46 results about "Restriction enzyme" patented technology

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.

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 invention discloses a dCAPS molecular marker primer linked with a brassica napus saline-alkaline tolerance gene BnaC03.RBGB3 and application of the dCAPS molecular marker primer. The primer comprises a forward primer and a reverse primer, and through PCR amplification and specific restriction enzyme digestion treatment, two genotypes of high resistance and high sensitivity of brassica napus saline-alkaline tolerance can be distinguished on agarose gel electrophoresis; the high-resistance genotype is represented by two electrophoretic bands (195bp and 23bp), and the high-sensitivity genotype is a band which cannot be digested by enzyme. The method disclosed by the invention is simple and convenient to operate and reliable in result, can be used for rapidly screening and identifying the saline-alkaline-tolerant variety of the brassica napus, and provides a powerful tool for breeding the saline-alkaline-tolerant variety of the brassica napus. By implementing the technology, not only is the breeding efficiency improved, but also a new thought and strategy are provided for molecular marker-assisted breeding of saline-alkaline tolerance of crops, and development and application of a saline-alkaline tolerance breeding technology of brassica napus and even other crops are promoted.

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.

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.

[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).

The invention belongs to the technical field of genetic engineering, and particularly relates to a universal bacterial artificial chromosome recombinant virus transfer vector and a construction method thereof, the transfer vector comprises a BAC amplification vector with pUC19-loxp-gpt-IRES-EGFP as a basic skeleton and pUC19-BAC as a functional sequence; according to the universal bacterial artificial chromosome recombinant virus transfer vector and the construction method thereof, a drug screening target gene reading frame and an EGFP fluorescent protein gene reading frame are connected through an IRES double-expression connection sequence, and double screening can be carried out; in the BAC construction process, screening genes and functional sequences are respectively constructed to a high-copy T vector, so that sufficient plasmid DNA can be conveniently copied and prepared. And through a proper restriction enzyme cutting site, introduction of various homologous arms is facilitated, the alignment is wide, simultaneous operation of various genomic sequences is facilitated, and the research efficiency is improved.

The invention belongs to the technical field of polypeptide preparation and biological medicine, and particularly relates to a preparation method of Jingzhaotoxin polypeptide IX (JZTX-IX) and application of Jingzhaotoxin polypeptide IX in treatment of diabetes mellitus. A chilobrachys jingzhao toxin polypeptide IX coding gene, a 6 * His tag nucleotide sequence, a thrombin restriction enzyme cutting site nucleotide sequence and an MBP hydrotropy tag nucleotide sequence are connected to a prokaryotic expression vector for prokaryotic expression, and the expressed polypeptide is purified to obtain the chilobrachys jingzhao toxin polypeptide IX protein. The target polypeptide with high purity, complete disulfide bond folding and uniform conformation is obtained, so that the high purity and biological activity of the target polypeptide are ensured. On the basis, the treatment activity of the JZTX-IX on diabetes mellitus especially type II diabetes mellitus is found and verified, and the JZTX-IX can remarkably reduce fasting blood glucose of mice with the type II diabetes mellitus, improve glucose tolerance and insulin sensitivity, relieve polydipsia and polyuria symptoms and promote insulin secretion and islet cell proliferation.

The application provides application of a CAPS molecular marker GW18 in identification of foxtail millet (Setaria italica) resistance to rice blast, and belongs to the technical field of molecular marker breeding. The application provides the application of the CAPS molecular marker GW18 in identification of the foxtail millet resistance to the rice blast, and G / T polymorphism exists at 34523782 bp of the 6th chromosome of the foxtail millet; when the polymorphism is G, the resistance to the rice blast cannot be recognized by a restriction enzyme, and is identified as the susceptibility to the rice blast; when the polymorphism site is T, the GW18 target sequence can be recognized by the restriction enzyme and is cut into two segments, and is identified as the resistance to the rice blast. The marker has a high co-separation rate with the disease resistance, a rapid, accurate and low-cost detection method, and can be used for rapid identification of foxtail millet germplasm resources resistant to the rice blast and molecular marker assisted breeding, and has important significance for improving breeding efficiency and guaranteeing safe production of the foxtail millet.

Various compositions and methods are disclosed in which a plurality of restriction enzymes, including methylation-sensitive and / or methylation-dependent restriction enzymes, are used for the analysis of cfDNA. Useful combinations can be inactivated by heating to 65° C., ideally for longer than 15 minutes. Restriction digestion of cfDNA can occur for 11 hours or less. Digestion may be followed by amplification and / or sequencing steps.

This application relates to a method and kit for the specific degradation of library contaminants based on a methylation-sensitive restriction endonuclease. The method includes: determining the recognition site of the methylation-sensitive restriction endonuclease, digesting library contaminants, fragment purification, verification, and quality control. In the library contaminant digestion step, the library reaction system is incubated at 35°C–39°C for 0.5–1.5 hours, followed by incubation at 60°C–70°C for 15–25 minutes to obtain the digested DNA library reaction system. This method selectively removes unmethylated DNA while having almost no effect on highly methylated DNA, avoiding non-specific degradation and effectively improving the purity of the target fragment.

The present invention discloses a composition, a kit and their applications for detecting lung cancer. The composition includes detection reagents for detecting the methylation status of one or more of 17 genes such as SIX2 and PANTR1. The detection reagents contain multiplex PCR pre-amplification primers, real-time fluorescence quantitative PCR primers and probes, and primers and probes for the internal reference ACTB, and are designed by nested PCR, and the primer sequences are SEQ ID No.1-90. The kit contains the above composition and also contains methylation-sensitive restriction enzymes and Exonuclease I. Among them, the real-time fluorescence quantitative PCR primers and probes for 17 genes and ACTB are分装 into 6 reaction tubes. The present invention combines methylation-sensitive restriction enzyme treatment, multiplex PCR pre-amplification and fluorescence quantitative PCR techniques to achieve high-sensitivity and high-specificity detection of lung cancer. The combined detection of 17 genes has an AUC of 0.91, is easy to operate, has low cost, is suitable for early screening and auxiliary diagnosis of lung cancer, and has good clinical application prospects. It should be noted that the word "分装" in the original text seems to be a misspelling. It should probably be "separately packaged" or something similar. The translation above keeps the original text as it is for that word.

The present application relates to a human BMP3 and NDRG4 gene methylation detection kit, the kit comprises a restriction enzyme reaction solution, the restriction enzyme reaction solution comprises a restriction enzyme buffer, a methylation-dependent restriction enzyme, and further comprises a fluorescent PCR reaction solution of primers and a partial double-stranded linear DNA probe designed for a CpG island of a BMP3 gene and / or a NDRG4 gene. The detection kit can realize the sequential restriction enzyme reaction and fluorescent PCR reaction in one tube to detect the methylation state of the BMP3 and NDRG4 genes, and has the characteristics of simple and rapid operation, high sensitivity, good specificity and easy automation.

The present disclosure relates to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcription of the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) separating the test cDNA fragments so as to form a test cDNA fragment map; and (f) comparing the test cDNA fragment map to a control cDNA fragment map wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment map comprises the control cDNA fragment map. Also disclosed are methods of quantifying and / or assessing the integrity of at least one mRNA molecule in a composition, processes of making a composition comprising at least one mRNA molecule and a primer pair for use in the methods of the invention.

The invention discloses a molecular marker for identifying an excellent haplotype of a corn ZmTAR4 gene and application of the molecular marker, and belongs to the technical field of plant molecular breeding and genetic detection. Specifically, the nucleotide sequence of the molecular marker is shown as SEQ ID NO.1, and a G / A polymorphic site exists at the 29bp position of the sequence. The invention further develops a primer pair for detecting the molecular marker, a detection product containing the primer pair is prepared, PCR amplification is carried out on corn genome DNA through the primer pair to obtain an amplification product, the amplification product is subjected to enzyme digestion through restriction enzyme, and the excellent haplotypes of the corn ZmTAR4 gene are distinguished according to the obtained enzyme digestion product. The molecular marker is reasonable in design, simple and convenient to operate and accurate in interpretation, is suitable for corn germplasm resource evaluation and molecular marker-assisted breeding, and has important application value in breeding practice for improving corn genetic transformation efficiency.

The application provides a Septin9 methylation detection composition and application thereof, and the composition comprises a methylation-dependent restriction enzyme, a capture oligonucleotide, a universal primer and a Septin9 methylation-specific primer; the capture oligonucleotide comprises a first universal sequence, a folding sequence and a binding capture sequence from 5' end to 3' end in sequence; the folding sequence is at least partially identical to a 5' end sequence of a Septin9 methylation site after the methylation-dependent restriction enzyme is cut; and the binding capture sequence is specifically bound to a fragment region where the detected Septin9 methylation site is located. The application is based on a methylation-dependent restriction enzyme and a universal primer fluorescence quantitative PCR technology, does not need bisulfite conversion, and realizes accurate and specific Septin9 methylation detection.

The invention provides a restriction enzyme combination and application thereof. Wherein the restriction endonucleases comprise a combination of restriction endonucleases EcoRV and restriction endonucleases Dral, the combination of the restriction endonucleases can effectively enrich cfDNA fragments where the lung cancer related mutation sites are located, so that the sensitivity of downstream analysis is improved, the mutation detection rate of the lung cancer related mutation sites reaches 95%, and the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result shows that the detection result is accurate. The method can be applied to various clinical and research fields, including tumor diagnosis, prenatal screening and disease monitoring.

The invention discloses a composition for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry and application of the composition. The invention relates to the field of gene detection, and provides a composition for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry, which comprises a target gene primer pair, a reference gene primer pair and an IIS type restriction enzyme. The composition can be used for detecting spinal muscular atrophy typing based on nucleic acid mass spectrometry, and the traditional technical method of nucleic acid mass spectrometry is improved, so that nucleic acid mass spectrometry detection does not need the steps of SAP treatment and single base extension reaction, the reaction time is shortened, the result is accurate and reliable, the steps are simple, and the cost is greatly reduced. The invention is of great significance to preparation of screening products suitable for large-scale screening of spinal muscular atrophy.

The present disclosure relates to the technical field of DNA fragmentation for technologies such as next generation sequencing (NGS) or any other technologies where fragmentation of DNA to a certain uniform size is required. In particular, the present disclosure relates to a variant of a Nucleoside Triphosphate (NTP)-dependent restriction enzyme (McrBC variants selected from McrBL68YC, McrBL68FC, and combinations of certain ratios thereof) for nucleic acid fragmentation for methods that require fragmentation of DNA.

The invention discloses a micropterus salmoides rhabdovirus gene engineering vaccine preparation method, which comprises: S1, recombinant plasmid construction: carrying out PCR amplification on a micropterus salmoides rhabdovirus G protein gene with a 6 * His tag, cloning the amplified gene between BamH I and Hind III restriction enzyme cutting sites of a pVL1393 vector to obtain a pVL1393-G-His recombinant plasmid, S2, preparing a transfection system, and S3, carrying out purification on the transfection system to obtain the micropterus salmoides rhabdovirus gene engineering vaccine. S3, recombinant baculovirus preparation and identification, S31, cell transfection, S32, P1-generation virus harvesting, and S33, virus identification, the G protein of the micropterus salmoides rhabdovirus (MSRV) is directionally expressed through a genetic engineering technology, a large number of pathogenic viruses do not need to be cultured, the biological safety risk is greatly reduced, meanwhile, the defect that a prokaryotic expression system lacks protein post-translational modification is overcome, and the method is suitable for industrial production. The sf9 insect cell is used as an expression host, so that the recombinant G protein can be correctly folded to form a functional structure domain, and the immunogenicity is remarkably improved.

The present application belongs to the technical field of plant species identification, and particularly relates to a detection and identification kit, method and application of Aglaia odorata. The kit contains an upstream primer with a nucleotide sequence of SEQ ID NO. 1, a downstream primer with a nucleotide sequence of SEQ ID NO. 2, and a restriction enzyme Sma I. The primer is used for PCR amplification of sample DNA, and the amplification product is digested by a restriction enzyme Sma I. The enzyme-digested product is subjected to agarose gel electrophoresis, and according to the number and size of specific electrophoretic bands, Aglaia odorata and its similar species can be distinguished and identified conveniently and quickly. The method has the advantages of intuitive results, high specificity, high identification efficiency, and avoidance of false positive amplification.

The invention discloses a preparation method and application of a magnetic protein enzymolysis material, and the preparation method comprises the following steps: growing ferroferric oxide nanoparticles on the surface of porous silica gel with large enough pore size and in pores by adopting an in-situ synthesis method, so that the ferroferric oxide nanoparticles have magnetism; immobilizing protease on the magnetic substrate to prepare a magnetic proteolysis material; putting the material into a protein solution to complete enzymolysis operation; taking out the magnetic protein enzymolysis material from the solution by magnetic separation; the obtained solution after enzymolysis is used for proteomics research. The method disclosed by the invention has the beneficial effects that the enzyme is fixed on the surface of a material in a form that protein forms a protein crown on the surface of the magnetic macroporous silica gel, and free collision and mutual contact among enzyme molecules are limited through a steric hindrance effect, so that the self-degradation probability of the enzyme is greatly reduced; the enzyme immobilization amount is increased, the enzymolysis efficiency is remarkably improved, and the enzymolysis time is remarkably shortened; the magnetically modified macroporous silicon balls can be quickly recovered from a reaction system through an external magnetic field and can be repeatedly used.

The invention discloses a bio-based antibacterial agent based on yeast fermentation and a preparation method of the bio-based antibacterial agent in the field of antibacterial agents. The bio-based antibacterial agent comprises the following components in parts by weight: 2-4 parts of modified chitosan, 1 part of modified polyethylene glycol, 0.2-0.3 part of pig gastric mucoprotein and 0.6-0.8 part of sodium alginate. According to the invention, a gene corresponding to an amino acid sequence of tachyplesin is optimized; according to the invention, a pYES2 / CT vector is used as a target gene, an EcoR I / Xho I restriction enzyme cutting site is introduced to a 5 ' / 3' end, it is ensured that the target gene can be accurately inserted into the pYES2 / CT vector, Tachyplesin-1 is a natural antibacterial peptide, a killing effect can be realized by destroying cell membranes of harmful bacteria and interfering metabolism of the harmful bacteria, and Tachyplesin breaks through intestinal environment limitation and accurately acts on the harmful bacteria through coating and functional modification of a modified vector.

The invention relates to the technical field of biological products, and particularly discloses an XhoI restriction enzyme freeze-drying protective agent and a preparation method and application thereof.The freeze-drying protective agent is composed of trehalose, polyvinylpyrrolidone, arginine, HSA and EDTA in parts by mass and volume, and a solvent is sterilized purified water. A co-amorphous substance is formed through interaction of all the components to construct a multi-level protection network for enzyme, in the freezing and drying process, hydroxyl of trehalose interacts with the surface of enzyme molecules through hydrogen bonds, the position of water molecules is effectively replaced, the enzyme is forced to maintain the natural hydration conformation of the enzyme, and therefore expansion and denaturation caused by dehydration are avoided. The amorphous state of the polyvinylpyrrolidone and the trehalose are in synergistic interaction, so that the glass transition strength of the mixture can be enhanced, the physical stability of the product can be improved, collapse can be prevented, the contact between enzyme molecules can be blocked through the huge steric hindrance effect, and the inactivation caused by aggregation can be effectively prevented.

The application provides a biotin-labeled exogenous circular DNA and a construction method and application in protein interaction thereof, and belongs to the technical field of gene interaction. The application provides a construction method of the biotin-labeled exogenous circular DNA, wherein a target sequence is amplified by using a biotin-labeled primer to obtain linear DNA containing a preset restriction enzyme cutting site; the obtained linear DNA is subjected to single enzyme cutting treatment by using a corresponding restriction endonuclease; and the DNA after enzyme cutting is subjected to a self-ligation reaction to generate closed circular DNA. The preparation process of the biotin-labeled circular DNA is stable and controllable, and has high repeatability; the biotin labeling of the circular DNA can be combined with a streptavidin system to realize high-affinity and specific enrichment of a DNA-protein complex; and the circular DNA can be stably delivered in cells and maintain the structural integrity of the circular DNA, thereby providing an effective means for truly reflecting the interaction between the circular DNA and the protein in a physiological environment.

The application discloses a preparation and transformation method suitable for different tissue protoplasts of soybean, and belongs to the technical field of biology. The method comprises the following steps: taking leaf, stem or root tissue of 10-12 day old soil culture yellowing soybean seedlings, chopping, and then carrying out light-free enzymolysis in a mixed enzyme solution; collecting precipitated cells after filtration and centrifugation; resuspending the precipitated cells to obtain a protoplast suspension; uniformly mixing the protoplast suspension with a plasmid, and adding 50% PEG transformation liquid to carry out transformation for 15 min under room temperature. The method optimizes an enzymolysis system and key parameters of a PEG transformation liquid, and obtains a protoplast preparation and transformation method suitable for multiple tissues and multiple genotypes of soybean, so that the problems of material limitation, low enzymolysis efficiency and poor transformation efficiency in the preparation and transformation process of soybean protoplasts in the prior art are solved.

The invention discloses a preparation method of a deer whole blood small molecule peptide, which comprises the following steps: constructing a special compound enzyme system for layering and distributed enzymolysis through specific recognition of a functional domain sequence, and solving the technical problem of poor accessibility of a restriction enzyme cutting site caused by a quadrupole structure of hemoglobin and a heme binding characteristic; the obtained deer whole blood small molecule peptide is good in color and luster, the yield of a 500-3000 Da peptide fragment is high, and meanwhile characteristic microelements such as iron and zinc are reserved.

The invention discloses a Ts-ELP temperature-sensitive self-assembly nano fusion protein, an antibiotic delivery system and a preparation method, and belongs to the technical field of nano preparations. The fusion protein comprises antibacterial peptide Ts and elastin-like polypeptide ELP which are sequentially connected. An MMP-9 restriction enzyme cutting site is introduced between the antibacterial peptide Ts and the elastin-like polypeptide ELP. Fusion protein and tigecycline (Tig) are self-assembled to form an antibiotic delivery system, the delivery system has Gram-negative bacterium surface lipopolysaccharide targeted recognition capability, and drug release is triggered through pH response and a phase separation mechanism in an infection microenvironment, so that the enrichment efficiency of tigecycline in a focus area is enhanced, and the drug delivery efficiency is improved. The antibiotic delivery system has the technical advantages of strong targeting property, enhanced antibacterial activity, low drug-resistant induction risk, good biological safety, simple preparation process, high yield and the like, can be used for treatment of multi-drug-resistant bacterium infection, and is especially suitable for prevention and treatment of carbapenem-resistant klebsiella pneumoniae (CRKP) and drug-resistant gram-negative bacterium infection related diseases.

The invention relates to a construction method of a database for discriminating proteolytic enzyme restriction enzyme cutting sites. Comprising the following steps: S1, acquiring family classification information and restriction enzyme cutting site data of proteolytic enzyme from an MEROPS database, acquiring sequence and function annotation data of substrate protein from a UniProt database, and acquiring three-dimensional structure data of the substrate protein from an AlphaFold database; s2, migrating and integrating the data in the S1 to the local; and S3, dividing the processed data into four correlative core data tables, and storing the four core data tables in a local database. According to the method disclosed by the invention, by combining MEROPS, UniProt and AlphaFold databases, the restriction enzyme cutting site of the proteolytic enzyme is accurately predicted by adopting an artificial intelligence algorithm. Compared with a traditional method, the method has the advantages that the restriction enzyme cutting sites can be recognized more efficiently and more accurately, and the protein function analysis speed and accuracy are remarkably improved.

This invention discloses an Ago-DNA polymerase simultaneous dual-cycle amplification system and its applications. The Ago-DNA polymerase simultaneous dual-cycle amplification system comprises: Ago enzyme, DNA polymerase, amplification template, dNTPs, MnCl2, and gDNA; the sequences of the amplification template and gDNA are obtained using the design method of this invention. This system can be applied to amplify and / or detect microRNA. This system enables integrated simultaneous dual-cycle amplification, simplifying the operation steps, reducing reaction complexity, avoiding aerosol contamination, reducing costs, and eliminating the requirement for specific sequences by restriction enzymes such as Cas and Nt.BstNBI. The amplification and / or detection methods using this system are highly versatile, accurate, and efficient, breaking through the bottlenecks of current molecular diagnostic technologies and holding promise for widespread application.