728 results about "Endonuclease" patented technology

To provide a method for detecting the presence of multiple specific nucleic acid sequences or single nucleotide polymorphisms in a temperature-dependent fashion.SOLUTION: Provided is a method, in which an invasive oligonucleotide and a primary probe oligo (a 5' flap oligonucleotide) hybridize to a target strand nucleic acid to form an invasive cleavage structure cleavable by a FEN-1 endonuclease to release a cleaved 5' flap (primary cleaved flap); in a secondary reaction, the primary cleaved flap from the primary reaction hybridizes to a FRET cassette 1, a hairpin oligonucleotide labeled with a fluorescent dye and a quencher molecule, to form a second invasive cleavage structure that is cleavable by a FEN-1 endonuclease at a site between the fluorophore and a quencher; and cleavage of FRET cassette 1 separates the quencher from the fluorophore, such that fluorescence signal from the fluorophore can be detected.SELECTED DRAWING: Figure 1

The present invention provides a method and a kit for detecting a target nucleic acid to be detected through a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a restriction endonuclease and a DNA polymerase, generating a reporter primer that is complementary to and pairs with the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by easy mutation of the target nucleic acid sequence to be detected.

The present invention discloses a Gs12-7 endonuclease variant and a gene editing system mediated thereby. Specifically, by means of a rational mutation strategy, two Gs12-7 mutants were constructed and compared, and it was found that after the 157th amino acid of the Gs12-7 endonuclease was mutated from Glu to Arg, the activity of this gene editing enzyme could be significantly improved, and this variant was designated as Gs12-7MAX. The present invention provides an efficient gene editing technology mediated by the CRISPR-Gs12-7MAX system, which has broad application prospects in the field of genome site-directed modification.

The invention relates to antarctic krill peptide with umami and an antarctic krill peptide mixture as well as a preparation method and application thereof. Specifically, the amino acid sequence of the euphausia superba peptide is shown as any one of SEQ ID NO: 1-5. The euphausia superba peptide mixture with delicate flavor is obtained by carrying out enzyme digestion treatment on an euphausia superba peptide solution by using incision enzyme, excision enzyme and freshness enhancing enzyme and harvesting supernate. Molecular docking simulation shows that the euphausia superba peptide disclosed by the invention can be combined with a delicate flavor receptor T1R1 / T1R3. The Antarctic krill peptide and the Antarctic krill peptide mixture can be used for development of umami seasonings, and a new idea is provided for development of marine seasonings.

Methods and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

Some aspects of this disclosure provide compositions, methods, systems, and kits for controlling the activity of RNA-programmable endonucleases, such as Cas9, or for controlling the activity of proteins comprising a Cas9 variant fused to a functional effector domain, such as a nuclease, nickase, recombinase, deaminase, transcriptional activator, transcriptional repressor, or epigenetic modifying domain. For example, the inventive proteins provided comprise a ligand-dependent intein, the presence of which inhibits one or more activities of the protein (e.g., gRNA binding, enzymatic activity, target DNA binding). The binding of a ligand to the intein results in self-excision of the intein, restoring the activity of the protein.

This invention relates to recombinant nucleic constructs comprising a DNA binding domain, an endonuclease and a reverse transcriptase and methods of use thereof for modifying nucleic acids in plants.

The invention relates to an endonuclease S2 mutant, a preparation method of the endonuclease S2 mutant and application of the endonuclease S2 mutant in antibody glycosylation transformation. The glycosidase S2 mutant comprises a carbohydrate binding module (CBM) defined by M677-D843 in SEQ ID NO: 1, and the amino acid sequence of the glycosidase S2 mutant comprises SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

Provided herein are novel TnpB polypeptides having RNA guided endonuclease activity. Also provided herein is a gene editing system comprising the TnpB polypeptide of the invention or a disarmed variant thereof, or a fusion polypeptide comprising the same, and a method of gene editing using the TnpB polypeptide of the invention or a disarmed variant thereof, or the fusion polypeptide or the gene editing system.

The invention belongs to the technical field of fish genetic identification, discloses a multi-site large yellow croaker wild domestication population identification system based on an agarose gel electrophoresis technology, and particularly discloses a group of InDel molecular markers for large yellow croaker wild and domestication population identification, and the InDel molecular markers comprise InDel11-5053756, InDel20-14004876, InDel3-28345321 and InDel21-17327392. The InDel molecular marker detection primer is used for amplification to obtain a PCR product, agarose gel can be used for detecting strip polymorphism, extra restriction enzyme digestion or complicated polyacrylamide gel electrophoresis operation is not needed, and identification of large yellow croaker wild and domesticated populations can be rapidly and accurately achieved.

The invention discloses a Vinck Cellulomicrobe and application thereof in degradation of corn straw and improvement of soil fertility, and belongs to the field of agricultural microbial technology and solid waste resource utilization. The bacterial strain can secrete a plurality of hydrolase and oxidase including AA10 family lytic polysaccharide monooxygenase, AA2 peroxidase, GH3 and GH1 beta-glucosidase, GH16 / GH17 beta-glucosan endonuclease and AA1 multi-copper oxidase. The bacterial strain has the advantages that the bacterial strain can secrete a plurality of hydrolase and oxidase; fermentation liquor is applied to soil according to the straw mass ratio of 1: 5, and the corn straw degradation rate can be increased under the condition that the mass ratio of soil to straw is 100: 1. Meanwhile, the microbial agent can synergistically improve soil nutrients and increase the contents of rapidly available phosphorus, rapidly available potassium, alkali-hydrolyzable nitrogen, soluble organic carbon and total phosphorus in soil. According to the microbial agent, a straw lignocellulose structure is effectively cracked through a multi-enzyme system synergistic effect, and the dual effects of accelerating straw degradation and improving soil fertility are achieved.

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

Provided herein are modified Staphylococcus aureus Cas9 nucleases and methods of using modified Staphylococcus aureus Cas9 nucleases. Also provided herein are chimeric nucleases comprising an I-Tevl nuclease domain, and a Staphylococcus aureus Cas9 and methods of using the chimeric nuclease. Also provided herein are methods to edit genes by administering a chimeric nuclease to a cell or organism without the use of a viral vector.

The invention discloses a molecular marker linked with yellow peel of bitter gourd and application of the molecular marker. It is found through analysis that mutation of a base G at the 199th site of SEQ ID NO.1 into A is a reason for formation of bitter gourd yellow peel. A specific primer is designed aiming at the mutation site, a bitter gourd genome is amplified by utilizing the primer, a PCR amplification product is subjected to incision enzyme XhoI enzyme digestion, and the bitter gourd variety with yellow peel can be identified according to the size of an enzyme digestion product. The method can accurately identify the peel color in the early growth stage of the bitter gourd, improves the breeding efficiency of the yellow peel variety of the bitter gourd, and has the advantages of high accuracy, simplicity and convenience in operation and the like.

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 and compositions for modulating a target genome and stable integration of a transgene of interest into the genome of a cell are disclosed. Specifically, provided herein comprises compositions comprising an RNA molecule comprising a reverse complement sequence comprising an exogenous sequence and homology arms, an RNA molecule comprising an endonuclease, and one or more guide RNAs for specific genomic integration of the exogenous sequence.

The invention relates to pichia pastoris engineering bacteria and a preparation method thereof, and a method for producing cellooligosaccharide / disaccharide by using corn straws or wheat straws, and belongs to the technical field of genetic engineering. A cellulose incision enzyme gene and a cellulose excision enzyme gene are transferred into pichia pastoris engineering bacteria. The preparation method comprises the following steps: respectively inserting a cellulose incision enzyme gene and a cellulose excision enzyme gene into plasmids to construct expression vectors; transferring the expression vector into a competent cell of pichia pastoris GS115; and screening and identifying to obtain successfully transformed pichia pastoris engineering bacteria. According to the present invention, the pichia pastoris heterologous expression cellulose incision enzyme and cellulose excision enzyme are adopted to construct the pichia pastoris engineering strain capable of degrading the plant straw into the cellooligosaccharide / disaccharide, such that the reliable chassis strain is provided for the production of the starch from the cellulose;

The present disclosure provides one or more engineered gene effectors and systems, compositions, and methods of use thereof, wherein the one or more engineered gene effectors can be used to effect regulation of a target gene in a cell (e.g., an endogenous target gene in a cell). The one or more engineered gene effectors can be operatively coupled to a heterologous endonuclease, such as a CRISPR / Cas protein.

The invention provides characterization and application of novel programmable endonuclease Argonaute (Ago), and particularly provides a detection system for directly detecting target nucleic acid molecules, the system comprises: (a) guide DNA (gDNA) and / or guide RNA (gRNA); (b) a programmable endonuclease Argonaute (Ago), wherein the programmable endonuclease Argonaute (Ago) is a programmable endonuclease Argonaute (Ago) which is used for guiding the shearing of the target DNA and / or the target RNA by taking the DNA and / or the RNA as a guide; (c) a reporter nucleic acid; wherein the target nucleic acid molecule is a target DNA and / or a target RNA, the invention also accidentally finds that the nuclease MaAgo provided by the invention can be used for independently detecting the target DNA or the target RNA and also can be used for simultaneously detecting the target DNA and the target RNA, and has a shearing characteristic different from that of the shearing DNA when the RNA is sheared, so that a new enzyme resource is expected to be provided for nucleic acid detection and in-vivo editing.

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 technical field of biology, and discloses a wheat bionic peptide and a biosynthesis method and application thereof, and the method comprises the following steps: carrying out amino acid sequence analysis on a protein sequence of a target wheat peptide, and determining a target nucleotide sequence corresponding to a coded same protein sequence according to codon degeneracy; optimizing the target nucleotide sequence according to password preference of escherichia coli to obtain a wheat bionic peptide nucleotide sequence; treating and combining the obtained wheat bionic peptide nucleotide sequence and a vector plasmid through restriction enzyme to construct a recombinant plasmid; transferring the recombinant plasmid into host bacteria, performing fermentation culture to induce protein expression, separating protein, and purifying to obtain the wheat biomimetic peptide. The wheat biomimetic peptide is obtained in a biosynthesis mode, and compared with plant extraction and chemical synthesis, the cost is reduced, and meanwhile, the safety of the product is guaranteed.

The invention belongs to the technical field of biology, and particularly discloses a DNA molecular cloning method based on GgeAgo and application of the DNA molecular cloning method. The method comprises the following steps: shearing a vector plasmid by a cleavage system; mixing a vector plasmid cleavage product with an exogenous fragment with a homologous region, assembling by adopting a recombination-based cloning method, and transforming escherichia coli to generate a recombinant plasmid. The cutting system comprises a guide nucleic acid molecule and a programmable endonuclease GgeAgo or / and a mutant thereof, the GgeAgo cuts plasmids under the guide of the guide nucleic acid molecule to linearize the plasmids, a to-be-inserted exogenous fragment with a homologous region is added, and after escherichia coli is transformed, the exogenous fragment is efficiently and accurately inserted into the target plasmids. The molecular cloning method provided by the invention is high in efficiency, short in time and simple and convenient to operate, and mutation caused by PCR is effectively reduced. The molecular cloning method provided by the invention provides a new thought for an Ago-based molecular cloning technology and a DNA synthesis and assembly technology.

Disclosed herein are methods to edit genes, for example by administering a chimeric nuclease to a cell or organism without the use of a viral vector. Some methods herein include editing DNA of a cell by contacting the cell with a nuclease comprising a Cas9 sequence, where the nuclease edits the DNA of the cell.

The present disclosure provides methods for editing a genomic target in a soybean plant through the use of viral-mediated delivery of at least one genome editing reagent. Also provided are methods of editing a grafted scion by expression in the rootstock of a meristem transport segment- tagged endonuclease, and delivery of additional genome editing reagents to the plant or to the scion by virus-mediated delivery. Also provided are viral vector systems for use in the methods herein, and soybean plants, seeds, and cells edited using the provided methods.

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 provides a simplified genome sequencing library construction method and a kit and related application thereof. The construction method of the simplified genome sequencing library comprises the following steps: (1) respectively carrying out enzyme digestion on a plurality of DNA (Deoxyribonucleic Acid) samples by using restriction endonuclease; wherein restriction endonucleases adopted by all the samples are different from one another, and the tail ends of specific DNA fragments of the samples are generated to serve as sample bar codes of the samples; (2) mixing the enzyme digestion products of the samples, constructing a DNA library, carrying out fragment sorting on the DNA library, and taking the selected library as a simplified genome sequencing library; or carrying out fragment sorting on the enzyme digestion product of each sample, and carrying out DNA library construction by using the selected fragments to obtain the simplified genome sequencing library. According to the method, the experimental operation process is simplified, the library building efficiency is improved, and meanwhile, the feasibility of the restriction endonuclease for recognizing 6 basic groups based on a third-generation long-read-long sequencing platform in the simplified genome sequencing technology is also explored.

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 present invention relates to a rapid detection of microbial-associated nuclease activity with chemically modified nuclease (e.g., endonuclease) substrates, and probes and compositions useful in detection assays.

The invention discloses a method for simultaneously determining gene point mutation and DNA methylation based on a CRISPR / Cas12a system. The method is characterized by comprising four steps of enzyme digestion, recognition, conversion and detection, wherein in the enzyme digestion step, methylation-sensitive restriction endonuclease (MSRE) is selected according to a target site sequence, unmethylated DNA is not hydrolyzed by MSRE and is kept complete, methylated DNA is hydrolyzed and broken under the action of MSRE, and the broken 3'end can be used as a primer P1 to trigger SDA1 amplification; wherein in the recognition step, a Flap primer and an Invading primer are designed according to a point mutation sequence, and an enzyme digestion product generated under the action of FEN1 enzyme can be used as a primer P2 to trigger SDA2 amplification; wherein in the conversion step, two different SDA amplification templates are designed according to sequences of P1 and P2, and amplification of SDA1 and SDA2 is triggered by enzyme digestion products P1 and P2 respectively, so that DNA methylation and gene mutation information are quickly converted into two different amplification products T1 and T2; wherein in the detection step, crRNA1 and crRNA2 are respectively designed as guide chains of a CRISPR / Cas12a reaction system according to sequences of T1 and T2, and synchronous detection of gene point mutation and DNA methylation is realized through'strong 'and'weak' of fluorescence signals. Based on a CRISPR / Cas12a system, the method solves the problem that a traditional detection method cannot realize simultaneous detection of gene point mutation and DNA methylation, can be used for scientific research analysis and clinical detection, and has a wide market prospect.