113 results about "Restriction enzyme" patented technology

The invention provides a preparation method, application and the like of large intestine expression recombinant A-type botulinum toxin (BONT / A). The recombinant BONT / A nucleic acid expression cassette comprises a tag, a restriction enzyme cutting site and a BONT / A nucleic acid sequence, and an exogenous protease recognition site is not introduced between a BONT / A light chain and a BONT / A heavy chain. The invention also provides a recombinant vector containing the nucleic acid expression cassette, a recombinant bacterium, a coded and expressed protein and the like, the prepared recombinant BONT / A does not introduce exogenous amino acid or only introduces two amino acids at one position, and the consistency of the recombinant BONT / A with the natural A-type botulinum toxin is ensured to the greatest extent. Through novel molecular design and preparation process, the recombinant reBONT / A with higher toxicity and low immunogenicity is efficiently obtained, and wide industrial application is facilitated.

The invention belongs to the field of molecular biological detection, and particularly relates to an FBXO32 gene SNP (Single Nucleotide Polymorphism) marker for rapidly detecting economic traits of Tibetan chicken as well as a detection method and application. It is found for the first time that G / A mutation at the 8916th site of the Tibetan chicken FBXO32 gene is closely related to whether the gene has an Hpa II restriction enzyme cutting site or not, and is closely related to main economic traits of Tibetan chicken. Based on the new discovery and the SNP site, the invention provides a method for rapidly detecting the SNP site and screening economic characters of Tibetan chicken. The method is simple to operate, convenient and fast, low in cost and reliable in result.

The invention belongs to the field of molecular biological detection, and particularly relates to a PLS3 gene SNP marker for rapidly detecting economic traits of Tibetan chicken, a detection method and application. It is found for the first time that G / A mutation at the 28469th site of the Tibetan chicken PLS3 gene is closely related to whether the gene has a BstZ17 I restriction enzyme cutting site or not, and is closely related to main economic traits of Tibetan chicken. Based on the new discovery and the SNP site, the invention provides a method for rapidly detecting the SNP site and screening economic characters of Tibetan chicken. The method is simple to operate, convenient and fast, low in cost and reliable in result.

The invention relates to the technical field of aquaculture fish vaccines, in particular to a fusion gene of grass carp NPM1a and II type reovirus VP38, recombinant protein, a composition and application. The nucleotide sequence of the fusion gene is as shown in SEQ ID NO: 1. The fusion gene is obtained by connecting grass carp NPM1a and II type reovirus VP38 through a linker sequence, a BamH restriction enzyme cutting site is added to the 5'end of the fusion sequence, and an XhoI restriction enzyme cutting site is added to the 3 'end of the fusion sequence; the sequence of the linker is as shown in SEQ ID NO: 2. The subunit vaccine of the fusion protein of the grass carp NPM1a and the II type reovirus VP38 is prepared, and indole-3-lactic acid is used as an immunopotentiator in a combined manner, so that the technical bottlenecks of low protection rate, poor intestinal mucosal barrier repairability and the like of the existing vaccine are overcome.

The invention discloses a micro-ring plasmid synthesis method, and belongs to the technical field of gene editing. Two pairs of primers are designed, eukaryotic expression plasmids containing selection markers and target fragments are used as templates for PCR amplification to obtain two target fragments, vector plasmids loxp-R6K with specific sequences and the target fragments form recombinant plasmids, the recombinant plasmids contain sequences such as the target fragments, restriction enzyme cutting sites and R6K replication start sites, and the recombinant plasmids are used for preparing the recombinant plasmids. The recombinant plasmid is transformed in Escherichia coli GT115 competent cells, the recombinant plasmid is verified to have the recombination efficiency of 81% after bacterial colony PCR amplification, a correctly cloned seed solution is screened and subjected to induced expression to form a bacterial solution containing a screening-label-free micro-plasmid, the micro-ring plasmid is separated and purified, and the screening-label-free micro-ring plasmid is obtained. The sequence of the microring plasmid only contains a target fragment and an R6K replication start site, the accuracy of the size sequence of the microring plasmid in a bacterial colony cultured and cloned by the microring plasmid is as high as 90%, and the microring plasmid can be efficiently and stably obtained.

The invention relates to the technical field of agriculture, and particularly provides a CAPS molecular marker of a rice salt-tolerant ST4 promoter region and application of the CAPS molecular marker. The method comprises the following steps: selecting leaves of rice to extract genome DNA (Deoxyribonucleic Acid) of the tissues, and then respectively carrying out PCR (Polymerase Chain Reaction) amplification by taking the DNA of the tissues as a template and taking corresponding sequences of ST4-PRO-F1 and ST4-PRO-R1 as specific primers to obtain amplified fragments; carrying out enzyme digestion on the amplified fragment by using Eco81I restriction enzyme, and carrying out electrophoresis after enzyme digestion; after the ST4 promoter fragment is subjected to electrophoresis, a single plant with only one 2162bp main band is a salt-tolerant single plant, and a single plant with 1366bp and 784bp bands appearing in electrophoresis is a salt-sensitive single plant. According to the invention, the rice variety improved by utilizing the wild rice ST4 gene can be rapidly bred, the current situations of much field work and large workload caused by carrying out a large amount of salt tolerance identification by utilizing a population material can be improved, and the method has important theoretical and practical significance for breeding a new variety of rice with salt tolerance character.

The invention relates to a method for detecting deletion in ITR to be detected contained in an AAV vector to be detected. The method comprises the following steps: cutting a fragment containing a sequence of the ITR to be detected from the AAV vector to be detected by using restriction enzyme, detecting the size of the fragment of an enzyme digestion product, and determining whether deletion exists in the ITR to be detected according to the size of the fragment.

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.

Methods for the processing and analysis of blood samples obtained with blood collection tubes that reduce contamination of cfDNA by genomic DNA but that inhibit digestion by methylation-sensitive and / or methylation-dependent restriction enzymes.

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 provides a method and a device for simulating a pulsed field gel electrophoresis band based on a sequencing technology. The method for simulating the pulsed field gel electrophoresis band based on the sequencing technology comprises the following steps: acquiring nanopore sequencing data of a biological sample to be analyzed, and assembling based on the nanopore sequencing data to obtain a genome sequence; according to the sequence characteristics of the restriction endonuclease, identifying a restriction enzyme cutting site corresponding to the restriction endonuclease from the genome sequence; when the restriction enzyme cutting site is the restriction enzyme cutting site subjected to methylation modification, removing the restriction enzyme cutting site to obtain a residual restriction enzyme cutting site; calculating the distance difference between two adjacent restriction enzyme cutting sites according to all residual restriction enzyme cutting sites, and taking the distance difference as the molecular weight of a simulated band; aiming at the molecular weight of each simulated strip, determining a strip position corresponding to the molecular weight; and drawing a simulated pulse field gel electrophoresis band result according to the position of each band and the molecular weight corresponding to the band position so as to accurately simulate the band result.

The present disclosure provides methods and compositions for preparing DNA molecules useful for determining the methylation status of selected regions of a DNA molecule, which utilize methylation-sensitive restriction enzymes and typically involve the amplification or selective enrichment of one or more target regions.

The invention discloses a high-efficiency expression method of porcine interleukin 22 (pIL-22) and application of the pIL-22 in resisting PEDV (porcine epidemic diarrhea virus) infection, and belongs to the technical field of biology. The high-efficiency expression method comprises the following steps: inserting a swine-derived interleukin 22 gene (SEQ ID NO: 1) into a pcDNA3.1 (+) vector restriction enzyme cutting site BamH I / Xho I to obtain a recombinant vector, mixing the recombinant vector and a transfection reagent, transfecting HEK-293 cells, collecting a cell suspension, ultrasonically crushing, filtering with a filter membrane to obtain a target protein, and finally purifying with a Protein A chromatographic column to obtain the swine-derived interleukin 22. (1) by adopting a mammalian cell expression system and a culture mode of suspension cells, not only can high-activity pIL-22 be expressed, but also the expression efficiency of the pIL-22 is greatly improved, and important guarantee and reference basis are provided for developing novel anti-PEDV drugs;

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.

To provide a preparation method of a unit DNA composition in which the mole number of a plurality of unit DNA are arrayed better, and a manufacturing method of a DNA connected body.SOLUTION: A preparation method of a unit DNA composition has: a process of preparing solution including a plurality of unit DNA, to which an additional sequence is connected, for each kind of the unit DNA; a process of, after preparation of each solution, measuring the concentration of the unit DNA in each solution in the state where an additional sequence is connected to the unit DNA, dispensing each solution on the basis of its result, and making the mole numbers of the unit DNA in each solution become closer to the same with each other. A manufacturing method of a DNA connected body has: a process of preparing a unit DNA composition; a process of preparing a vector DNA; a process of removing each additional sequence from the unit DNA in which an additional sequence in solution after preparation is connected by using restriction enzyme; and a process of connecting the vector DNA and each unit DNA with each other after the removal process.SELECTED DRAWING: None

The invention belongs to the technical field of plant species identification, and particularly relates to a detection kit for identifying cymbidium tigrinum, a detection method and application. The kit contains an upstream primer with a nucleotide sequence as shown in SEQ ID NO.1, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.2 and a restriction enzyme AgeI. The primer is adopted to carry out PCR amplification on sample DNA, and an amplification product is subjected to AgeI enzyme digestion and then is subjected to agarose gel electrophoresis. And the cymbidium tigrinum can obtain two bands of 436bp and 106bp, so that the distinguishing and identification of the cymbidium tigrinum and the similar species thereof can be conveniently and quickly realized according to the number and the size of the bands. The method has the advantages of visual result, high specificity, high identification efficiency, capability of avoiding false positive amplification and the like.

This application belongs to the field of biotechnology and provides the use of a cytokine-like protein of Bombyx mori as an active ingredient of a drug for inhibiting Bombyx mori nucleopolyhedrovirus. The amino acid sequence of the cytokine-like protein of Bombyx mori is shown as SEQ ID NO: 1, and this protein can inhibit the replication of Bombyx mori nucleopolyhedrovirus. In addition, this application also provides a cytokine-like protein of Bombyx mori, which is obtained by amplifying a cytokine-like plasmid of Bombyx mori with a PCR primer set to obtain a cytokine-like amplification product of Bombyx mori, then double-digesting the cytokine-like amplification product and the cytokine-like plasmid of Bombyx mori with restriction enzymes respectively, purifying the digested products and then ligating them with T4 ligase, transforming Escherichia coli DH5α competent cells, screening positive clones, and extracting plasmids.

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.

Molecular cloning method using Type IIS restriction enzymes The invention is addressed to a cloning method for obtaining a destination nucleic acid vector comprising "n" inserts, wherein "n" is an integer of at least 1, and each insert comprises a sequence of interest "S" and, wherein the method comprises contacting in a reaction mixture: (i) a receiving backbone, wherein: (a) one end of the receiving backbone comprises a palindromic overhang and the other end comprises a non-palindromic overhang, or (b) each end of the receiving backbone comprises a different palindromic overhang; (ii) the n inserts, wherein each insert comprises two different overhangs, wherein the overhangs comprised in the n inserts are different overhangs, and each one is complementary only to another overhang comprised in another insert or to an overhang of the receiving backbone, and is non-palindromic, except the insert's overhang(s) complementary to the palindromic overhang(s) of the receiving backbone, and (iii) a DNA ligase; wherein the molar ratio of the receiving backbone and the insert(s) comprising a palindromic overhang is from 1:0.1 to 1:1.9 (receiving backbone:insert), and the molar ratio of the receiving backbone and the remaining inserts is from 1:1.1 to 1:3 (receiving backbone:insert). In a particular embodiment, at least one of the n inserts and the receiving backbone are obtained upon digestion of one or more polynucleotides and a receiving vector with Type IIS restriction enzyme(s).

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 utility model discloses a kind of fermentation enzyme preparation production purification equipment, including storage bucket body, still including fixed installation in the feed inlet on the storage bucket body, the spiral groove being opened on the feed inlet, heating assembly being installed on the feed inlet, adjusting assembly and filter plate component being installed on the storage bucket body, first transmission component being installed on the adjusting assembly, current-limiting component being installed on transmission component, driving component being installed on the storage bucket body, second transmission component being installed on the driving component and push plate being installed on the driving component. By setting spiral groove, when enzyme liquid pours into feed inlet, enzyme liquid can be effectively limited to directly fall, avoid its direct drop into the inside of storage bucket body and cause splashing, this can greatly reduce the case that enzyme liquid splashes to the barrel wall of storage bucket body, barrel top, reduce enzyme liquid residual amount, both reduce raw material waste, and reduce subsequent equipment cleaning burden, save maintenance time.

The present invention discloses a preparation method and application of a soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein. The present invention belongs to the field of biomedical technology. The preparation method includes the following steps: cloning the coding sequences of the extracellular region of BAFF-R3 and the adiponectin globular domain; connecting the extracellular region of BAFF-R3 and the adiponectin globular domain through a peptide chain, and inserting the expression sequence of the eBR3-gAD fusion protein into a plasmid using restriction enzymes and DNA ligase; transfecting the above plasmid into cells and screening out a cell line with stable expression; culturing the above cell line, collecting the supernatant, and purifying by gel filtration and chromatography to obtain the soluble B lymphocyte stimulator receptor 3 and adiponectin globular domain fusion protein. The preparation method of this novel fusion protein has the advantages of high specificity, high expression level, and simple purification.