41 results about "Dna breakage" patented technology

Factors affecting the fragmentation pattern of cell-free DNA (e.g., plasma DNA) and the applications, including those in molecular diagnostics, of the analysis of cell-free DNA fragmentation patterns are described. Various applications can use a property of a fragmentation pattern to determine a proportional contribution of a particular tissue type, to determine a genotype of a particular tissue type (e.g., fetal tissue in a maternal sample or tumor tissue in a sample from a cancer patient), and / or to identify preferred ending positions for a particular tissue type, which may then be used to determine a proportional contribution of a particular tissue type.

The invention discloses a traceless modification method of bacillus subtilis genome. The method disclosed by the invention is used for carrying out traceless modification on the bacillus subtilis genome by utilizing an upp positive-negative selection system. The method disclosed by the invention utilizes a ComK expression system, so that the bacillus subtilis has an excellent dsDNA (double-stranded deoxyribonucleic acid) conversion efficiency which can be up to 3-5*10<3>cfu / microgram dsDNA. Meanwhile, an exogenous endonuclease I-SceI expression system is utilized to bring double-stranded DNA breakage into the genome, so that the genetic recombination efficiency in negative selection molecules is obviously improved and can be up to 8*10<-4>. According to the method disclosed by the invention, iterative modification can be performed on a plurality of target nucleotide sequences in the bacillus subtilis genome, and the steps of introducing gene mutation to the genome, knocking out target gene sequences from the genome, deleting a large fragment of genomic sequences and the like are included.

The invention provides for a method of producing a mutant somatic human cell line of cells comprising a genomic mutation of interest (MOI) at a predefined genomic site of interest (GOI) in close proximity to a genomic target site, which comprises: a) providing a guide RNA (gRNA) comprising a tracrRNA in conjunction with crRNA including an oligonucleotide sequence that hybridizes with the target site; b) providing an RNA-guided endonuclease which catalyzes the DNA break at the target site upon hybridizing with the gRNA; c) introducing the gRNA into the cells in the presence of the endonuclease to obtain a repertoire of cells comprising a variety of genomic mutations at the target site; d) selecting a cell from said repertoire which comprises a MOI; wherein the cell is haploid for the genomic locus of the target site; and e) expanding the cell to obtain the mutant cell line. The invention further provides for a mutant human somatic cell line obtainable by such method; and libraries of mutant human somatic cell lines of isogenic cells with a variety of genomic mutations at different predefined genomic target sites.

The present invention relates to the treatment of cancer with compounds that inhibit the activity of endo-exonuclease. Endo-exonuclease has been shown to be necessary for the repair of damaged DNA. Compounds that inhibit the activity of endo-exonuclease have been shown to be particularly effective for treating cancer when used in combination with drugs that induce DNA breaks such as cisplatin and mitomycin C. These compounds have a synergistic effect when used in combination for inhibiting tumour growth. The invention includes pharmaceutical compositions for inhibiting tumour growth comprising a compound that inhibits endo-exonuclease activity. These pharmaceutical compositions preferably include compounds that induce DNA breaks. The invention includes methods of treating cancer with these pharmaceutical compositions and uses of these compositions to treat cancer. The preferred compounds that inhibit the activity of endo-exonuclease have low toxicity. One such compound is pentamidine. The invention also includes a method for diagnosing cancer and monitoring its progression. This aspect of the invention involves isolating serum from a patient; measuring the concentration of endo-exonuclease in said serum and determining whether said concentration is above a predetermined mean.

The invention discloses a high-temperature combined method for extracting genomic DNA of fish enteric microorganisms. The high-temperature combined method includes the steps that firstly, the walls of various fish enteric microorganisms are properly broken through a lysozyme method and an ultrasonic method at the same time, wherein the ultrasonic physical wall breaking condition ranges from 150 w to 250 w, ultrasonic treatment is carried out for 2-3 s and then carried out again after an interval of 5 s, and the process is repeated 50-60 times; secondly, the sample obtained after ultrasonic treatment and lysozyme are jointly incubated for 30 min in a water-bath constant-temperature oscillator at the temperature of 55-65 DEG C, and the mixture and RNase A are jointly incubated at the temperature of 27-32 DEG C. According to the method, the walls of the various microorganisms are properly broken under the proper and mild ultrasonic condition, and the genomic DNA is prevented from being broken and degraded. As the specific temperature range for incubating the lysozyme and the sample and the specific temperature range for incubating the RNase A and the sample are set, the splitting decomposition effect of the lysozyme can be improved, and it can be avoided that the genomic DNA is degraded by DNA enzymes in tissues and cells. Operation is easy, the extracted genomic DNA is complete and low in degradation rate, and the fish enteric microorganism structure can be truly reflected by the types of the microorganisms identified according to the extracted DNA.

Methods of editing a target genomic region(s), methods of repairing of a DNA break via a HDR pathway, methods of inhibiting or suppressing repair of a DNA break via a NHEJ pathway, and methods of modifying expression of a gene(s) or protein(s) comprise administering to one or more cells that include one or more target genomic regions, a genome editing system and a DNA protein-kinase (DNAPK) inhibitor disclosed herein. Kits and compositions for editing a target gene comprise a genome editing system and a DNAPK inhibitor disclosed herein.

It is an object of the present invention to increase the biomass quantity of plants by causing an exogenous gene to be carried in plant cells. An exogenous gene that promotes double-stranded DNA breakage is introduced into the cells of a plant, and the plant is grown.

The invention relates to a method for detecting a PAS (Poly A Site) of an RNA (Ribose Nucleic Acid), and provides a method for analyzing APA (Alternative Polyadenylation) based on 3T-seq (TT-mRNA Terminal Sequencing) within a whole-genome range. The method for analyzing APA comprises the following steps: preparing magnetic beads containing oligo dT, i.e., mixing an oligo dT primer modified by 5' end biotin and the magnetic beads with streptavidin; mixing the magnetic beads with total RNA and screening RNA containing Poly A; performing reverse transcription, synthesizing the second strand of a double-stranded cDNA and breaking the double-stranded cDNA; removing the Poly A structure; after terminal modification, connecting a sequencing linker; and recovering libraries. According to the method for analyzing APA, RNA horizontal operation is reduced, a Poly A sequence is horizontally removed from the DNA, and the influence of the Poly structure on sequencing is eliminated; double-stranded DNA breaking enzyme is selected for treatment, and on the premise that the quality of the libraries is not affected, the DNA is broken horizontally; the experiment process is simplified, the experiment difficulty and cost are lowered and the method for analyzing APA is wide in application range.

The invention relates to the technical field of molecular biology and particularly relates to a novel methylation library-establishing kit and an application thereof. The novel methylation library-establishing kit at least comprises a bisulfate conversion reagent, a sticky end joint, ligase, a DNA extending reagent and a DNA amplifying reagent. The novel methylation library-establishing kit can beapplied to a sample with a low initial mass (as low as 6pg) and suitable for genome DNA with relatively poor mass extracted from FFPE samples and the like, so that the loss of sample information caused by DNA breakage during drug treatment is greatly reduced; and a double-chain DNA joint with a sticky end can be directly introduced by virtue of relatively economic and practical common ligase, sothat the accuracy of data can be improved, the diversity of samples can be preserved to a great extent, and the library deviation property caused by human factors can be reduced.

The invention relates to a method for screening and evaluating genetic toxicity of industrial wastewater with complex ingredients. Gemmiparous broad bean seeds are cultivated in a suspension manner in to-be-detected wastewater solution prepared with tap water by means of gradient dilution; after two weeks, root tips are cut down and placed in proteinase K solution, and vacuumizing is carried out. Root tip cell nucleuses are separated, and the single-cell gel electrophoresis (SCGE) technique is used for separating DNA fragments. A fluorescence microscope is used for shooting comet pictures. A cometscore image-analysis system is used for measuring the tail length and the tail moment of the comet. Then, an SPASS program is used for statistic analysis. The method promotes proteinase K to penetrate into root tip meristem cell nucleuses by means of vacuumizing, accelerates degradation of DNA crosslinked protein, thus removes retardation of DNA-protein cross-linking on DNA fragments, and facilitates revelation and accurate evaluation of the true level of NDA breakage and damage inducted by the to-be-detected wastewater.

The invention relates to the technical field of molecular biology and in particular relates to a novel methylation library construction method and an application thereof. The novel methylation libraryconstruction method provided by the invention is suitable for single-strand connection, amplification or library construction of a DNA sample treated by using bisulfite. Due to the adoption of a method for bisulfite treatment and then adhesive end joint connection, sample information loss caused by DNA breakage resulted from drug treatment is greatly reduced.

The invention provides sgRNA, a CREBRF point mutation-type Bama miniature pig constructed by the sgRNA, and application. The sgRNA comprises a nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ IDNO: 2. According to the invention, a scheme of combining CRISPR / Cas9 with homologous recombination is adopted, point mutation of CREBRF gene is carried out, the designed gRNA has specific binding property on a target site near the 1370th site of the CEREBRF gene, the CRISPR / Cas9 can be guided to carry out specific recognition cutting near the target site, double-stranded DNA breakage is formed, homologous recombination repair with exogenous single-stranded DNA as a template is promoted, and therefore the point mutation is introduced. The mutant Bama miniature pig prepared by the method has a typical obesity phenotype and can be used as a model organism to be applied to the field of research and development of medicines for treating obesity and diabetes.

The invention relates to the field of fish sperm preservation solutions, in particular to a pseudobagrus sperm preservation solution for solving the problem of pseudobagrus large scale reproduction and a preparation method of the pseudobagrus sperm preservation solution. The sperm preservation solution is prepared from sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium bicarbonate, glucose and penicillin and is prepared by weighing the raw materials, adding the raw materials in a plastic bottle or a glass bottle, and preparing into a solution with distilled water. The prepared sperm preservation solution is subjected to sperm chromatin detection with a flow cytometer, spermatids are circled on an FSC / SSC graph, and a door is set on an FL1(FITC) / FL3(ECD) scatter diagram for analysis; and a DNA fragment index at is calculated through a formula to evaluate sperm DNA quality. The sperm preservation solution provided by the invention can be preserved for 10 days in a refrigeration tank at the temperature of 4 DEG C, 98 percent or more of sperms keep exuberant vigor and can still be taken as sperms for semination, and the fertilization rate is 95 percent orabove.

The invention relates to the technical field of anticancer drugs, in particular to a ruthenium complex anticancer drug and a preparation method thereof. The ruthenium complex anticancer drug is activated through redox, inserted into tumor cell DNA, and coordinated with DNA to undergo a crosslinking reaction, so as to prevent DNA replication and DNA breakage. Due to the introduction of a planar structure ligand, namely, 1,8-naphthalic anhydride, the binding capacity of the drug to DNA is enhanced by 14.17 times, and the stability and anti-tumor activity of the anticancer drug are greatly improved.

The invention discloses a novel tetracyclonaphthooxazole derivative. The structure of the novel tetracyclonaphthooxazole derivative is represented by general formula (I) shown in the specification. The invention also discloses a preparation method of the tetracyclonaphthooxazole derivative. The method allows the tetracyclonaphthooxazole derivative to be innovatively synthesized through using a carbon hydrogen activation serial reaction with amino substituted quinone as a reaction substrate. The method has the advantages of simplicity, easy implementation and high efficiency. The synthetic tetracyclonaphthooxazole derivative has a similar structure with a traditional DNA clastogen, can inhibit cell proliferation and growth, and has antibacterial, antifungal, anti-HIV and antitumor bioactivity. The invention also discloses an application of above compounds as a cell proliferation and growth inhibitor, and an application of the compounds in the preparation of antitumor medicines.

The invention discloses a novel tetracyclonaphthooxazole derivative. The structure of the novel tetracyclonaphthooxazole derivative is represented by general formula (I) shown in the specification. The invention also discloses a preparation method of the tetracyclonaphthooxazole derivative. The method allows the tetracyclonaphthooxazole derivative to be innovatively synthesized through using a carbon hydrogen activation serial reaction with amino substituted quinone as a reaction substrate. The method has the advantages of simplicity, easy implementation and high efficiency. The synthetic tetracyclonaphthooxazole derivative has a similar structure with a traditional DNA clastogen, can inhibit cell proliferation and growth, and has antibacterial, antifungal, anti-HIV and antitumor bioactivity. The invention also discloses an application of above compounds as a cell proliferation and growth inhibitor, and an application of the compounds in the preparation of antitumor medicines.

The invention relates to a method for rapid detection of in vitro DNA break damage strength by plasma, and the method is used for determining the damage strength of plasma mutagenesis methods including atmospheric and room temperature plasma (ARTP) on DNA. The method includes the steps of: 1) treating DNA coated microspheres by plasma mutagenesis method, and setting a control group without plasma treatment; 2) performing washing, and then dyeing the DNA coated microspheres with a dye; 3) determining the fluorescence value of the dyed DNA coated microspheres by a fluorescence detector; and 4) calculating the DNA relative content of the control group and the plasma mutagenesis treated microspheres according to the fluorescence value, and judging the gene damage strength of plasma. The invention utilizes DNA coated microspheres to establish a rapid detection method for in vitro DNA damage strength so as to realize direct and effective evaluation of plasma caused damage strength to DNA.

The invention belongs to the technical field of engineering, and particularly relates to a bacterial genome multi-editing method and application thereof. The bacterial genome multi-editing method based on double-stranded DNA recombinant engineering comprises the following steps: (1) mixing dsDNA substrates targeting different target sequences, and introducing the mixed dsDNA substrates into host competent cells with recombinase plasmids; (2) recovering the competent cells at the temperature lower than the optimum growth temperature; dNTPs with the final concentration of 10 nM is added in the resuscitation process; and (3) screening out a single colony with a marker from the recovered competent cells, and identifying. The method has the advantages that the substrate preparation cost is low, the length of the inserted gene is not limited, the method is not dependent on double-stranded DNA breakage, a plurality of guide RNA expression vectors do not need to be constructed, and a restrictive repair system of host bacteria does not need to be inactivated.

A method of modifying the genome of an organism, wherein the modification method includes modifying the genome of the organism by using in a cell of the organism a protein having an optimal temperature for double-stranded DNA breakage activity in an ordinary temperature region.

Provided is a genome shuffling technology suited to creating a useful next-generation plant body.Genome shuffling is performed using a polypeptide comprising a first polypeptide region for double-stranded DNA breakage activity and a second polypeptide region for intertissue migration activity.

The invention provides a DNA sample treating method, a sequencing method and a kit. The method for treating a DNA sample comprises the following steps: performing fragmentation, terminal repair and dAtail addition on DNA in a first premixed system to obtain a first product, wherein the first premixed system comprises premixed enzyme, and the premixed enzyme comprises DNA fragmentation enzyme, polynucleotide kinase and DNA polymerase. Based on test experiments and an optimized mixed enzyme system, multiple enzymes are mixed in the same reaction system, DNA breakage, terminal repair and dA tailaddition are realized in one step, the convenience and rapidness are achieved, the operation time of sample treatment before loading is saved, and therefore the industrialization is facilitated.

Provided is a genome shuffling technology suited to creating a useful next-generation plant body.Genome shuffling is performed using a polypeptide comprising a first polypeptide region for double-stranded DNA breakage activity and a second polypeptide region for intertissue migration activity.

The invention relates to a method for detecting a PAS (Poly A Site) of an RNA (Ribose Nucleic Acid), and provides a method for analyzing APA (Alternative Polyadenylation) based on 3T-seq (TT-mRNA Terminal Sequencing) within a whole-genome range. The method for analyzing APA comprises the following steps: preparing magnetic beads containing oligo dT, i.e., mixing an oligo dT primer modified by 5' end biotin and the magnetic beads with streptavidin; mixing the magnetic beads with total RNA and screening RNA containing Poly A; performing reverse transcription, synthesizing the second strand of a double-stranded cDNA and breaking the double-stranded cDNA; removing the Poly A structure; after terminal modification, connecting a sequencing linker; and recovering libraries. According to the method for analyzing APA, RNA horizontal operation is reduced, a Poly A sequence is horizontally removed from the DNA, and the influence of the Poly structure on sequencing is eliminated; double-stranded DNA breaking enzyme is selected for treatment, and on the premise that the quality of the libraries is not affected, the DNA is broken horizontally; the experiment process is simplified, the experiment difficulty and cost are lowered and the method for analyzing APA is wide in application range.

The invention discloses a new application of astragalus polysaccharide, and belongs to the technical field of biology medicine. A basis is provided for preventing and treating myoblastosis through radix astragali. The inventor of the invention finds that the astragalus polysaccharide can obviously reinforce the proliferation activity of human BM-MSCs in formaldehyde environment, and can reduce DNAfragmentation and DPCs formation of human BM-MSCs induced by formaldehyde so as to have good protection effects. The astragalus polysaccharide can promote DNA injury repair factors of XPA, XPC, ERCC1, RPA1 and RPA2 mRNA and protein expression, and improve DNA damage repair capacity. The inventor finds that the astragalus polysaccharide can reinforce the proliferation activity of human BM-MSCs inthe formaldehyde environment for the first time, can reduce the DNA fragmentation effects of the human BM-MSCs in the formaldehyde environment and can reduce the DPCs formation of the human BM-MSCs inthe formaldehyde environment.

The invention discloses a high-temperature combined method for extracting genomic DNA of fish enteric microorganisms. The high-temperature combined method includes the steps that firstly, the walls of various fish enteric microorganisms are properly broken through a lysozyme method and an ultrasonic method at the same time, wherein the ultrasonic physical wall breaking condition ranges from 150 w to 250 w, ultrasonic treatment is carried out for 2-3 s and then carried out again after an interval of 5 s, and the process is repeated 50-60 times; secondly, the sample obtained after ultrasonic treatment and lysozyme are jointly incubated for 30 min in a water-bath constant-temperature oscillator at the temperature of 55-65 DEG C, and the mixture and RNase A are jointly incubated at the temperature of 27-32 DEG C. According to the method, the walls of the various microorganisms are properly broken under the proper and mild ultrasonic condition, and the genomic DNA is prevented from being broken and degraded. As the specific temperature range for incubating the lysozyme and the sample and the specific temperature range for incubating the RNase A and the sample are set, the splitting decomposition effect of the lysozyme can be improved, and it can be avoided that the genomic DNA is degraded by DNA enzymes in tissues and cells. Operation is easy, the extracted genomic DNA is complete and low in degradation rate, and the fish enteric microorganism structure can be truly reflected by the types of the microorganisms identified according to the extracted DNA.

The present invention relates to the technical field of anticancer drugs, and more specifically to a ruthenium complex anticancer drug and a preparation method thereof. The ruthenium complex anticancer drug is activated by redox, inserted into tumor cell DNA, and at the same time coordinates the DNA to undergo cross-linking reaction, prevent DNA replication, and cause DNA breakage. Due to the introduction of planar structure ligand 1,8‑naphthalene dicarboxylic anhydride, the drug’s ability to bind to DNA has been enhanced by 14.17 times, which greatly improves the stability and anticancer properties of anticancer drugs. tumor activity.