of movable transposable elements are disclosed, the target genes relating to disease pathologies commonly found in large Mendelian genomics projects, and the movable transposable elements relating to genetic diseases, cancer, and aging. The method comprises: (i) contacting a sample containing genomic DNA with a DNA endonuclease for an effective amount of time to cleave the genomic DNA into fragments, the genomic DNA being uncrosslinked; (ii) subjecting the fragments obtained from step (b) to a DNA ligase to obtain circularized DNA; (iii) subjecting the circularized DNA to reverse PCR amplification containing a reverse primer, wherein the reverse primer is designed to match a expected wild-type sequence near a suspected mutant locus in the gene; and (iv) sequencing the amplified products. Exemplary conditions include Bardet-Biedel syndrome; severe upper and lower limb defects; retinitis pigmentosa; syndromic microcephaly; spastic paraplegia; and atypical hemolytic uremic syndrome.

The invention relates to the technical field of molecular biology, in particular to a methylation marker for diagnosis of diabetic nephropathy. Specifically, the methylation marker comprises a P site of a CAT gene promoter region and / or an E site of a CAT gene exon 1 region. Based on a detection system combining methylation sensitive restriction enzyme with quantitative PCR, the kit has the advantages of simplicity and convenience in operation, good repeatability, accuracy in quantification and easiness in standardization, and is suitable for low-invasive samples such as peripheral blood. By effectively distinguishing pure type 2 diabetes patients from diabetic nephropathy combined patients, a reliable technical means is provided for risk stratification, early intervention and dynamic monitoring of diabetic people, and the method has good clinical application prospects and popularization value.

The present disclosure relates generally to (CRISPR) RNA (crRNA) for the precision silencing of transcripts. In some embodiments, the crRNA are enriched for guanosine (G) nucleotides at key spacer positions, which is useful in enhancing the silencing efficacy of otherwise inefficient crRNA, thereby expanding the targeting spectrum of Cas13 endonucleases, e.g., Cas13b and Cas13d. In other embodiments, the crRNA comprise a spacer sequence having at least one nucleotide mismatch relative to the target RNA sequence, wherein the target RNA sequence is a wild-type transcript and / or a variant transcript (e.g., a transcript comprising a single nucleotide variant (SNV)). The present disclosure also provides RNA editing systems comprising the crRNA described herein in complex a Cas13 effector protein and a target RNA sequence, methods for the selective targeting of transcripts encoding proteins that are difficult to target, or are not amenable to pharmacological targeting, e.g., oncogenic fusion transcripts or oncogenic transcripts comprising single nucleotide variant(s), and methods for the design and selection of potent crRNA.

Provided are polynucleotides comprising a mobile bacterial island nucleotide sequence (B-INS) comprising one or more modifications (a modified B-INS) that include deletion or modification of restriction enzyme recognition sites to inhibit restriction enzyme cleavage of the B-INS, and an insertion of at least one cargo sequence into the B-INS. The B-INS is packaged within a bacterium into a phage-like particle that contains a bacteriophage capsid, tail and tail fiber proteins. The phage-like particle function as an antibacterial drone "ABD" and infects bacteria. The cargo sequence is introduced into bacteria infected by the ABD and kills the infected bacteria or inhibits growth of infected bacteria.

A primer pair, method, and use for rapidly identifying newborn nude mice. On the basis that a deletion mutation occurs in the base G at position 337 of a coding region of the Foxn1 gene in nude mice, a primer pair introducing an enzyme digestion site is designed according to sequence characteristics. Using the genome of a mouse to be tested as a template, the Foxn1 gene is amplified according to a PCR-RFLP method. The PCR product is then digested with a SmaI restriction endonuclease, and the digested product is genotyped by agarose gel electrophoresis. In addition, a reaction system that can be combined with lateral flow technology is screened, thereby achieving instant detection of the newborn nude mice. The method has the characteristics of simple operation, short time consumption, low cost, instant detection, etc.

The invention discloses recombinant poria cocos chitin endonuclease as well as a gene, a preparation method and application thereof. The nucleotide sequence of the gene is shown as SEQ ID NO.1 in a sequence table. According to the nucleotide sequence as shown in SEQ ID NO.1, high-level recombinant secretory expression of target protein in pichia pastoris host bacteria can be realized by taking pichia pastoris inducible expression plasmid pPICZ alpha A as a carrier. Under a shake flask fermentation condition, the average secretory expression quantity can reach 300.3 mg / L. The recombinant enzyme obtained by nickel ion affinity chromatography purification not only can effectively hydrolyze colloidal chitin to generate chitosan oligosaccharide (COS), but also can directly degrade chitin-containing raw materials such as shrimps, crabs and the like to generate COS. In addition, the enzyme also shows the activity of hydrolyzing polysaccharides such as cellulose, pectin, agar and the like, and has important potential values in the fields of functional chitosan oligosaccharide preparation, biological energy production, medicine development, feed, environmental protection, agricultural application and the like.

The invention discloses a dCAPS molecular marker for identifying sweet potato pulp color characters and application of the dCAPS molecular marker, and belongs to the technical field of sweet potato breeding. According to the dCAPS molecular marker provided by the invention, on the basis of SNP variation sites, mismatched bases are introduced to form restriction endonuclease restriction enzyme cutting sites, the sweet potato pulp color can be judged by utilizing PCR-restriction enzyme cutting-polyacrylamide gel electrophoretic separation, and then sweet potato varieties with high and low carotenoid contents can be identified according to the sweet potato pulp color. The method has important guiding significance and application value for assisting sweet potato carotenoid content related molecular breeding research.

An engineered transcriptional modulator (ETM) comprising: (a) at least one epigenetic effector domain; operably linked to (b) an endonuclease.

This disclosure provides endonuclease enzymes having distinctive domain characteristics, as well as methods for using such enzymes or variants thereof. More specifically, this disclosure provides an engineered base editing system comprising a sequence-defined deaminase base editor.

This invention relates to the field of biomedical technology, specifically to a heart-spleen synergistic regulatory composition based on Chinese yam complex peptides, its preparation method, and its application in improving heart-spleen dysfunction. The method involves first gelatinizing Chinese yam, then sequentially performing primary endopeptidation with papain and secondary fine exopeptidation with flavor protease, followed by separation via a 1000 Da ultrafiltration membrane to directionally prepare a Chinese yam complex peptide with a peptide fraction of at least 90% having a molecular weight less than 1000 Da. This complex peptide is then combined with extracts of ginseng, lotus seed, longan pulp, and tangerine peel in a specific ratio. The yam peptides obtained by this invention can self-assemble with macromolecular hydrophobic components such as ginsenosides in the system to form microscopic peptide-saponin complexes, acting as biological delivery carriers and significantly improving the transmembrane absorption rate of macromolecular active ingredients in individuals with weak spleen and stomach. Simultaneously, the two-step enzymatic hydrolysis process achieves the simultaneous retention of highly active small molecules and precise debittering.

The invention discloses an sgRNA composition, a vector and a method for synchronous editing of pig SLA-DMA and SLA-DMB genes, and belongs to the field of gene editing. Editing targets capable of being specifically recognized by Cas9 endonuclease are designed for SLA-DMA and SLA-DMB genes of pigs, sgRNAs corresponding to the targets are connected in series with tRNA-sgRNA-gRNA scaffold as a minimum repetitive unit, and then a carrier capable of achieving synchronous expression of the four sgRNAs and targeting the SLA-DMA and SLA-DMB genes at the same time is constructed. When the vector is transfected into porcine cells, the SLA-DMA and SLA-DMB genes can be subjected to synchronous mutation, and an important tool is provided for researching a synergistic effect mechanism of the porcine SLA-DMA and SLA-DMB genes and pig gene editing breeding.

The present disclosure provides a method for detecting the methylation and mutation status of a DNA sample, specifically, a method for simultaneously detecting the methylation and mutation status of DNA molecules in a sample, the method comprising treating the sample with a methylation-sensitive restriction enzyme, performing hybrid capture on amplification products of the restriction enzyme-treated sample and a sample that has not been subjected to enzyme cleavage treatment with a probe set including a mutation capture probe and a methylation capture probe, and sequencing the captured products.

An isolated peptide for regulating intracellular protein degradation that includes a linker sequence fused to a C-end degron peptide and, optionally, a caging molecule bonded to the carboxyl group of a C -terminal alanine residue. Also disclosed is a method for temporal control of protein degradation that relies on fusing a protein of interest at its C- terminus to the degron peptide having a caging molecule, and a method for high-fidelity gene editing that utilizes an RNA-dependent endonuclease modified at the C -terminus with the caged isolated peptide. A fusion protein for high-fidelity gene editing is further provided.

The invention provides a method for producing a partially closed deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease a

This invention provides a method for preparing low-bitter rice protein peptides, relating to the fields of food processing and bioengineering. The method involves preparing rice protein slurry, followed by ultra-fine pulverization using an ultra-high pressure, high-energy fluid mill. Through the synergistic effect of extreme shearing, cavitation, and impaction, the dense protein structure is efficiently disrupted, exposing enzyme cleavage sites and directionally reconstructing hydrophobic ends. Subsequently, a combination of endopeptides and exopeptides is added for enzymatic hydrolysis. The endopeptides achieve efficient hydrolysis, while the exopeptides precisely remove terminal hydrophobic amino acids, controlling bitterness generation at its source. This invention overcomes the technical bottlenecks of low efficiency and high bitterness associated with traditional enzymatic hydrolysis. The resulting rice protein peptides have a degree of hydrolysis exceeding 11%, a protein recovery rate exceeding 55%, and a bitterness value far lower than commercially available products. Furthermore, the process is continuous and the conditions are mild, making it suitable for high-end fields such as infant formula, sports nutrition foods, and special medical foods.

(1) An artificial nucleic acid molecule comprising (a) at least one of a 3'-untranslated region (3'UTR) element and / or (b) a 5'-untranslated region (5'UTR) element, and (2) a poly(A) tail is described. The artificial nucleic acid molecule may further comprise a polypeptide comprising a reverse transcriptase (RT) domain and optionally an endonuclease domain for genome modification. A system comprising the artificial nucleic acid molecule and a method of using the system are also described.

This invention belongs to the fields of biotechnology and genomics, and relates to a method and kit for capturing plant chromatin conformation. The method includes the following steps: (1) cross-linking and fixing the chromatin of a plant sample; (2) digesting the fixed chromatin using an enzyme digestion combination containing restriction endonuclease AluI and restriction endonuclease HypCh4V; (3) ligating the enzyme fragments generated in step (2) at spatially adjacent locations to form ligation products; (4) enriching the ligation products and constructing sequencing libraries using the enriched ligation products. This invention uses a combination of AluI and HypCh4V enzymes to replace traditional single-enzyme digestion, which significantly improves data resolution while reducing technical bias. It can achieve chromatin interaction mapping at a resolution of 500 bp in plants and has broad application prospects in the study of fine tissue characteristics at the single-gene level in plants.

Cosmetic composition comprising an endolysin derived from a Staphylococcus aureus phage and a mineral filler. The present invention relates to a composition, particularly a cosmetic one, comprising, in a physiologically acceptable medium, at least one endolysin derived from a Staphylococcus aureus phage and at least one mineral filler selected from hydrophilic fumed silicas and spherical porous silica particles. It also relates to the use of such a composition to prevent and / or treat a skin disorder related to colonization by Staphylococcus aureus in an individual in need, and in particular to prevent and / or treat acne and / or eczema in an individual in need, and a non-therapeutic cosmetic process for the care of keratinous materials, particularly skin, comprising at least one step of topical application of such a composition to said keratinous materials.

Mutant Cas9 proteins are described that have one or more mutations in a Keap1 degron sequence of the Cas9 protein. The one or more mutations can increase the half-life of the mutant proteins. Nucleic acid sequences and constructs comprising a sequence that encode a mutant Cas9 protein, as well as methods of enhancing CRISPR efficiency by administering one or more guide RNAs (gRNAs) to a cell comprising the mutant Cas9 proteins are also disclosed herein.

[Problem] To provide: an adapter for binding to polynucleotide fragments that is used for preparing a genome-wide gRNA library for genome editing systems; a gRNA library that is obtained by using the adapter; and uses of the gRNA library. [Solution] An adapter having a structure described in A). A) A structure having i) to iii). i) A polynucleotide chain having sequences i)-1 to i)-3. i)-1: Sequence corresponding to the forward primer. i)-2: Sequence of the recognition site of a restriction enzyme capable of excising a gRNA spacer sequence from the 5' end of a polynucleotide fragment bound to the adapter. i)-3: Sequence of the recognition site of a restriction enzyme capable of cleaving an adapter having the structure of A) from the polynucleotide fragment. ii) A polynucleotide chain having the sequence complementary to i). iii) A single-stranded polynucleotide chain for formation of a loop connecting the 5' end of i) and the 3' end of ii) of the double strand resulting from the binding of i) and ii).

This invention relates to an identification method and kit for overlapping PCR with enzyme digestion. The method includes selecting a corresponding restriction endonuclease using N4N5X or N4N5Y as the last three bases of the introduced restriction site; if no corresponding restriction endonuclease is available, selecting a corresponding restriction endonuclease using N5X or N5Y as the last two bases of the introduced restriction site; amplifying to obtain a universal fragment primary PCR product; designing PCR amplification primers based on the restriction endonuclease and universal primers to obtain the primary PCR product of the template to be tested; and performing enzyme digestion and genotyping on the final product of the overlapping PCR amplification. This invention utilizes the DOPCR method to identify SNPs / InDels. Compared with existing methods that create restriction sites, it adds an overlapping PCR step, resulting in significant differences in the digested fragments. It eliminates the need for PAGE electrophoresis; detection can be completed using ordinary electrophoresis, making it low-cost and rapid.

The invention discloses an integrated micro-fluidic chip which comprises a chip main body consisting of a reaction layer, a channel layer and a detection layer which are sequentially sealed from top to bottom in the vertical direction, the chip main body internally comprises a first fluid channel network and a second fluid channel network; the first fluid channel network comprises a first reaction chamber, a first mixing unit, a first transfer sample injection unit, a first digital detection unit and a fluid channel for connecting the structural units, which are communicated in sequence; the first fluid channel network and the second fluid channel network have the same structure and size, and the first fluid channel network and the second fluid channel network are arranged in a mirror symmetry mode along the central symmetry axis of the chip body. According to the chip disclosed by the invention, methylation sensitive incision enzyme digestion treatment, reagent mixing and digital amplification detection can be integrated on a closed micro-fluidic platform, and'sample input-result output 'is realized for the first time aiming at DNA methylation quantitative detection.