187 results about "Large fragment" patented technology

The invention provides a method for establishing a humanized rat drug evaluation animal model. According to the method, a multidrug resistance gene 1 (Abcb1)-knocked-out genetically engineered rat is obtained through a microinjection method by virtue of a CRISPR / Cas9 gene knockout technology and 153kb bacterial artificial chromosome (BAC) fragments containing a humanized Abcb1 promoter and cDNA is simultaneously inoculated into the rat genome through the microinjection method by virtue of a large fragment transgenic technology to obtain a transgenic rat capable of stably expressing human Abcb1 and the genetically engineered rat and the transgenic rat are hybridized to establish the humanized rat drug evaluation animal model. RT-PCR analysis shows that Abcb1 expression profiles of humanized Abcb1 rat are significantly different from those of the rat endogenous Abcb1. The method has the beneficial effects that the humanized rat capable of expressing human Abcb1 is obtained and the rat is used for expressing human Abcb1 genes and has closer expression profiles to those of human so that the model can be well used for the efficacy evaluation of newly developed drugs.

The invention relates to a cell line gene knock-out method for obtaining large fragment deletion through a CRISPR / Cas9 system, and belongs to the field of genetic engineering and genetic modification. A pX458 vector is modified, so that the pX458 vector is provided with DsRed2 and ECFP (Enhanced Cyan Fluorescence Protein); then, a plurality of specific sgRNA sites are designed by aiming at a target gene and are connected into the modified vector; after the cell line transfection, cell groups are sorted by a flow cytometry; single cells subjected to genome editing can be very fast obtained; then, a single-cell DNA (Deoxyribonucleic Acid) sequence is subjected to PCR (Polymerase Chain Reaction) amplification; and single cells with large fragment deletion can be picked out from the single cells through gel electrophoresis. The CRISPR / Cas9 system, the flow cytometry single-cell sorting and fluorescent protein screening on the expression vector are combined, so that the positive monoclonal cells with the large fragment deletion can be obtained in a short time; and the work efficiency of the cell line gene knock-out is greatly improved.

The invention belongs to the technical field of plant genetic engineering and specifically relates to a method for directionally knocking out miRNA (micro Ribonucleic Acid) of paddy rice. Aiming at solving the technical problems, a method and a detection means are provided for directionally modifying miRNA sites of plants. The invention discloses a method for obtaining miRNA mutants of the paddy rice; specific operation of the method comprises: knocking out single miRNA, knocking out a plurality of miRNAs or knocking out large fragments of the miRNA by adopting a CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats- associated 9) method. The method provided by the invention is suitable for creation and screening of directionally knocking out the miRNA of the paddy rice.

The invention discloses a preparing method for MC3R gene knockout pigs. The preparing method for MC3R gene knockout pigs includes the steps that a CRISPR / Cas9 system is adopted, the CRISPR / Cas9 system comprises an encoding gene of gRNA1 and an encoding gene of gRNA2, and the target sequence of a MC3R gene identified by the gRNA1 is a sequence 1 in a sequence table; the target sequence of the MC3R gene identified by the gRNA2 is a sequence 2 in a sequence table. An experiment shows that the MC3R gene knockout efficiency is 29.16% with the preparing method for MC3R gene knockout pigs; a target gene can be rapidly and efficiently knocked out in a large-fragment mode, exogenous gene fragments are not left, and the preparing method can be used for researching the action mode and mechanism of the MC3R gene, and can be further used for animal breeding.

The invention relates to a preparation method of an SD rat T cell-deficient genetic model, and belongs to the technical fields of genetic engineering and genetic modification. Specific knockout on a key gene Lck, which is in charge of controlling development of rat T cells, is achieved in a rat by virtue of a CRISPR-Cas9 system, so that the SD rat T cell-deficient genetic model is obtained. According to the preparation method provided by the invention, two specific targeting sites, which are targeted to the rat Lck gene, are determined for the first time, and experiments prove that the specific targeting sites are quite high in shear efficiency; two sgRNAs are simultaneously adopted to conduct targeting on the rat Lck gene, and the large-fragment deficient gene knockout rat is obtained, so that in one aspect, the targeted gent completely loses functions, and in the other aspect, subsequent detection is also facilitated. The constructed T cell-deficient SD rat animal model is great significance to immunity and disease researches.

The functional analysis of genes frequently requires the manipulation of large genomic regions. A yeast-bacteria shuttle vector is described, that can be used to clone large regions of DNA by homologous recombination. The important feature of present invention is the presence of the a bacterial replication origin, which allows large DNA insert capacity. The utility of this vector lies in its ability to isolate, manipulate and maintain large fragments in bacteria and yeast, allowing for mutagenesis by yeast genetics and simplified preparation of plasmid DNA in bacteria.

The invention discloses a method for obtaining label-free transgenic plant. The method is implemented by a CRISPR / Cas9 or CRISPR / Cpf1 system, and a vector of the CRISPR / Cas9 or CRISPR / Cpf1 system comprises a gene edited protein gene, a sgRNA of targeting the vector and a specific temporal and spatial expression gene promoter. The specific temporal and spatial expression gene promoter is not expressed during callus period, and the gene edited protein gene is expressed after genetic transformation is completed. The method provided by the invention has simple operation, and mainly aims at cropsor economic plants, especially plants that are perennial and vegetative propagated; large fragments of transgenic components are lost by splicing the vector, and the significance is great.

The invention discloses a chip and method for capturing target sequences of tumor susceptibility genes, and a mutation detection method. The chip is a liquid chip which binds with probe groups capable of simultaneously capturing at least 5, preferably at least at least 10, preferably at least at least 20, preferably at least at least 30, preferably at least at least 50, preferably at least at least 80, preferably at least at least 100, preferably at least at least a10, or preferably all of 115 genetic tumor susceptibility genes as shown in a table 1. The mutation detection method comprises a step of capturing the target sequences of the genetic tumor susceptibility genes by using the liquid chip and a step of carrying out sequencing by using G2 high-flux sequencing technology so as to find out mutation sites. The mutation detection method has the advantages of a wide application scope, high efficiency, comprehensiveness and easy operation, can detect replacement of a single base, insertion or deletion of a single base / multiple bases and deletion / amplification of large fragments and is capable of realizing high-efficiency comprehensive detection of common tumor susceptibility gene mutation.

Improved liquid chromatography systems having components made of titanium, stainless steel, or organic polymeric material are useful in the separation of polynucleotide fragments, particularly large fragments of double-stranded polynucleotides, by Matched Ion Polynucleotide Chromatography (MIPC). The titanium, stainless steel, or polymeric components are treated so that they do not release multivalent cations into aqueous solutions flowing through the chromatography system. Alternatively, or in addition to utilizing materials made of the components listed above, a multivalent cation capture resin placed upstream of the separation column can be employed to remove multivalent ions from the system. The multivalent cation capture resin can be contained in a guard disk, a guard column, or a guard cartridge. Novel methods for separating mixtures of polynucleotide fragments into fractions based on their molecular weight by Matched Ion Polynucleotide Chromatography and slalom chromatography utilize the liquid chromatographic systems described above.

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.

A fragmentation warhead comprising manual selection means for generating larger fragments versus smaller fragments upon detonation. The warhead includes a generally cylindrically shaped fragmenting metal outer warhead within which lies a generally cylindrically shaped explosive charge. Cylindrically arranged ring mechanisms within the warhead may be rotated to select desired fragmentation patterns.

An engineered nitrile hydratase-producing bacterium and its construction method as well as its applications, wherein the engineered nitrile hydratase-producing bacterium is a mutant strain of an original nitrile hydratase-producing bacterium strain obtained by knocking-out or inhibiting the amidase gene in the original strain. The construction method of the engineered bacterium is to block the expression of the amidase gene by inserting the large fragment of a recombinant suicide plasmid carrying an amidase gene fragment into a wild-type strain through the homologous recombination between the recombinant suicide plasmid and the amidase gene of the wild-type strain. Compared to the corresponding wild-type bacterium strain, both the cell growth and the nitrile hydratase expression of the engineered nitrile hydratase-producing bacterium according to the invention are increased. In the process of catalyzing the hydration of acrylonitrile to produce acrylamide, the yield of the product, acrylamide, is significantly increased, while the yield of the by-product acrylic acid is significantly decreased. The engineered nitrile hydratase-producing bacterium of the present invention has wide application prospect in the production of acrylamide by microbiological process.

The invention discloses a rapid assembling method of multi-fragment DNA yeast. The rapid assembling method comprises the following steps: (1) performing co-transformation on a plurality of DNA molecules with homologous arms, and linearized yeast shuttle vectors, so as to obtain yeast; (2) eluting all the transformed yeast colonies on a whole screening culture plate, centrifuging, and discarding the supernate to obtain transformed yeast cells; (3) extracting plasmid DNA of the yeast cells obtained in step (2), transforming the competent cells of colon bacillus; and (4) screening colon bacillus, and cloning to obtain large-fragment DNA. The method for assembling large-fragment DNA molecules is fast in speed, simple, convenient and feasible, high in success rate, low in cost, high in efficiency, easy to operate, beneficial in enlarging the industrialization scale and wide in application, and a plurality of small-fragment DNA molecules can be assembled into one large-fragment DNA molecule.

The present invention provides a method for detecting genomic structure variation based on nanopore sequencing, which includes performing nanopore sequencing data quality control and genomic mapping;excavating within-alignment reads and split reads information; defining structural variation and typing; and performing multi-sample data variation type integration. The method of the invention can detect the variation of mutants and wild-type materials, the variation between extremely resistant materials, transgenic events and the location of inserted fragments; can also organically integrate theresults of a large number of samples and assist in the correction of variation sites; provide rich structural variation data for natural resource groups for subsequent GWAS analysis; not only can detect common large fragment structural variations, but also have high accuracy and precision in the detection of chimeric mutations, and clusters the reads of the typed structural mutations to determinehomozygous and heterozygous structural variations.

The invention relates to the DNA synthesis field, and concretely relates to large fragment assembly of DNA and especially a carrier used for DNA fragment assembly. The invention provides a Gibson assembly carrier, a preparation method therefor and applications thereof.

The invention discloses a kiwi fruit InDel molecular marker and a screening method and application thereof. Two different cold-proof Actinidia arguta genome DNAs are used as research subjects, resequencing of four variety genes is carried out, InDel primers are designed according to resequencing data, and PCR (polymerase chain reaction) amplification, agarose electrophoresis and non-denaturing polyacrylamide gel electrophoresis are carried out; polymorphism detection is carried in Actinidia arguta to screen out 14 pairs of InDel primers, the 14 pairs of InDel primers are applicable to the genetic diversity analysis for Actinidia chinensis, Actinidia deliciosa and the like and are also applicable to the researches, such as hybrid offspring authenticity identification, genetic map construction, and molecule-assisted breeding. The kiwi fruit InDel primers according to the embodiment of the invention has good mutation stability and low detection difficulty, allow InDel insertion / loss of large fragments, and allows agarose analysis, with steps that may be simplified.

The dynamically configurable controlled fragmentation insert mechanism of this invention includes an assembly of three or more sleeves with differing through hole patterns thereon, that fit inside the shell casing. The individual sleeves can move independently of one another and a simple pinning mechanism holds the parts in place for a selected configuration. The warfighter can realign the insert sleeves by to create different geometric patterns of holes, each designed to engage a different target set with optimally sized fragments. The aligned patterns of holes creates individual geometric shapes that focus high-velocity jets to cut into the steel shell casing to correlate to the through-holes in the aligned patterned sleeves. Realigning the insert sleeves changes the through-hole pattern to produce different fragment sizes and mass distributions. To defeat light armored vehicles for instance, a warfighter can deploy a sleeve hole pattern to produce larger fragments with greater penetrating power, while to engage enemy troops for instance, a warfighter can “dial in” another hole pattern through the fuze assembly to otherwise produce a much larger number of smaller, lighter fragments.

The invention discloses an optimized enterotoxigenic escherichia coli (ETEC)-producing polyvalent antigen gene sequence and the application thereof in preventing weaned piglet diarrhea, and belongs to the technical field of biology. The polyvalent antigen gene sequence is synthesized by a large fragment according to the preference of the lactococcus lactis codon, and the synthesized polyvalent antigen gene sequence contains six antigen genes, namely common ETEC dominant serotype F4<+> primary structure protein FaeG causing the weaned piglet diarrhea, the receptor binding domain RBD of F18<+>, and toxins Stx2e and STa mutant, LTB subunit and STb, and molecular peptides CO1 and YadA31 genes targeting the M cells and the intestinal cells, respectively; the genes are connected by GGGGS. The genes are applicable to constructing a lactic acid bacterium living-vector vaccine and remarkably improving the secretory expression quantity of the target protein, and have excellent immunogenicity and protection effect; the genes also are suitable for high-efficiency expression in the escherichia coli; experiments prove that the inclusion body of the genes has excellent immunocompetence and can be taken as the vaccine for preventing the weaned piglets from F4<+> and F18<+> ETEC infection.

The invention discloses a porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of a cell and attenuation via drug screening and an application thereof. The attenuated vaccine strain is prepared through the following steps: based on a porcine pseudorabies virus variant strain (named as strain HeN1, of which the microbial preservation serial No. is CGMCC No. 6656), firstly, carrying out low-temperature passage and screening on a Vero cell to obtain large fragments of deleted viruses including gI, gE, Us9, Us2 and part of inverted repeated sequence which exist in zone US through, and then making the TK gene thereof partially deleted through drug screening. The gene-deleted attenuated vaccine strain is named as strain PRV TP, of which the microbial preservation serial No. is CGMCC No. 12300. A live vaccine or an inactivated vaccine (a single vaccine or combined vaccine) can be prepared from the attenuated vaccine strain disclosed by the invention, and can prevent porcine pseudorabies effectively, and a reagent for diagnosing or treating porcine pseudorabies can be prepared from the attenuated vaccine strain too. According to the porcine pseudorabies virus gene deletion attenuated vaccine strain for passage via low temperature of a cell and attenuation via drug screening and the application thereof, the porcine pseudorabies attenuated vaccine strain PRV TP has the advantages of good safety, efficient protection, convenient differential diagnosis and the like.

The invention provides a method for synthesizing DNA fragments and assembling synthetic genes in Escherichia coli through a one-step method. The method mainly comprises the following steps: constructing LIC vector plasmid LIC-A; preparing a LIC-A linear vector; constructing a receptor vector puc-Kana for assembling the DNA fragments; selecting target genes, and dividing small fragments of about 300bp for synthesizing; and assembling multiple small-fragment DNA into large-fragment genes by utilizing a Jinmen cloning reaction. The method is simple in operation, the operations of cloning and sequencing are not needed to be repeatedly performed, direct annealing is realized, the cloning efficiency is high, luminous report genes such as sfgfp and gfp are used, the screening method is intuitive, the error rate of the synthesis method is extremely low, the period of the synthetic genes is short, the cost is greatly saved, and the time is saved. Moreover, the method is suitable for synthesizing various genes and is particularly effective for large-gene synthesis.

The invention discloses a data storage method and system, electronic equipment and a computer readable storage medium. The method comprises the steps of acquiring parameter values used for representing the stored data volume of a current storage system; determining the size of a target fragment positively related to the parameter value; determining the number of target fragments based on the sizeof the to-be-written data and the size of the target fragments; and creating a target fragment in the target disk according to the target fragment size and the target fragment number, and storing theto-be-written data by using the target fragment. The size of the target fragment positively related to the current storage system is determined based on the parameter value representing the stored data size of the current storage system, it is guaranteed that when the stored data size is large, the large fragment size is determined, the fragment number is relatively reduced, the expenditure of metadata operation is effectively reduced, and the performance of the system is improved; and when the stored data volume is small, the relatively small fragment size is determined, so that the large data management granularity caused by the large fragment is avoided, and the flexibility of the system and data management is improved.

A method of updating a clone data map associated with a plurality of nodes of a computer system is disclosed. The clone data map includes node identification data and clone location data. A node failure event of a failed node of the computer system that supports a primary clone is detected. The clone data map is updated such that a secondary clone stored at a node other than the failed node is marked as a new primary clone. In addition, clone data maps may be used to perform node load balancing by placing a substantially similar number of primary clones on each node of a node cluster or may be used to increase or decrease a number of nodes of the node cluster. Further, data fragments that have a heavy usage or a large fragment size may be reduced in size by performing one or more data fragment split operations.

The invention discloses an LAMP (loop-mediated isothermal amplification) kit for rapid detection of Shigella. The kit is composed of an LAMP reaction solution, a standard positive template, and a negative quality control standard. The LAMP reaction solution contains a Bst DNA polymerase large fragment, primers, an LAMP10*buffer, a dNTPs solution, an MgSO4 solution and betaine. The primers include forward and reverse primers. The kit disclosed in the invention has the advantages of good specificity, high sensitivity, rapidness and convenience, high repeatability, and judgeable results obtained by naked eyes, etc., can perform rapid qualitative detection on Shigella in industrial food, and can be used to replace the continuously used traditional culture method and the serological diagnostic method.

The invention belongs to the fields of soil microorganisms, biochemistry and molecular biology and relates to a method for separation and purification of a large-fragment DNA from soil. The method is used for indirect separation and purification of a large-fragment DNA having molecular weight more than 30kb from various types of soil, wherein the large-fragment DNA is used for construction of a metagenomic library, or is used for separation of soil microbial gene clusters. The method solves the problem that separation of microbial cells from soil and preparation of large-fragment soil DNA having high purity and satisfying various biological study demands are realized difficultly by the existing indirect method, and is an efficient method for extraction of soil microorganism DNA having a large fragment and high purity.

The invention discloses an LAMP (loop-mediated isothermal amplification) kit for rapid detection of staphylococcus aureus. The LAMP kit for rapid detection of staphylococcus aureus comprises LAMP reaction fluid, a standard positive template and a negative quality control standard. The LAMP reaction fluid contains large fragments in Bst DNA (deoxyribonucleic acid) polymerase, primers, LAMP 10Xbuffer, dNTPs (deoxyribonucleotide triphosphates) solution, MgSO4 solution and betaine. The primers include forward primers and reverse primers. The LAMP kit for rapid detection of staphylococcus aureus has the advantages that the LAMP kit is high in specificity, high in sensitivity, high in speed, simple and high in reliability, results are recognizable to naked eyes, and the like. The LAMP kit is applicable to rapid qualitative detection of staphylococcus aureus in industrial foods, and can be a substitute for commonly used traditional culture methods and serodiagnosis methods.

The invention discloses a new method for gene injection for a somatic cell nuclear transfer reconstructed embryo. The method comprises the following steps: target gene preparation, somatic cell nuclear transfer and a microinjection technology. An expression vector is constructed after a target gene is obtained, the cell nucleus of an oocyte is removed by virtue of micromanipulation, then a somatic cell is injected in the space of the denucleated oocyte, then an exogenous gene is directly injected in the somatic cell, the reconstructed embryo is constructed through one-step electric fusion and activation, and the embryo is transferred to obtain a transgenic progeny. According to the method disclosed by the invention, large-fragment cell gene transfection and screening are avoided, the transgenosis efficiency is increased, the time is saved, and the transgenosis application range is expanded. Importantly, the somatic cell is injected in a transpanent zone during a nuclear transfer operation process, thus microinjection for the exogenous target gene is facilitated.

The application discloses a building method of DNA large-fragment library for Pacbio platform and an assay kit. The building method includes subjecting broken DNA large fragments to VII enzyme treatment, sequentially carrying out primary DNA damage repairing and terminal repairing treatment, linker splicing treatment, exonuclease ExoIII and ExoVII double-digest treatment, subjecting products obtained by double-digest treatment to fragment separation through nucleic acid electrophoresis and a fragment recovery system, subjecting products obtained by the fragment separation to secondary DNA damage repairing, so as to obtain the DNA large-fragment library adaptable to the Pacbio platform. According to the building method of the DNA large-fragment library, the whole building process is carriedout without Pacbio library-building assay kit, library building cost is lowered greatly, and a material foundation is laid for promotion and application of the third-generation sequencing and Pacbionucleic acid sequencing platform.

The invention discloses an equine infectious anemia virus (EIAV) gene transfer vector, a constructed method and an application thereof. The vector of the invention comprises a CMV / R / U5 promoter, a 5' non-translated region homing sequence, a partial 5' gag gene coding region sequence, a central polypurine sequence, an Rev reaction element of EIAV, a CMVIE / chicken beta-actin promoter, a reporter gene, WPRE of WHBV, a polyclone locus, a poly (A) signal before EIAV 3'LTR, complete 3'LTR and partial sequence of eukaryon expression vector. The vector of the invention possesses common advantages of lentiviral vector, lacks auxiliary genes of all the wild-type virus, can carry one or a plurality of selected genes to transmit to target cells, can insert with target DNA sequence with large fragments and can lead the foreign genes and reporter genes to express at a high level in the cells.

The invention relates to a double-plasmid expression system capable of producing a virus-like particle packing large-fragment RNA, which belongs to the field of biotechnology. The double-plasmid expression system capable of expressing the virus-like particle containing large-fragment RNA consists of a plasmid PET-MC and a plasmid pACYC-X, wherein the PET-MC is constructed by integrating pET-28(b) with mature enzyme protein of phage MS2 and capsid protein cDNA, and the pACYC-X is constructed by integrating a 19mer packing site of the phage MS2 and the cDNA of target RNA. The invention has the advantage that the virus-like particle expressed by the double-plasmid expression system contains long-fragment RNA. Different target fragments to be amplified by virus are constructed together to form a chimera which is packed into the virus-like particle, thus the virus-like particle can be taken as a multi-target nucleic acid standard and a quality control material applicable to simultaneous detection of a plurality of viruses for the same specimen so as to save cost and simplify operation procedures.

The invention discloses an LAMP (loop-mediated isothermal amplification) kit for rapid detection of Listeria monocytogenes in food industry. The kit consists of an LAMP reaction solution, a standard positive template, and a negative quality control standard. The LAMP reaction solution contains a Bst DNA polymerase large fragment, primers, an LAMP 10*buffer, a dNTPs solution, an MgSO4 solution and betaine. The primers include forward and reverse primers. The kit disclosed in the invention has the advantages of good specificity, high sensitivity, rapidness and convenience, high repeatability, and judgeable results obtained by naked eyes, etc., can perform rapid qualitative detection on Listeria monocytogenes in industrial food, and can replace the continuously used traditional culture method and the serological diagnostic method.