35 results about "Restriction site" patented technology

A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.

This invention belongs to the field of molecular biology detection, specifically relating to a rapid detection method for economic traits of Tibetan chickens. FBXO32 Gene SNP markers, detection methods, and applications. This invention is the first to discover: Tibetan chickens... FBXO32 The G / A mutation at position 8916 of the gene is closely related to the presence or absence of the Hpa II restriction site, and is also closely related to the main economic traits of Tibetan chickens. Based on this new discovery and the SNP site, this invention provides a method for rapidly detecting this SNP site and screening for economic traits in Tibetan chickens. The method is simple, convenient, fast, low-cost, and provides reliable results.

The invention provides a preparation method and application of a CBD-AL fusion recombinant protein, a variant Z region obtained by site-directed mutagenesis of a B region of SPA is connected in series with a B structural domain of a protein L, codon optimization is carried out, the front end of CBD is introduced into a BamHI restriction enzyme cutting site, a cysteine sequence, a termination codon TAA and an EcoRI restriction enzyme cutting site are added to the tail end of the B region of the recombinant protein L, and the CBD-AL fusion recombinant protein is obtained. BamHI and EcoRI are subjected to double enzyme digestion and then are connected to a PET28a vector subjected to double enzyme digestion, a recombinant protein CBD-AL gene expression vector of a structural domain of strongest binding IgG of protein A and protein L connected with CBD is constructed, the recombinant protein CBD-AL gene expression vector is transferred into an expression strain for amplification culture, an inducer is added for induction, bacterial sludge is collected, purification is performed by cellulose affinity chromatography, and the recombinant protein CBD-AL gene expression vector is obtained. A binding protein which has a cellulose binding function and can bind all types of immunoglobulins and subtypes thereof is prepared.

This invention relates to an identification method and kit for overlapping PCR with enzyme digestion. The method includes selecting a corresponding restriction endonuclease using N4N5X or N4N5Y as the last three bases of the introduced restriction site; if no corresponding restriction endonuclease is available, selecting a corresponding restriction endonuclease using N5X or N5Y as the last two bases of the introduced restriction site; amplifying to obtain a universal fragment primary PCR product; designing PCR amplification primers based on the restriction endonuclease and universal primers to obtain the primary PCR product of the template to be tested; and performing enzyme digestion and genotyping on the final product of the overlapping PCR amplification. This invention utilizes the DOPCR method to identify SNPs / InDels. Compared with existing methods that create restriction sites, it adds an overlapping PCR step, resulting in significant differences in the digested fragments. It eliminates the need for PAGE electrophoresis; detection can be completed using ordinary electrophoresis, making it low-cost and rapid.

A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.

The invention relates to a Pseudomonas sp. strain for use in the production of a recombinant protein characterised in that said strain comprises a nucleotide sequence encoding a phi15 RNA polymerase. The invention further relates to a plasmid, capable of integrating or replicating in Pseudomonas sp., comprising a phi15 promoter sequence operably linked to a nucleotide comprising one or more restriction sites for the insertion of a nucleotide sequence encoding a recombinant protein, or operably linked to a nucleotide sequence encoding a recombinant protein.

The invention discloses a virus-mediated plant high-flux mixed pool gene editing method which comprises the following steps: designing corresponding sgRNA according to a target gene, and adding a joint AAAC / TAGA connected with a carrier PaqC I restriction enzyme cutting site at two ends of the sgRNA; the method comprises the following steps: annealing designed sgRNA to synthesize double strands, and connecting the double strands to a carrier skeleton pGY345 subjected to PaqC I enzyme digestion through a T4 enzyme to construct a mixed library of sgRNA expression vectors; sgRNA expression vectors and TRV1 vectors in the mixed library are transformed into agrobacterium tumefaciens, a TRV virus vector mixed solution carrying different sgRNAs is constructed, and spraying inoculation is carried out; and observing the movement condition of the virus vector of the inoculated plant, and completing plant sgRNA typing and editing identification. By optimizing the OD value of the infection liquid and the inoculation method, the coverage degree of editing targets is remarkably improved, and multi-target mutant population creation can be carried out at the same time.

The invention discloses an efficient circular RNA (Ribonucleic Acid) preparation method and application thereof, and relates to the technical field of biology. The preparation method comprises the following steps: preparing a transcription vector of the circular RNA, and the transcription vector comprises the following structural elements: a promoter, a cyclization sequencer-3'intron / exon, a target sequence, a cyclization sequencer-5 'intron / exon and a linearization enzyme cutting site; the intron / exon sequence of the cyclization sequence 3'is selected from any one of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 7, and the intron / exon sequence of the cyclization sequence 5 'is selected from any one of SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 9. According to the preparation method of the circular RNA, provided by the invention, the cyclization proportion of the circular RNA is increased, and the preparation efficiency of the circular RNA is improved.

The invention discloses a urate oxidase gene derived from aspergillus cristatus and application thereof, and the urate oxidase gene is characterized in that a natural urate oxidase gene coding region sequence is obtained from the aspergillus cristatus, and the gene is optimized according to the codon preference of escherichia coli to obtain an optimized sequence. Connecting the optimized gene to a carrier through NdeI and BamHI restriction enzyme cutting sites, and constructing a wild type uox recombinant plasmid; the method comprises the following steps: replacing a sequence of uox of aspergillus cristatus for coding 243-site tyrosine (Y) with a sequence for coding lysine (K) through site-specific mutagenesis, constructing mutant Y243K recombinant plasmids, respectively transforming the recombinant plasmids into competent cells, culturing, and adding IPTG to induce target protein expression. And collecting the induced thalli, carrying out ultrasonication and centrifugation to collect the supernatant so as to obtain a crude enzyme, and purifying the crude enzyme through a nickel affinity column so as to obtain the purified UOX protein. Uric acid is used as a substrate to measure the enzyme activity of the purified UOX, and the result shows that the specific enzyme activity of the mutant Y243K is obviously higher than that of a wild type, and the activity is improved by 2.75 times, so that the mutant Y243K has higher application value.

This invention provides a research model and construction method for calcium ion imaging of ASH neurons in nematodes, belonging to the field of biological model construction technology. The construction method includes the following steps: nematode culture; construction of a transgenic probe plasmid: obtaining the ASH neuron-specific promoter sra-6 using PCR technology, and connecting HindIII and BamHI restriction sites to both ends of the promoter; amplifying YC3.60 using PCR technology; then inserting the promoter sequence into the pPD95.75 plasmid, and then inserting YC3.60 after the promoter. Transgenic microinjection of nematodes is used, followed by screening to obtain nematodes with ASH neurons carrying green fluorescent protein. In this application, YC3.60 is transferred into nematodes via transgenic means. Using a promoter specifically expressed in ASH neurons to link the gene of this fluorescent probe protein into the nematode, the fluorescent probe can be specifically expressed in the neuron. The constructed nematode model can be used for neuronal structure and functional imaging studies, as well as neuropharmacological efficacy analysis.

The invention discloses a duck plague virus UL7 gene deleted strain DPV-UL7null as well as a construction method and application thereof. The method comprises the following steps: (1) by taking a pEP-Kan-S plasmid as a template, amplifying a Kan resistance gene segment with homologous sequences at two ends of a UL7 gene and an I-SceI restriction enzyme cutting site by utilizing a primer; (2) electrically transforming the target fragment of the UL7 gene to a competent state; after the electroporation is finished, culturing and identifying bacterial colonies; (3) knocking out the Kan gene through homologous recombination, so as to obtain the correct infectious clone pDPV-UL7null of the duck plague virus UL7 gene deleted strain; and (4) carrying out virus rescue to obtain the UL7 gene deletion virus. According to the duck plague virus UL7 gene deleted strain DPV-UL7null disclosed by the invention, partial UL7 gene is deleted, so that the amino acid sequence of UL7 protein is not expressed, the DPV virulence is obviously reduced, the pathogenicity to ducks is reduced, and the multiplication capacity in duck bodies is weakened.

The invention relates to the technical field of viruses, in particular to a construction method and application of a porcine parvovirus full-length infectious clone carrying a stable genetic marker. The method comprises the following steps: cloning a complete genome of a porcine parvovirus SXND18 strain to a plasmid pBluescript II SK (+) through an In-fusion seamless cloning technology, so as to obtain a recombinant plasmid pSXND18; meanwhile, a new EcoRI restriction enzyme cutting site is introduced into a genome by utilizing an overlapping PCR (Polymerase Chain Reaction) technology and is used as a genetic marker for identifying a wild virus and a rescue virus; and mixing the recombinant plasmid with a transfection reagent, and transfecting ST cells to obtain the rescue virus rSXND18. The porcine parvovirus reverse genetic operating system established by the invention can be used for research in the directions of porcine parvovirus virulence analysis, cross-species transmission mechanism and the like, and also lays a foundation for research on novel vaccines of the porcine parvovirus.

The invention provides an engineering bacterium of a recombinant soluble TRAIL protein, an expression and purification method and application thereof, and belongs to the technical field of gene engineering. The method comprises the following steps: inserting a TRAIL gene between Nde I and Xho I restriction enzyme cutting sites of a pET-32a (+) vector to construct a recombinant plasmid; and transferring the recombinant plasmid into escherichia coli BL21 (DE3) to obtain an engineering bacterium for expressing the recombinant soluble TRAIL protein. The invention further provides an expression and purification method of the recombinant soluble TRAIL protein based on the engineering bacterium, the yield of the recombinant soluble TRAIL protein is improved by optimizing induction conditions, the recombinant soluble TRAIL protein with high purity and high biological activity is obtained by optimizing purification conditions, and support is provided for clinical application of the recombinant soluble TRAIL protein.

The invention provides construction and application of a medical intermediate P29 eukaryotic expression engineering bacterium, and belongs to the technical field of biosynthesis, the engineering bacterium takes pichia pastoris as a chassis cell and contains a recombinant plasmid for expressing a fusion protein, and a gene sequence of the fusion protein contains a protection sequence-enzyme cutting site-P29 sequence structure. Wherein the protection sequence is derived from collagen and is specifically shown as SEQ ID NO. 2. According to the invention, a medical intermediate P29 eukaryotic expression engineering bacterium is successfully constructed by using pichia pastoris, and the engineering bacterium can realize efficient and low-cost production of the intermediate P29, and provides a substrate and a raw material for chemical synthesis of the semeglutide.

A recombinant polynucleotide suitable for use in a display vector is provided. The recombinant polynucleotide includes from 5′ to 3′: a first nucleic acid sequence (or insert) encoding an amino acid sequence to be displayed on a surface; a first pre-selected restriction site; a second nucleic acid sequence encoding a surface peptide capable of being displayed on the surface; and a second pre-selected restriction site. Corresponding display vectors that can be converted into expression vectors in a high-throughput fashion, as well as methods of use thereof, are also provided.

A target polynucleotide is expanded. In respect of each nucleotide in the target polynucleotide, the target polynucleotide comprises clock nucleotides and at least one signal nucleotide in a predetermined order. The clock nucleotides have a predetermined sequence common to each nucleotide in the target polynucleotide. The at least one signal nucleotide is characteristic of the identity of the respective nucleotide in the target polynucleotide. During translocation of the expanded polynucleotide through a nanopore, electrical measurements dependent on the polynucleotide within the pore are made, to derive an analysis signal. Clock signals derived from the clock nucleotides are identified. Relative to the positions of the identified clock signals, nucleotide signals derived from the least one signal nucleotide are derived to analyse the target polynucleotide. The predetermined sequence of the clock nucleotides comprises a restriction site for a restriction enzyme and at least one further nucleotide that extends the predetermined sequence.

A recombinant polynucleotide suitable for use in a display vector is provided. The recombinant polynucleotide includes from 5′ to 3′: a first nucleic acid sequence (or insert) encoding an amino acid sequence to be displayed on a surface; a first pre-selected restriction site; a second nucleic acid sequence encoding a surface peptide capable of being displayed on the surface; and a second pre-selected restriction site. Corresponding display vectors that can be converted into expression vectors in a high-throughput fashion, as well as methods of use thereof, are also provided.

A system for the analysis of MTHFR gene polymorphisms (C677T & A1298C) in glucose intolerance during pregnancy, consisting of: a) a sampling unit configured for taking peripheral blood samples from pregnant women; b) a sample storage unit configured to store blood samples in EDTA tubes at 4 °C; c) a DNA extraction unit configured to isolate genomic DNA from blood samples using the Qiagen kit and to store isolated samples at -20 °C; d) a PCR amplification unit configured to amplify the MTHFR gene regions C677T and A1298C using specific primer sequences in a reaction volume of 15 µl; e) a restriction digestion unit configured to digest amplified PCR products with the restriction enzymes HInfi and Mbo II by RFLP for 16 hours at 37 °C; f) an electrophoresis analysis unit configured to analyze digested RFLP products by polyacrylamide gel electrophoresis (PAGE) to identify restriction sites in amplified DNA products; and g) a data analysis unit configured to calculate genotype and allele frequencies using the Hardy-Weinberg equilibrium and the chi-square test.

The invention develops a colorimetric detection method for salmonella based on DNAWalker driving double signal amplification. The detection principle is as follows: target DNA triggers AP hybridization to form Y-type connection with CP, an Nt.BbvCI restriction enzyme cutting site is exposed, DNAWalker is released through restriction enzyme cutting circulation, and target circulation amplification is realized. Meanwhile, PDANS (palladium-iron-nano particles) is synthesized through Fe < 2 + > / H2O2 Fenton reaction, a pH indicator is loaded through silver nano particle modification (PDANS (at) Ag), and a signal is enhanced. An amplified product is combined with PDANS (at) Ag through a magnetic bead sandwich structure, remarkable color change is caused, and quantitative detection can be performed through a smart phone App (such as' Color Grab ') or a microwell plate reader. The method is easy and convenient to operate, high in sensitivity and suitable for on-site rapid identification of salmonella.

According to the molecular marker method for rapidly identifying the southern and northern kelp culture population, the nucleotide sequence of the specific molecular marker of the northern strain is SEQ ID NO: 1, and the nucleotide sequence of the specific molecular marker of the southern strain is SEQ ID NO: 2. Compared with amplified fragment length polymorphism, restriction site amplification polymorphism, microsatellite DNA marking and the like, the method is more convenient, only simple PCR amplification needs to be carried out, distinguishing can be carried out according to sequencing data, and tedious experiment steps are omitted. The molecular marker provided by the invention can be quickly identified only through simple PCR amplification and next-generation sequencing, so that the detection cost is relatively low. The development of the specific molecular marker not only can avoid seedling mixing in production, but also can lay a foundation for evaluation and identification of subsequent new strains.

The invention relates to a production method and application of a heat-resistant broad-spectrum pH type recombinant polysaccharide cleavage monooxygenase rLPMO9 preparation based on aspergillus niger, which can effectively solve the production problem of the heat-resistant broad-spectrum pH type recombinant polysaccharide cleavage monooxygenase rLPMO9 preparation and realize the application in degrading lignocellulose materials. An amino acid sequence is encoded by an LPMO9 gene of an aspergillus niger HKS11 strain and consists of 412 amino acid residues, a catalytic activity center of the enzyme consists of His21, His103, Tyr183 and Cu < 2 + >, the gene for encoding the enzyme is cloned between EcoRI and NotI restriction enzyme cutting sites of an expression vector pPIC9K, so that a target gene sequence is connected with the expression vector pPIC9K, and an expression recombinant plasmid pPIC9K-LPMO9 is obtained; and after PmeI linearization treatment, transforming to a Pichia pastoris GS115 host cell by an electroporation method to obtain a recombinant yeast engineering strain, and fermenting and centrifuging to obtain the recombinant yeast engineering strain. The method is simple and easy to operate, the enzymolysis cost is reduced, and technical support is provided for efficient and energy-saving conversion of lignocellulose.

The present application relates to the technical field of aquaculture fish vaccine, in particular to a grass carp NPM1a and type II reovirus VP38 fusion gene, recombinant protein, composition and application. The nucleotide sequence of the fusion gene is shown as SEQ ID NO: 1. The fusion gene is obtained by connecting grass carp NPM1a and type II reovirus VP38 through a linker sequence, a BamH restriction site is added at the 5' end of the fusion sequence, and a XhoI restriction site is added at the 3' end. The linker sequence is shown as SEQ ID NO: 2. By preparing a subunit vaccine of grass carp NPM1a and type II reovirus VP38 fusion protein, and jointly using indole-3-lactic acid as an immune enhancer, the technical bottlenecks of low protection rate of existing vaccines and poor intestinal mucosal barrier repair are overcome.

The invention provides a fusion protein which is characterized in that the fusion protein comprises the following elements from the 5'end to the 3 'end: 5'-MBP-Furin restriction enzyme cutting site-rhIL-7-3 ', and a method for in-vitro expression and purification of recombinant human interleukin 7 in mammalian cells, and the method comprises the following steps: a, providing a first expression vector, the first expression vector comprises nucleic acid for coding the fusion protein; b, providing a second expression vector, wherein the second expression vector comprises nucleic acid for coding furin; c, co-transfecting the first expression vector and the second expression vector to mammalian cells, and performing fermentation culture to enable the cells to express recombinant human IL-7 protein; d, centrifuging to obtain fermentation supernate from the step c; and e, purifying by using POROS XS and CHT chromatographic methods to obtain the rhIL-7 fragment.

The present invention relates to a method and system for assembling DNA. In particular, it allows for efficient and ordered assembly of a target nucleic acid from a number of extended oligonucleotides that contain repeating units of Type IIB restriction enzyme restriction sites and fragment sequences of interest. The method uses Type IIB enzymes to digest the extended oligonucleotides in a manner which produces units of the sequences of interest with single stranded overhangs, referred to as sticky ends, that can then anneal and be ligated together in an order determined by the sticky ends to give the target sequence.

The invention relates to the technical field of plant genetic transformation, and provides a plant genetic transformation system based on direct delivery, a genetic transformation method and application. An original vector pMKV057 is directionally modified to obtain a modified vector, and the modification method comprises the following steps: replacing an LUC expression cassette of the vector with a modular expression cassette integrated with an epitope tag; the method comprises the following steps: designing multiple cloning sites on a vector, introducing restriction enzyme cutting sites HindIII and EcoRI or HindIII and XbaI, and inserting a Kozak sequence into a 5'end upstream adjacent region of a nucleotide sequence of the multiple cloning sites to obtain a modified vector. And transforming the modified vector into agrobacterium tumefaciens to carry out plant genetic transformation. Through the technical scheme, the problems of low plant genetic transformation efficiency and long transformation time in related technologies are solved.

The invention provides a T vector plasmid fused with a Lacz gene and suitable for blue-white spot screening and preparation of the T vector plasmid, and belongs to the field of cloning vectors, and a preparation method of the T vector plasmid comprises the steps that a plasmid containing the Lacz gene serves as a starting plasmid, a gene segment with two XcmI restriction enzyme cutting sites is fused into the Lacz gene, and then a target plasmid is obtained. The T vector plasmid provided by the invention is extremely simple and convenient to use, when the plasmid is applied to T vector clone construction, only white colonies need to be selected for PCR verification, a large number of positive clones can be screened, the preparation cost of a single transformation vector can be reduced to 0.15 yuan per time and is far lower than that of an existing commercial T vector, and the T vector has extremely high commercial value.

Cloning vectors and methods useful for building multigenic and / or polycistronic constructs for delivery to cells and / or organisms are disclosed. The vectors are genetically engineered to comprise unique acceptor domains that independently enable the sequential or iterative addition of compliant DNA and / or RNA inserts. Insertion of a first nucleotide insert into an acceptor r domain destroys a complementary restriction site a 3'end of the first insert segment while the 3' restriction site is regenerated at the insertion point of the cloning vector to form a circular vector with a first insert. A subsequent cleavage at a 3' end of the first insert creates an insertion point for a second insert segment, and the iterative assembly of additional insert segments proceeds with each subsequent cleavage at a 3' end of a growing linear chain of a desired number of inserts.

The present invention is related to a nucleic acid molecule, which is also referred to as third nucleic acid molecule, wherein the third nucleic acid molecule comprises(1) a nucleic acid molecule comprising the following elements:(a) optionally, a first part of a genome of a virus;(b) a nucleotide sequence, preferably a genomic nucleotide sequence, or a transcription unit;(c) a regulatory nucleic acid sequence which has a regulatory activity in a prokaryote;(d) exactly one site-specific recombination site;(e) a nucleotide sequence providing for a negative selection marker;(f) a bacterial nucleotide sequence unit comprising (i) bacterial nucleotide sequences for conditional replication and (ii) a nucleotide sequence providing for a positive selection marker;(g) optionally a first restriction site; or(2) a nucleic acid molecule comprising a nucleotide sequence according to SEQ ID NO: 6; or(3) a nucleic acid molecule identical or similar to the nucleic acid molecule contained in the organism deposited with the DSMZ under the Budapest treaty under accession number DSM 23754, wherein preferably the nucleic acid molecule contained in the organism is a heterologous nucleic acid molecule;wherein the third nucleic acid molecule is either a linear or a circular molecule.

The present application relates to a method for analyzing the similarity of at least two samples of a plurality of samples comprising genomic DNA. The method comprises the following steps: a) providing a plurality of samples comprising genomic DNA; b) performing a deterministic restriction site whole genome amplification (DRS-WGA) of the genomic DNA separately for each sample; c) preparing a massively parallel sequencing library from each product of DRS-WGA using a no-fragmentation, sequencing adapter / WGA fusion primer PCR reaction; d) performing low-pass whole genome sequencing of the massively parallel sequencing library at an average coverage depth of less than 1x; e) aligning the reads of each sample obtained in step d) to a reference genome; f) extracting the allele content at a plurality of polymorphic loci for each sample; g) calculating a pairwise similarity score locus for at least two samples based on the measured allele content at the plurality of loci; h) determining the similarity of at least two samples based on the similarity score, the method being used for non-invasive prenatal testing or diagnosis.

The invention belongs to the technical field of protein purification, and particularly relates to a carrier for protein expression and a protein preparation method. The construction mode of the vector is as follows: a sequence for coding a 6His-Twin StrepII-MBP tag, a sequence for coding a TEV restriction enzyme cutting site, a sequence for coding a linker and a sequence for coding a target protein are sequentially inserted between polynucleotide restriction enzyme cutting sites of a vector plasmid according to a sequence. Efficient purification of the target protein can be realized by utilizing the carrier and a series of column chromatography separation (including a nickel column, an MBP column and a Strep II column). The preparation method of the protein has the advantages of easiness in purification of the protein, wide application range, easiness in mass production and the like, and has a very good application prospect.