89 results about "Targeted Mutation" patented technology

Methods are provided for diagnosis and prognosis of disease by analyzing expression of a set of genes obtained from single cell analysis. Classification allows optimization of treatment, and determination of whether on whether to proceed with a specific therapy, and how to optimize dose, choice of treatment, and the like. Single cell analysis also provides for the identification and development of therapies which target mutations and/or pathways in disease-state cells.

The invention relates to chimeric endonucleases, comprising an endonuclease and a heterologous DNA binding domain, as well as methods of targeted integration, targeted deletion or targeted mutation of polynucleotides using chimeric endonucleases.

The invention discloses a molecular genetic improvement method for lowering rice seed holding by targeted modification on rice seed holding gene qSH1 by using a CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. The method comprises the following steps: 1) selecting an appropriate target; 2) establishing a vector containing the target spot sequence; 3) establishing a recombinant vector containing the target spot sequence by utilizing the vector; 4) introducing the recombinant vector into receptor rice to obtain a transgenic positive plant; 5) obtaining a targeted-mutation mutant plant by utilizing the transgenic positive plant; 6) carrying out additive-generation growth on the mutant plant to obtain a transgenic-component-free homozygous mutant plant; and 7) carrying out seed holding investigation by using the homozygous mutant plant to obtain the plant with obviously lower seed holding as the seed-holding-improved plant. The method has the advantages of high directionality, small genetic background changes and the like, can avoid the risks caused by genetic transformation, and can culture the new species and new combination of transgenic-component-free rice with obviously lower seed holding.

The invention relates to chimeric endonucleases, comprising a endonuclease and a heterologous DNA binding domain comprising one or more Zn₂C₆ zinc fmgers, as well as methods of targeted integration, targeted deletion or targeted mutation of polynucleotides using chimeric endonucleases.

Provided are optimized endonucleases, as well as methods of targeted integration, targeted deletion or targeted mutation of [polynucleotides using optimized endonucleases.

The invention discloses an FFPE reference product for gene detection and a preparation method and application of the FFPE reference product. The preparation method comprises the following steps that S1, cell culture is performed on a tumor cell line, and cell pellets are collected; S2, formalin fixation is performed on the cell pellets, then sepharose gel wraps the cell pellets to form cell aggregates, and the cell aggregate are prepared into cell paraffin blocks; S3, genomic DNA of the tumor cell line in the cell paraffin blocks is extracted, and genetic mutation frequency determination is performed on the genomic DNA of the tumor cell line; S4, the genomic DNA of the tumor cell line containing a target mutation site is mixed into a DNA mixture of a target mutation frequency, and the DNAmixture is the FFPE reference product for gene detection. According to the technical scheme, formalin fixation and paraffin wrapping are performed on the cells cultured by tumor cell line, so that thesituation of clinical samples can be better simulated.

The invention belongs to the field of biotechnology, and particularly relates to a qRT-PCR method for identifying Indian variant of novel coronavirus. According to the method, in order to improve the detection sensitivity and specificity, a TaqMan probe is introduced; in order to reduce the cost, two pairs of primers are changed into one pair and a half of primers, that is, two upstream primers respectively target a mutation site and an original non-mutation site, and one downstream primer is shared by the two upstream primers. in order to improve the sensitivity of mutation detection, mutation is introduced to the third site in the 5' direction of the mutation site of the upstream mutation primer, and by reducing the matching degree of the mutation primer and non-mutated virus nucleic acid and the matching degree of the non-mutated primer and the mutation virus nucleic acid in the reaction system, the amplification curve of the mutation virus nucleic acid amplified by mutation primer in the reaction system is earlier than the amplification curve of the non-mutation nucleic acid, and in the same way, the amplification curve of the non-mutation nucleic acid amplified by the non-mutation primer is earlier than the amplification curve of the mutated nucleic acid.

Compounds and compositions are presented that inhibit K-Ras, and especially mutant K-Ras. Certain compounds preferentially or even selectively inhibit specific forms of mutant K-Ras, and particularly the G12D mutant form.

The invention relates to a gene site-directed mutation method based on seamless cloning, a kit, and applications of the gene site-directed mutation method based on seamless cloning. The gene site-directed mutation method based on seamless cloning comprises following steps: 5' terminal forward and reverse mutation primers containing complementary homologous arm sequences are designed based on the sequences of mutation sites, wherein the mutation sites may be at any places of homologous arms; 5' terminal forward and reverse mutation primers containing another complementary homologous arm are designed respectively at plasmid non-mutation site zones, PCR amplification is adopted to obtain two segments, wherein one end of each segment contains mutation sites, and more than 500bp difference is formed; a mutation primer with the same pair number of that of sites is designed based on mutation site number; homologous recombinase is adopted for one-step seamless splicing of 2 to 5 segments, andconstruction of an annular plasmide with target mutation sites is realized. Compared with classic overlap extension PCR, the gene site-directed mutation method possesses following advantages: the genesite-directed mutation method is rapid and simple, can be used for single or multiple site, continuous or discontinuous mutation of any sites of a plasmid smaller than 40kb, and mutation efficiency is 100%.

The invention discloses a transgenic vector for target mutation of primordial germ cells, a method for preparing the transgenic vector and application thereof. The transgenic vector, the method and the application have the advantages that the vector pTol2 (UAS: Cas9) is introduced into zebrafish embryos at unicellular stages to obtain strains Tg (UAS: Cas9) and strains Tg (kop: KalTA4), and Cas9 genes can be efficiently, specifically and continuously expressed in the primordial germ cells of zebrafishes by hybrid embryos obtained after fishes of the strains Tg (UAS: Cas9) and female fishes of the strains Tg (kop: KalTA4) are hybridized; the hybrid embryos of the male fishes of the strains Tg (UAS: Cas9) and the female fishes of the strains (kop: KalTA4) are used as receptor embryos when mutation strains are about to be prepared, somatic mutation can be effectively reduced after gRNA [guide RNA (ribonucleic acid)] of target genes is directly injected, accordingly, the survival rate of the injected P0-generation embryos can be increased, P0-generation adult fishes can effectively pass on mutation, and accordingly the transgenic vector and the method have broad application prospects.

The invention is suitable for the technical field of computer application, and provides a program testing method and device. The program testing method comprises the steps: obtaining a to-be-tested program and program information thereof; constructing a generation mode of a test case corresponding to the to-be-tested program according to the program information, and generating the test case according to the generation mode; performing variation processing on the test cases according to program information to obtain a plurality of variation cases; selecting a target variation case for testing the to-be-tested program from all variation cases; and testing the to-be-tested program according to the target variation case to obtain a test result. A test case corresponding to a to-be-tested program is generated according to program information of the to-be-tested program; the test cases are mutated to obtain the multiple mutation cases, and the mutation case with the highest case coverage rate is determined from the multiple mutation cases to serve as the target mutation case to test the to-be-tested program; and the code coverage rate when the to-be-tested program is tested is effectively increased, and the comprehensiveness of the test process and the test result is guaranteed.

The invention discloses an application of ascorbic acid oxidase RIP5 to regulating and controlling of drought resistance of rice. An RIP5 gene knockout mutant is constructed by targeted mutation of anRIP5 gene through a GRISPR/Cas9 system, and the inventor finds that the drought resistance of plants can be effectively improved by inhibiting the expression quantity and/or activity of the RIP5 protein, that is, the ascorbic acid oxidase gene RIP5 of rice can negatively regulate and control the drought resistance of the plants. The invention also provides a gRNA target sequence for editing the ascorbic acid oxidase gene RIP5 of the rice, and the gRNA target sequence cooperates with a CRISPR/Cas9 gene editing system to realize targeted mutation of the coding gene of the RIP5 protein, so thatthe drought resistance of the rice can be regulated and controlled. The research of the invention provides new gene targets and resources for drought resistance genetic breeding of the plants, and knockout mutant plants obtained by editing the gene have important application value.

The invention discloses a molecular breeding method for thickening intermuscular thorns of erythroculter ilishaeformis and megalobrama amblycephala, and belongs to the technical field of aquatic organism breeding. According to the molecular breeding method, based on a technical means of gene editing, the F0 generation of targeted mutation erythroculter ilishaeformis and megalobrama amblycephala mstn genes is obtained, and mutant individuals with mstn deletion and increased intermuscular bony area are obtained through passage. According to the invention, a parent method for obtaining erythroculter ilishaeformis and megalobrama amblycephala with increased intermuscular stabbing area by utilizing a gene editing technology is provided for the first time. The method is beneficial to large-scalecultivation of wild erythroculter ilishaeformis and megalobrama amblycephala which have thick intermuscular thorns and can be inherited in production. Being different from a transgenic method, the method can be applied to artificial breeding, overcomes the difficulty that intermuscular thorns are small and difficult to process in production, does not need to worry about the influence of transgenic food on people, is convenient for people to find the intermuscular thorns more easily when people eat erythroculter ilishaeformis and megalobrama amblycephala, and is easy to popularize in production.

The invention belongs to the technical field of aquatic organism breeding, and discloses a molecular breeding method for thickening intermuscular bones of silver carp and bighead carp. The molecular breeding method provided by the invention is based on a technical means of gene editing, an F0 generation of targeted mutation silver carp and bighead carp mstn (myostatin) genes is obtained, and mutant individuals with deleted mstn and increased intermuscular ossification areas are obtained through passage. In the invention, a parent method for acquiring silver carp and bighead carp with increasedintermuscular ossification areas by utilizing a gene editing technology is put forward for the first time. According to the method, wild silver carp and bighead carp which have thick intermuscular bones and can be inherited can be cultivated on a large scale in production; different from a transgenic method, the method can be applied to artificial breeding, the difficulty that intermuscular bonesare fine and small and difficult to process in production is overcome, people do not need to worry about the influence of transgenic food on people, people can find intermuscular bones more easily when eating silver carp and bighead carp, and the method is easy to popularize in production.

The invention provides a method for carrying out directional evolution on a gene promoter. The method comprises the following steps: amplifying the promoter by adopting an error-prone PCR technology, thus obtaining a group of promoter sequences with high mutation frequency; removing harmful mutation by adopting a DNA shuffling technology, and collecting beneficial mutation, thus obtaining the promoter after shuffling; forming an expression cassette by adopting the recombinant promoter and a galactosidase gene, transforming host cells by adopting the expression cassette, and carrying out blue and white spot screening and enzyme activity determination, thus obtaining the target mutation promoter. With the method provided by the invention, the functional region of the gene promoter does not need to be analyzed, the cost is low, the operation is efficient, rapid, easy and convenient, the success rate is high, and the method is suitable for carrying out directional evolution on gene promoters of escherichia coli or other bacteria, fungi and mammalian cells.

The invention discloses a molecular breeding method for thickening intermuscular bones of grass carps and black carps, and belongs to the technical field of aquatic organism breeding. The molecular breeding method provided by the invention is based on a technical means of gene editing, obtains an F0 generation of grass carp and black carp mstn genes subjected to targeted mutation, and obtains mutant individuals with mstn deletion and increased intermuscular bony area through passage. In the invention, a parent method for obtaining grass carp and black carp with increased intermuscular boning area by utilizing a gene editing technology is provided for the first time. According to the method, wild grass carps and black carps which have thick inherited intermuscular bones can be cultivated ona large scale in production, different from a transgenic method, the method can be applied to artificial breeding, overcomes the difficulty that intermuscular thorns are small and difficult to process in production, does not need to worry about the influence of transgenic food on people, is convenient for people to find the intermuscular thorns more easily when eating grass carps and black carps,and is easy to promote in production.

Methods are provided for diagnosis and prognosis of disease by analyzing expression of a set of genes obtained from single cell analysis. Classification allows optimization of treatment, and determination of whether on whether to proceed with a specific therapy, and how to optimize dose, choice of treatment, and the like. Single cell analysis also provides for the identification and development of therapies which target mutations and/or pathways in disease-state cells.

An interleukin-2 (IL-2) mutation gene with the base sequence shown by SEQ ID No.1 is prepared from natural IL-2 gene through overlapped PCR for targeted mutation to the base on the gene. Its advantages are less mutant site, high protein expression level, and no change to amino acid sequence of natural protein.

Ultra-sensitive assays for the detection of mutations, e.g., from blood-based sources of tumor genetic material (circulating tumor cells or plasma), or other settings in which limiting amounts of DNA,e.g., tumor DNA, is available. The assay is exemplified in the estrogen receptor, but is broadly customizable to target mutations in other genes.

The invention discloses a probe pool for detecting NTRK-1-2-3 fusion gene variation based on an NGS method and a kit thereof. The probe pool is selected from at least one of probes with nucleic acid sequences as shown in SEQ ID NO: 1-74, and the kit is suitable for detecting the NTRK-1-2-3 fusion gene variation of FFPE and fresh tissues based on an NGS method. The capture probe is designed according to the highly related region of the NTRK-1-2-3 fusion gene variation, the coverage rate is high and uniform, and the specificity of fusion detection is improved. The library conversion rate is high, the RNA usage amount is low, and the sequencing cost is low. According to the present invention, the hot spot targeting mutant gene is covered, the probe capture specificity is high, and in addition, one product can meet the detection requirements of a variety of pan-cancer NTRK-1-2-3 fusion genes, can solve the clinical sample preparation problem, and can reduce the complex problem of repeatedwork of experimental workers.

Transgenic non-human animals and methods of producing non-human transgenic animals comprising within their genome a conditional targeted mutation in a VEGFR, e.g., VEGFR1 or VEGFR2, gene are provided along with methods and vectors used to produce such animals or cells.

The invention provides sgRNA of a targeted Tmc1 mutant (c.1235T > A; p.M412K), an expression vector, a CRISPRs-CasRx system, a kit and application of the sgRNA, and belongs to the technical field of genetic engineering. The sequence of sgRNA of the targeted Tmc1 mutant is shown as SEQIDNO.3; and CasRx containing the sgRNA can enable a Tmc1Bth transcript to be reduced by 82% under the condition that wild Tmc1 + is not interfered, CasRx based on an AAV-PHP.eB carrier is injected into inner ears of newborn Beethoven mice, it is found that Tmc1Bth is reduced by 70% within two weeks, and miss-target is not detected in a whole transcriptome. The sgRNA targeted Tmc1 mutant provided by the invention is high in specificity, and can improve the survival rate of Tmc1Bth mutant mouse hair cells, so that the form of cilia bundles is recovered, the mechanical transduction current is reduced, and progressive hearing loss is remarkably improved.