168 results about "Transposase" patented technology

Provided herein are methods, compositions, and kits for detecting analytes of interest from biological samples using tagmentation. The method comprises: (a) hybridising a nucleic acid analyte to a capture probe on an array, wherein the capture probe comprises: (i) a spatial barcode and (ii) a capture domain; (b) extending the capture probe using the nucleic acid analyte as a template, thereby generating a barcoded nucleic acid molecule on the array, wherein the barcoded nucleic acid molecule comprises a complement of the sequence of the nucleic acid analyte; and (c) tagmenting the barcoded nucleic acid molecule or a derivative thereof using a transposome comprising a transposase to insert a transposon end sequence into the barcoded nucleic acid molecule or the derivative thereof, thereby generating a barcoded nucleic acid fragment, wherein the barcoded nucleic acid fragment comprises: (i) the barcode, (ii) the transposon end sequence, and (iii) at least a portion of a sequence of the nucleic acid analyte or a complement thereof.

The invention discloses a high-flux and rapid chromatin immunoprecipitation sequencing method based on a HiBiT tag and a Tn5 enzyme, and belongs to the technical field of biology. According to the method, the high affinity characteristic of a HiBiT tag system is combined with a Tn5 enzyme in-situ library building technology, firstly, a DNA binding protein recombinant expression strain containing a HiBiT tag is constructed and subjected to induced expression, after formaldehyde crosslinking and cell lysis, an Anti-HiBiT antibody is used for rapid immunoprecipitation, and a Tn5 enzyme library is constructed; tn5 transposase is directly loaded on the magnetic beads combined with the protein and DNA fragment compound to complete fragmentation and linker connection, so that the construction of a next-generation sequencing library is quickly carried out, and the total consumed time from immunoprecipitation to the completion of library construction is less than or equal to 5 hours. Meanwhile, automatic treatment is realized through a micro experiment board (the reaction system is less than or equal to 200 microliters) and a liquid workstation, the experiment cost and the sample dosage are reduced, 48 transcription factors and 48 control samples can be synchronously treated, the efficiency is greatly improved, and an efficient tool is provided for microbial transcription regulation and control research.

The invention relates to the technical field of gene engineering, in particular to a transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on a Tol2 transposition subsystem and a construction method of the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry. The preparation method comprises the following steps: S1, constructing a Tol2 transposon expression vector pTol2-ef1a-tjp1a-P2A-mCherry which contains an ef1a promoter, a tjp1a gene, a P2A peptide sequence and an mCherry reporter gene; s2, carrying out in-vitro transcription to prepare Tol2 transposase mRNA; s3, mixing the expression vector with Tol2 transposase mRNA, and microinjecting the mixture into the single-cell stage embryo of the zebra fish; s4, performing fluorescence screening on the F0 generation embryos surviving after injection to obtain the Founder fish with positive transgenosis; s5, after the F0-generation positive fish is bred to be sexually mature, an F1 generation is obtained through mating, transgenic positive individuals with stable inheritance are screened out after identification, and a transgenic zebrafish strain is established. According to the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on the Tol2 transposon system and the construction method of the transgenic zebrafish system pTo12-efla-tjp1a-P2A-mCherry based on the Tol2 transposon system, efficient integration of exogenous genes is achieved by utilizing the Tol2 transposon system, and the transgenic zebrafish line capable of being stably inherited to the F1 generation is successfully obtained.

The invention relates to the technical field of sequencing, in particular to a Tn5 transposase-based next-generation sequencing method and a DNA library rapid construction kit, and particularly provides a Tn5 transposase-based next-generation sequencing DNA library rapid construction kit, a next-generation sequencing method and a next-generation sequencing DNA library construction method, and the kit comprises a reagent for constructing a next-generation sequencing DNA library. The Tn5 transposase is adopted for double-end Index library building sequencing, the library building process can be simplified, high efficiency and accuracy of high-throughput sequencing can be ensured, DNA fragmentation and linker addition are achieved in a single step by means of the unique characteristics of the Tn5 transposase, the sequencing efficiency and data management flexibility are improved by introducing the double-end Index, and the method is suitable for large-scale popularization and application. Each Index sequence ensures high specificity and traceability, meanwhile, the sequencing method can effectively work under the condition of low initial template quantity, and the application range is expanded.

This disclosure provides improvised reagents and methods for inserting a transgene into the genome of a living host cell. Sleeping beauty (SB) transposase is used in protein form rather than as a vector. This has been made possible by using rational mutagenesis in a particular region of the SB crystal structure to develop variant SB transposase protein that is more soluble. The changes increase solubility while maintaining transposase activity, thereby adapting the variant transposase for the purpose of promoting recombinant integration of a transgene into a target cell when used in protein form rather than as a polynucleotide vector. The modified transposase is highly soluble in electroporation buffer and thermostable during storage. When introduced into a host cell, it promotes integration of a transgene into the genome of the cell in a dose-dependent manner. It is degraded within 48 hours, thereby rapidly clearing transposase activity from the host cell.

This application relates to the field of molecular biology, and more specifically to isolated transposases and their use. More specifically to nucleic acids and nucleic acid constructs encoding transposases, nucleic acid sets and nucleic acid set constructs, and compositions, recombinant vectors, recombinant host cells and kits comprising transposases. More specifically to methods for introducing exogenous nucleic acid fragments into the genome of host cells, methods for editing the genome of host cells, and methods for obtaining host cells containing exogenous nucleic acid fragments in their genomes. More specifically to the use of transposases, nucleic acids and nucleic acid constructs, nucleic acid sets and nucleic acid set constructs, compositions, recombinant vectors, or recombinant host cells for introducing exogenous nucleic acid fragment genes into the genome of host cells, or for preparing drugs or formulations for gene therapy, cell therapy, genome research, or stem cell induction and post-induction differentiation.

Single-cell multi-omics by co-encapsulating a single cell with two beads, the first an RNA barcoding bead having barcoded mRNA capture primer oligonucleotides attached on the bead surface; and the second a DNA barcoding bead having two types of oligonucleotides releasably attached to the surface: (1) barcoded adapter oligonucleotides that are complementary to oligonucleotides bound to the transposase that are eventually incorporated into gDNA fragments and (2) polyadenylated barcoded oligonucleotides containing the same barcode sequence as the adapters. In addition, integrated analysis of RNA and protein, including intracellular protein, from individual cells using similar co-encapsulation of a single cell, an RNA barcoding bead, and with / without a specific or non-specific protein binding bead in a microwell, to avoid protein fixation by first lysing the cell to liberate intracellular contents, and then capturing protein either on a solid surface or in solution with barcoded affinity reagents.

The present disclosure relates generally to transposase domains, in particular transposase domains comprising an amino terminal deletion, as well as transposase domains that form obligate heterodimers and transposase domains comprising a DNA targeting domain.

The invention discloses a method and system for synchronously detecting host chromatin openness and in-vivo microbiome based on transposase, and belongs to the technical field of biological sequencing data analysis. According to the method, transposase is used for selectively fragmenting an open chromatin region of a host, and a microbial genome is almost randomly cut, so that synchronous enrichment of host and microbial DNA is realized; after high-throughput sequencing library construction and double-end sequencing, sequencing data is split into host source and non-host source reads through bioinformatics analysis, host chromatin state and microorganism composition are analyzed respectively, and a microorganism-host epigenetic regulation network is constructed; the invention further provides a matched DNA sequencing library and an analysis system, multi-scene research of infectious diseases, intestinal microecology, tumor microenvironment and the like is supported, a public database can be reanalyzed, and potential microbial interaction signals are mined. According to the method, the host-microorganism interaction research efficiency is remarkably improved, and a high-sensitivity and integrated technical scheme is provided for epigenetic regulation mechanism analysis.

The application discloses a kind of chicken primordial germ cell transfection methods, including the following transfection steps: S1, the separation of chicken primordial germ cell is carried out, and grouping culture in culture medium, S2, according to the proportion of 3.5:1 of pPB-GFP transposon plasmid and transposase plasmid mPB dosage Two are mixed, S3, first dilute plasmid with opti-MEN, then dilute transfection reagent with opti-MEN, plasmid diluent and transfection reagent diluent are mixed to obtain compound, the transfection operation of chicken primordial germ cell is carried out by the culture medium of different serum adding amount and adding time in the application, the influence of different serum adding amount and different serum adding time on chicken primordial germ cell transfection efficiency can be understood, then the increase and decrease of the fluorescence brightness in chicken primordial germ cell after transfection are recorded, the growth of chicken primordial germ cell after transfection is understood, so that the most suitable culture medium can be selected, serum adding condition is optimized, and more efficient chicken primordial germ cell transfection can be realized.

Provided are a truncated chromatin opening element and a combination thereof, an expression system comprising the chromatin opening element or the combination thereof and applied to eukaryotic cells, and use thereof. The chromatin opening elements are truncated sequences of two chromatin opening elements from different sources, respectively, and can be used alone or in combination. The chromatin opening element significantly shortens the effective sequence, thereby facilitating the integration of a vector comprising the element or the combination thereof with other elements, such as integration with ITR, achieving semi-site-specific integration in the presence of the PiggyBac transposase, and then using a bulkpool rapid screening process to shorten the protein production and cell strain construction cycle by about 4-5 weeks while maintaining comparable yields.

The invention belongs to the technical field of transgenic and biomedical research tools, and particularly relates to a method for constructing a transgenic fish system based on a Tol2 transposon system, which comprises the following steps: constructing a myo6b-lhfpl5b-DsRed plasmid by using myo6b as a promoter, lhfpl5b as a target gene and DsRed as a red fluorescent protein tag through a microinjection method, and constructing a Tol2 transposon system-based transgenic fish system. Co-injecting the plasmid containing the Tol2 transposase mRNA and the Tol2 transposase mRNA into a fertilized egg of an AB zebra fish strain, and screening generation by generation to obtain a transgenic fish strain with stable inheritance MET channel gene lhfpl5b marked hair cells. The spatio-temporal dynamic visualization of the living whole fish to the lhfpl5b is realized, the deafness-causing molecular pathway is clarified, and an in-vivo platform is provided for drug screening and intervention strategies.

The application discloses a Buster transposon system and application thereof, and the system comprises AnBT, CrBT, HvBT, LcBT and LlBT transposons and corresponding transposases, the nucleotide sequences of the AnBT, CrBT, HvBT, LcBT and LlBT transposons are shown in SEQ ID No. 1-5, and the transposases are wild type or enhanced type. The application obtains key elements such as target site repeat (TSD) and terminal inverted repeat (TIR) of the AnBT, CrBT, HvBT, LcBT and LlBT transposons and corresponding transposase sequences through molecular reconstitution, and constructs a set of gene transfer vector systems. The AnBT, CrBT, HvBT, LcBT and LlBT vector systems can effectively mediate gene transfer through cell verification, and have great application potential in transgenic animal preparation and gene therapy.

Provided are a single-cell chromatin accessibility and transcriptome combined sequencing method and application thereof. The method comprises: treating a cell nucleus using a transposase to obtain gDNA of a chromatin open region connected with a first specific tag sequence; treating the cell nucleus using a transcriptome capture sequence to obtain cDNA connected with a second specific tag sequence; generating a droplet based on a droplet microfluidic to seal the cell nucleus and a first microbead in the droplet, wherein the gDNA and the cDNA are captured by the first microbead, and wherein more than one cell nucleus is sealed in the droplet; and sequencing the gDNA and the cDNA to obtain combined information of single-cell chromatin accessibility and transcriptome according to the first specific tag sequence and the second specific tag.

PIGGYBAC® transposases engineered to increase stability in a cell. The engineered PIGGYBAC® transposases are useful for stably transforming cells, cell line development, genome modification, and improving titer of recombinant proteins, among other uses.

The present disclosure provides systems and methods for transposing a cargo nucleotide sequence into a target nucleic acid site in a target nucleic acid. These systems and methods may comprise a double-stranded nucleic acid comprising the cargo nucleotide sequence, wherein the cargo nucleotide sequence interacts with a transposase recognition complex, an effector complex comprising an effector and at least one engineered guide polynucleotide that hybridizes to the target nucleic acid, and the transposase recognition complex wherein the transposase recognition complex recruits the cargo nucleotide to the target nucleic acid site.

Provided herein are methods and systems for detecting a target nucleic acid sequence. The method comprises contacting an oligonucleotide comprising the target nucleic acid sequence with a transposon complex; inserting one or more T7 RNA promoters into the oligonucleotide using the transposase; and (c) amplifying the target nucleic acid sequence. The transposon complex may comprise a transposase and a transposon sequence comprising one or more T7 RNA promoters. The target nucleic sequence may be amplified by generating RNA oligonucleotides comprising the target nucleic acid sequence via transcription from the inserted one or more T7 RNA promoters. The amplified target nucleic acid may be detected using a CRISPR Cas13-based detection system.

Fusion proteins and methods for conditionally fine-tuning transposase protein stability and activity for clinical applications are provided herein

The present invention provides methods, compositions and kits for using a transposase and a transposon end for generating extensive fragmentation and 5′-tagging of double-stranded target DNA in vitro, then using a DNA polymerase for generating 5′- and 3′-tagged single-stranded DNA fragments without performing a PCR amplification reaction, wherein the first tag on 5′-ends exhibits the sequence of the transferred transposon end and optionally, an additional arbitrary sequence, and the second tag on 3′-ends exhibits a different sequence from the sequence exhibited by the first tag. The method is useful for generating 5′- and 3′-tagged DNA fragments for use in a variety of processes, including processes for metagenomic analysis of DNA in environmental samples, copy number variation (CNV) analysis of DNA, and comparative genomic sequencing (CGS), including massively parallel DNA sequencing (so-called “next generation sequencing”).

The invention relates to a novel functional transposase system. More specifically, the invention relates to a DNA transposon comprising a heterologous polynucleotide flanked by transposon flanking regions and to a recombinant transposase of Acyrthosiphon pisum (AP) or Aphis craccivora (AC) comprising an amino acid sequence of at least 90% sequence identity with SEQ ID NO: 4 fused to a heterologous nuclear localization signal, optionally comprising at least one activity improving mutation and to a polynucleotide or an expression vector encoding said DNA transposon or transposase as well as an expression system comprising the transposase / transposon pair. The invention also relates to methods for stably integrating a polynucleotide into cells or stably expressing a protein of interest using the transposase / transposon pair.

Methods and compositions for modifying the coding sequence of endogenous genes using rare-cutting endonucleases and transposases. The methods and compositions described herein can be used to modify the coding sequence of endogenous genes.

To provide more active hyperactive transposons and transposases.SOLUTION: The present invention relates to a polypeptide comprising a piggyBac transposase or a fragment or a derivative thereof having a transposase function comprising at least one amino acid substitution. Further, the present invention relates to a transposable element comprising a piggyBac or piggyBac-like left repeat sequence and left internal repeat sequence, where the left internal repeat sequence comprises at least one nucleotide modification. Furthermore, the present invention relates to a kit comprising the transposase and / or transposable element. In addition, the present invention relates to a targeting system comprising the transposase and / or transposable element.SELECTED DRAWING: None

The application discloses a method for analyzing cell epigenomics in multiple dimensions, which comprises the following steps: embedding different linker sequences in cells by using ChiTag transposase and conventional Tn5 transposase respectively, and realizing the joint analysis of chromatin open region information and specific protein binding sequence information at the cell level. The method has important application prospects in the research of cell population heterogeneity related to development and / or diseases, the drawing of cell atlas, the analysis of tumor cells with different clinical characteristics, the clinical research of tumor cell evolution and / or metastasis and the like.

The invention provides a primer for detecting bordetella pertussis through RPA (recombinase polymerase amplification)-lateral flow immunochromatography. The primer comprises specific primer pairs NAT-F and NAT-R which are designed aiming at transposase genes of a bordetella pertussis IS1663 family, and specific primer pairs NAT-F and NAT-R which are designed aiming at transposase genes of a bordetella pertussis IS1663 family, the invention also provides corresponding probes, which comprise a gold nanoparticle binding probe, a quality control line capture probe and a bordetella pertussis IS1663 capture probe. Meanwhile, the invention further provides application of the primer and the probe in preparation of a pertussis bordetella detection product, a specific detection kit and a specific detection method. According to the invention, the specific primer and probe are designed aiming at the transposase gene of the pertussis bordetella IS110-like element IS1663 family, and the rapid, high-sensitivity and high-specificity detection of pertussis bordetella is realized by fusing the rapid isothermal amplification technology of RPA and the visual detection advantage of lateral flow immunochromatography, no complex instrument or professional operation is needed, and the kit has the advantages of high sensitivity, high specificity, high sensitivity and high specificity. The method is suitable for multi-scene early convenient diagnosis.

The present invention refers to hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB). The invention further refers to corresponding nucleic acids producing these variants, to a gene transfer system for stably introducing nucleic acid(s) into the DNA of a cell by using these hyperactive variants of a transposase of the transposon system Sleeping Beauty (SB) and to transposons used in the inventive gene transfer system, comprising a nucleic acid sequence with flanking repeats (IRs and / or RSDs). Furthermore, applications of these transposase variants, the transpsoson, or the gene transfer system are also disclosed such as gene therapy, insertional mutagenesis, gene discovery (including genome mapping), mobilization of genes, library screening, or functional analysis of genomes in vivo and in vitro. Finally, pharmaceutical compositions and kits are also encompassed.

The application discloses a kind of plant whole genome RNA-chromatin interaction detection methods, the method includes the interaction state of fixed RNA and chromatin, preparation double link, obtain nuclear, chromatin DNA fragment, RNA / linker / DNA chimera, RNA-DNA double-stranded complex in turn;Tn5 transposase fragmentization cuts double-stranded complex;PCR amplification forms DNA library.The application applies Tn5 transposase to the original RADICL-seq method, successfully to RNA-DNA complex is cut and library, greatly simplifies the process, experimental procedure is reduced by at least half, experimental period is shortened from 4-5 days to 3 days, the fragment size of DNA library produced is between 250bp-400bp, increase the length of final effective RNA / DNA fragment pair, thereby greatly improve the efficiency when aligning to plant genome in subsequent bioinformatics analysis, provide effective means for plant whole genome RNA-chromatin interaction detection.

The application provides a 5mCpG high-throughput detection method TnM-seq based on TnM transposase, which adopts transposase TnM including an MBD domain to detect 5mCpG in situ, and comprises the following steps: after pretreatment of cells to be detected, the cells are incubated with transposase TnM; DNA in the target sample after transposition is recovered and purified; the purified DNA is subjected to PCR library construction; and the library construction product is subjected to high-throughput sequencing. The new transposase TnM newly designed and synthesized is used, combined with high-throughput sequencing, to realize convenient, fast, low-cost and high-sensitivity detection of 5mCpG in a sample. Based on the transposase TnM, a multi-omics technology HAM-seq for simultaneously detecting histone modification H3K9me3, chromatin accessibility and DNA methylation modification 5mCpG in the same sample is further developed, and after being combined with a commercial single-cell microfluidic platform, an epigenome and gene expression four-omics technology EpiX-seq for single-cell analysis in a complex tissue is obtained.

The application belongs to the technical field of bioengineering, and discloses a gene engineering bacterium of acetobacter pasteurii with high temperature resistance, a construction method and application thereof. Acetobacter pasteurianus The gene engineering bacterium is constructed by using the acetobacter pasteurii (ATCC 10137) as a starting strain and knocking out the transposase gene in the genome of the acetobacter pasteurii (ATCC 10137) by a homologous recombination method. TnsR The gene engineering bacterium with high temperature resistance is obtained. TnsR The deletion of the gene is beneficial to maintaining the stability of the genome, so that the strain can maintain better growth and metabolic capacity under high-temperature pressure. Experimental results show that the biomass, survival rate and acetic acid yield of the gene engineering strain under the condition of 40 DEG C high temperature are significantly better than those of the wild-type strain. When the gene engineering strain is applied to traditional solid-state food vinegar brewing, the yield of organic acids such as acetic acid is significantly improved, the content of ester flavor substances is greatly improved, and the flavor quality of food vinegar is effectively improved. The application provides an effective technical means for solving the problem that high temperature in summer leads to abnormal fermentation and yield reduction of food vinegar.

This disclosure generally relates to fusion proteins comprising TAL Arrays targeting a repetitive element and a transposase domain comprising amino terminal deletions, as well as dual cysteine rich domains (CRD), for targeting site-specific transposition into ribosomal DNA (rDNA) repeats or LINE1 repetitive elements, polynucleotides and vectors encoding the fusion proteins, and methods of use therefor.