86 results about "Dna cleavage" patented technology

A composition and method of cleaving a target DNA and isolating a DNA sequence of interest, directed by a targeting oligonucleotide (“ON”) including a DNA binding agent (stable or unstable), is disclosed. The targeting ON binds to the target DNA before or during DNA cleavage. After cleavage, the isolation of the DNA fragment of interest is facilitated by the affinity tag on the targeting ON or an affinity tag attached using either ligation or polymerase extension method.

Methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell are disclosed. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof. Further disclosed are chimeric restriction endonucleases.

The present invention pertains to engineered T-cells, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered T-cells of the invention are characterized in that the expression of beta 2-microglobulin (B2M) and / or class II major histocompatibility complex transactivator (CIITA) is inhibited, e.g., by using rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding B2M and / or CIITA, or by using nucleic acid molecules which inhibit the expression of B2M and / or CIITA. In order to further render the T-cell non-alloreactive, at least one gene encoding a component of the T-cell receptor is inactivated, e.g., by using a rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding said TCR component. In addition, expression of immunosuppressive polypeptide can be performed on those modified T-cells in order to prolong the survival of these modified T cells in host organism. Such modified T-cell is particularly suitable for allogeneic transplantations, especially because it reduces both the risk of rejection by the host's immune system and the risk of developing graft versus host disease. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer, infections and auto-immune diseases.

The present invention pertains to engineered T-cells, method for their preparation and their use as medicament, particularly for immunotherapy. The engineered T-cells of the invention are characterized in that the expression of beta 2-microglobulin (B2M) and / or class I I major histocompatibility complex transactivator (CIITA) is inhibited, e.g., by using rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding B2M and / or CIITA, or by using nucleic acid molecules which inhibit the expression of B2M and / or CIITA. In order to further render the T-cell non-alloreactive, at least one gene encoding a component of the T-cell receptor is inactivated, e.g., by using a rare-cutting endonucleases able to selectively inactivating by DNA cleavage the gene encoding said TCR component. In addition, expression of immunosuppressive polypeptide can be performed on those modified T-cells in order to prolong the survival of these modified T cells in host organism. Such modified T-cell is particularly suitable for allogeneic transplantations, especially because it reduces both the risk of rejection by the host's immune system and the risk of developing graft versus host disease. The invention opens the way to standard and affordable adoptive immunotherapy strategies using T-Cells for treating cancer, infections and auto-immune diseases.

An engineered highly specific DNA-cleavage enzyme delivers a site-specific nick in a double stranded DNA, to cleave one DNA strand within its target site while leaving the opposing DNA strand intact. The engineered enzyme provides the ability to induce a gene conversion event in a mammalian cell. An engineered sequence-specific nickase derived from a LAGLIDADG homing endonuclease is altered by a single amino acid residue, wherein the amino acid residue is involved in the polarization of solvent molecules and acid-base catalysis in the active site without affecting direct contacts between the enzyme and either the bound DNA or bound metal ions. Engineered, site-specific nickase variants, such as of I-AniI and other homing endonucleases, are particularly useful in targeted genome engineering as well as therapeutic, targeted gene repair.

The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease / DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9 / sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

Process for the hydrolytic ring cleavage of thiazolidine derivatives to give 2-aminomercaptan derivatives and carbonyl compounds, which comprisesbringing an aqueous solution of the thiazolidine derivative into contact with an acidic cation exchanger in the H+ form, giving a solution L1 which contains the carbonyl compound andeluting the cation exchanger with a suitable eluent, giving a solution L2 which contains the 2-aminomercaptan derivative.

The invention discloses application of novel ScCas12a protein in nucleic acid detection. It is found through research that the novel ScCas12a protein with DNA cleavage activity can be used for nucleicacid detection, gene editing and gene modification as a novel CRISPR / ScCas12a system and provide new necessary tool selection for Cas12a-based gene editing, modification and molecular detection. Theinvention also provides a novel nucleic acid detection system comprising the ScCas12a protein and gRNA and a kit. The molecular detection with high sensitivity and high precision can be achieved at room temperature of 25-37 DEG C, the detection specificity is good, the sensitivity is high, the cost is low, the operation is convenient and fast, the application range is wide, and the application prospect in nucleic acid detection is good.

A method of detecting an enzyme-mediated DNA cleavage reaction in a fluorometric assay is provided. The method can be used to detect DNA cleavage caused by restriction endonucleases, retroviral integrase enzymes, DNases, RNases, or enzymes utilized in other strand separating processes in molecular biology.

The invention discloses a high-extraction-purity, good-integrity and high-yield extraction method of apostichopus japonicus body-wall total RNA (Ribonucleic Acid). The method comprises the following steps of: quickly freezing apostichopus japonicus body-wall tissue in liquid nitrogen; grinding and putting the frozen tissue in lysate to homogenate, centrifugate and take supernatant fluid; adding chloroform to the supernatant fluid and centrifugating to take the supernatant fluid to another centrifuge tube; then, adding a high-salt solution and isopropyl alcohol and filtering precipitation with ethanol; dissolving the precipitation with DEPC (diethypyrocarbonate) treated water to have constant volume; sequentially adding a DNA enzyme buffer solution without RNA enzymes, DNA enzymes without RNA enzymes and an RNA enzyme inhibitor to the dissolved solution to mix; carrying out a water bath at 37 DEG C to obtain DNA lysate; adding phenol and chloroform in a ratio of 5:1 to the DNA lysate to mix, centrifugate and take supernatant fluid; adding a glycogen solution, a potassium acetate solution and pre-cooling ethanol to the supernatant fluid; mixing and staying over night; centrifugating to discard the supernatant fluid; washing and dying the precipitation with ethanol; dissolving the solution with the DEPC treated water to have constant volume of 20 mu l; and storing at 80 DEG C below zero.

The invention provides a peculiar PCR detection kit of Cryptosporidium and micro Cryptosporidium, comprising a DNA lysis solution, a PCR reaction solution, a peculiar primer of the Cryptosporidium and the micro Cryptosporidium and positive control genomic DNA of the micro Cryptosporidium. According to the diagnosing sequence for analyzing Cryptosporidium and species peculiarities, a Cryptosporidium and species peculiar primer are designed by utilizing a peculiar detection method of PCR, the sensibility of the primer is improved by optimizing and amplifying conditions, the amplifying result can be directly observed by electrophoretic analysis. The kit of the invention has strict design, simple and easy operation, high sensibility and strong peculiarity, can simultaneously identify the Cryptosporidium infection of Cryptosporidium and species peculiarities so as to obtain an accurate and objective judgment result.

Compositions and methods are provided that relate to a modified DNA cleaving enzyme having at least 35% amino acid sequence identity with T7 Endo I. The modifed enzyme includes two catalytic centers separated by a beta-bridge where the beta-bridge contains at least one mutation having an effect of altering enzyme cleavage activity compared to the unmodified enzyme. Activities associated with the modified DNA cleaving enzyme that can be modulated in different reaction conditions include at least one of: (a) non-sequence specific nicking activity; (b) cleaving the second strand of a duplex DNA at a preexisting nick site to produce a linear duplex with a single strand overhang; (c) non-sequence specific DNA cleavage; (d) cleaving DNA flanking a mismatch; and (e) cleavage at a cruciform structure in a DNA duplex.

This invention discloses a kind of measuring PRVHS fluorescence quantitative PCR reagent box and application. The reagent box has a) DNA cracking liquid, b)Taq DNA polymerization enzyme, c) pG-HB-contained nucleoside series of standard positive template, d)the fluorescence quantitative reaction liquid containing positive and negative primer and fluorescence probe series. Advantages: simple and easy for operation, fast and accurate quantitation and so on.

The present invention relates to an Argonaute protein mutant which lacks DNA cleavage activity, but has DNA binding activity. The mutation of the mutant is located in a PIWI domain. The invention alsorelates to application of the protein mutant particularly in the enrichment of target DNA and in the construction of a sequencing library. Therefore, the present invention also relates to a method for enriching the target DNA. The method comprises the steps of: (a) designing a leader sequence for a specific sequence in the target DNA; (b) combining the mutant, the leader sequence and the target DNA according to the present invention to obtain a mutant-leader sequence-target DNA ternary complex; (c) capturing the mutant-leader sequence-target DNA ternary complex through a capture medium; and (d) isolating the target DNA from the captured mutant-leader sequence-target DNA ternary complex to obtain enriched target DNA.

The invention relates to the technical field of biological medicine, and particularly relates to novel bleomycin derivatives prepared from streptomycete flavoviridis SB9026 (having a preservation number of CCTCCM 2011292) and the anti-tumour activity thereof. The bleomycin derivatives disclosed by the invention have a chemical structure general formula shown in a formula (I), wherein R1 is selected from H, alkyl, amino, alkyl, acyl, sulfonyl, halogenated alkyl, heter-alkyl, hydroxyl alkyl, amino-alkyl or -(CH2)n-C(NH2)=NH, wherein n is equal to 1-6; and R2 is selected from H or -OH. Simultaneously, according to the invention, a preliminary test is performed on the biological activities of two specific bleomycin derivatives, and the preliminary test found that the DNA (deoxyribonucleic acid) cracking activity of the derivative compound BLM S (compound 1) is equivalent to the activity of the known bleomycin A2, and the DNA cracking activity of the derivative compound 6'-dehydroxylated-BLM S (compound 2) is at least 10 times as high as the activity of the bleomycin A2.

The presently claimed invention offers programmable and precise regulation of Cas9 functions by utilizing a set of compact Cas9 derivatives created by deleting conserved HNH and / or REC-C domains based on the structural information across variant class 2 CRISPR effectors. In addition, a novel strategy for engineering the dimeric gRNA-guided nuclease by splitting the mini-dSaCas9 and fusing the FokI domain right after the split point is claimed to increase the on-target DNA cleavage efficiency and potentially reduce the off-target effect because of a closer proximity of dimeric FokI nuclease to the target sequence. By combining the optimized and compact gRNA expression cassette and the downsized SaCas9 derivatives, the entire CRISPR / Cas system with different effector domains for transactivation, DNA cleavage and base editing is loaded into a single AAV virus. Such an all-in-one AAV-CRISPR / Cas9 system will be particularly appealing in biomedical applications that require safe and efficient delivery in vivo.

This invention discloses a fluorescent PCR test kit for quantitatively detecting Chlamydia trachomatis, which is composed of DNA lysis solution, fluorescent quantitative PCR reaction solution, positive standard template and negative standard sample. The fluorescent quantitative PCR reaction solution comprises forward and reverse primers and fluorescent probes. The test kit has such advantages as high specificity, high sensitivity, high accuracy, simple operation and good repeatability.

Methods of modifying, repairing, attenuating and inactivating a gene or other chromosomal DNA in a cell are disclosed. Also disclosed are methods of treating or prophylaxis of a genetic disease in an individual in need thereof. Further disclosed are chimeric restriction endonucleases.

The invention discloses an application of an electrochemiluminescence biosensor based on cytosine (C)-containing cyclic DNA sequences where in-situ reduced Ag nano-clusters are enriched as signal probes and adopting dual amplification strategy to detection of target thrombin. According to the technical scheme, DNA cleavage enzyme with hairpin DNA recognition capability and catalysis effect is designed, when target thrombin exists, hairpin DNA is opened, and substrate DNA is cleaved under the action of Zn<2+>; a large quantity of cytosine (C)-containing cyclic DNA sequences are aggregated through HCR (hybridization chain reaction), in-situ reduction of AgNO3 is performed on the electrode surface by NaBH4, a large quantity of Ag nano-clusters are formed, and the electrochemiluminescence biosensor with dual amplification effect is prepared. The sensor is subjected to luminescence detection, and a linear relation is formed between light-emitting signals and concentration of a to-be-testedsample. The Ag nano-cluster signal probes and the DNA dual amplification technology are combined for rapid and high-sensitivity detection of thrombin, and the electrochemiluminescence biosensor has great application potential in early clinical analysis and detection.

The invention provides a constant temperature amplification detection kit and a method for detecting a food allergen crustacean gene. The kit comprises DNA lysis solution and constant temperature amplification reaction liquid, wherein the constant temperature amplification reaction liquid comprises a forward peripheral primer, a backward peripheral primer, two probes, a cross-species amplification primer, 1*Thermol buffer, MgSO4, dNTPs, Bst DNA polymerase and sterile double-distilled water. The invention also provides the method for detecting the food allergen crustacean gene by using the kit. The method comprises the steps of extracting DNA from a sample to be detected with the DNA lysis solution, performing amplification reaction and detecting. The method for detecting the food allergen crustacean gene has the advantages of accuracy, flexibility and capability of rapidly detecting the food allergen crustacean gene and is suitable for field rapid detection.

The invention discloses a fluorescent quantitative PCR (polymerase chain reaction) kit for quickly detecting gonococcus, relates to a technique for detecting a pathogen gene causing various human diseases, and is applicable to qualitative / quantitative detection of gonococcus. The kit comprises a DNA (deoxyribonucleic acid) lysate, a fluorescent quantitative PCR reaction solution, a standard positive template PMD (polarization mode dispersion) 18-T-NG and a negative quality control standard substance, wherein the fluorescent quantitative PCR reaction solution contains a gonococcus specific primer pair and a fluorescent probe. The kit disclosed by the invention has the advantages of accurate quantification, high detection speed, high specificity, high sensitivity, simple operating steps and high repeatability. The kit can be used for quickly detecting gonococcus qualitatively / quantitatively and can be substituted for the traditional culturing method which is still in use until today.

The invention provides a kit for the specific PCR (polymerase chain reaction) detection of pneumocystis, which comprises DNA lysate, a PCR reaction solution, positive control and negative control. The invention provides a detection method of the kit for the specific PCR detection of pneumocystis, which comprises the steps of sample pretreatment, sample DNA extraction, PCR amplification and PCR product observation. The kit for the PCR detection, disclosed by the invention has rigorous design; meanwhile, the detection method has the advantages of simpleness and rapidness in operation, high sensitivity, strong specificity and accuracy and objectivity in result determination.

The invention relates to the technical field of biology, and discloses a LAMP visual rapid detection kit of silkworm pathogenic micro spore worms and a detection method of the LAMP visual rapid detection kit of the silkworm pathogenic micro spore worms. The kit comprises four LAMP primers, deoxyribonucleic acid (DNA) lysate liquid, LAMP reaction liquid, positive reference products, negative reference products and color development liquid to form a detection reaction system. Under the constant temperature of 60 DEG C to 65 DEG C, formwork DNA is rapidly amplified, developed dye is added to the formwork DNA, and identification results are observed through naked eyes under natural light. If the color of reaction products changes into green, the fact that samples to be tested contain the silkworm pathogenic micro spore worms is confirmed, and if the color of reaction products changes into orange, the fact that the samples to be tested do not contain the silkworm pathogenic micro spore worms is confirmed. The kit can detect current various silkworm pathogenic micro spore worms fast and flexibly, is easy to operate, and low in cost, and needs no special or complex instruments, and reaction results are easy to judge, and specificity is strong. The kit can meet the urgent needs of disease surveillance, on-site emergency situation and the detection of production samples, and can be widely popularized and applied easily.

The invention provides supramolecular metal complexes as DNA cleaving agents. In the complexes, charge is transferred from one light absorbing metal (e.g. Ru or Os) to an electron accepting metal (e.g. Rh) via a bridging π-acceptor ligand. A bioactive metal-to-metal charge transfer state capable of cleaving DNA is thus generated. The complexes function when irradiated with low energy visible light with or without molecular oxygen.

The invention discloses a PCR detection reagent box of animal toxoplasmosis; the box contains DNA lysate, red blood cell lysate, white blood cell lysate, PCR reaction liquid and toxoplasmosis gene DNA positive control liquid; the 529bp repeated DNA segment of toxoplasmin is taken as genetic mark and special primer is taken to optimize the PCR reaction conditions such as the magnesium ion consistency, the anneal temperature and the regulation of the cycle number. The invention can detect the toxoplasmosis infection of pig, dog and cat in a fast special sensible way; the operation is convenient and the sensibility is high; the invention can be used for diagnosing the toxoplasmosis of pig, dog and cat and detecting and testing the drug effect appraisal of the selection of the toxoplasmosis medicines.

The problem to be solved in the present invention is to provide a simplified and efficiently improved DNA recombination method.The above problem can be solved with the present method for preparing a recombinant DNA by inserting a DNA fragment of interest into a vector DNA, the method comprising the step of carrying out the following reactions at the same reacting location at the substantially simultaneouse time: (a) a reaction for simultaneously cleaving a site of the vector for inserting the fragment and a DNA containing the fragment in the presence of a restriction enzyme whose DNA recognition site and DNA cleavage site are discrete; and (b) a reaction for inserting the fragment into the vector in the presence of a DNA ligase.

The invention discloses a method and kit for extracting sperm cell DNA from a sexual assault case check sample by differential splitting decomposition. The kit comprises a box body, a DNA extraction bin and a DNA amplification bin are arranged in the box body, and a sexual assault check sample splitting decomposition tube for extracting target DNA, a first storage tube for storing a mild splittingdecomposition solution and a second storage tube for storing protease K, a third storage tube used for a sperm cell splitting decomposition and a fourth storage tube for storing reducing agent are placed in the DNA extraction bin. According to the method and kit for extracting sperm cell DNA from the sexual assault case check sample by differential splitting decomposition, the reagents needed forDNA splitting decomposition extraction and amplification are packed in the kit, the DNA extraction and amplification is convenient, a legal medical expert can conveniently indentify the DNA, and theidentification efficiency is improved; and a mild splitting decomposition environment is provided for the extraction of sperm cell DNA, the residual epithelial cell DNA in the sperm cell precipitationsolution is effectively removed, the pure sperm cell DNA is obtained, and the identification accuracy is improved.

This invention discloses a fluorescent PCR test kit for quantitatively detecting Ureaplasma parvum, which is composed of DNA lysis solution, fluorescent quantitative PCR reaction solution, positive standard template pU-UP and negative standard sample. The fluorescent quantitative PCR reaction solution comprises a pair of specific primers of Ureaplasma parvum and fluorescent probes. The test kit has such advantages as high specificity, high sensitivity, high accuracy, simple operation and good repeatability. The test kit can be used for detecting four serotypes of Ureaplasma parvum quantitatively and qualitatively, and can differentiate Ureaplasma parvum and Ureaplasma urealyticum.

The invention relates to a biosensor, composition and kit comprising an allosteric transcription factor and a CRISPR / Cas12a system, and related methods and application thereof. The mechanism of the biosensor is that a small molecule detection tool mediated by the allosteric transcription factor and the CRISPR / Cas12a is adopted; through combination of a target small molecule and the allosteric transcription factor, affinity between the allosteric transcription factor and double-stranded DNA is changed; and cis-cleavage activity and / or trans-single-stranded DNA cleavage activity of CRISPR / Cas12aare / is adopted for cutting a dsDNA probe and / or a ssDNA probe so as to convert a target small molecule signal into a corresponding optical signal. With the biosensor, the composition, the kit and therelated methods in the invention, time and cost can be saved when small molecules are detected in vitro, sensitivity is high, and the potential of high-throughput detection is achieved.