251 results about "Transposase" patented technology

The present disclosure relates to materials and methods for spatially analyzing nucleic acids that have been fragmented with a transposase enzyme, alone or in combination with other types of analytes.

The present disclosure relates to materials and methods for spatially analyzing nucleic acids that have been fragmented with a transposase enzyme, alone or in combination with other types of analytes.

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 provides a method for obtaining engineered recombinant mammalian cells and application of the engineered recombinant mammalian cells, and relates to the technical field of biology. The method comprises introducing into a mammalian cell a minicircle DNA comprising a transposon and a polynucleotide encoding a transposase, the transposon comprising a fragment encoding an antigen binding domain. The method has the advantages of shortening the preparation period of recombinant mammalian cells, simplifying the process and reducing the cost.

The present invention provides engineered transposase polypeptides useful in preparation of next generation sequencing libraries, as well as compositions, methods of utilizing these engineered polypeptides, and polynucleotides encoding the engineered transposases. The present application further relates to methods for using engineered transposases, e.g., in the preparation of nucleic acid libraries for nucleic acid sequencing. More specifically, the invention pertains to methods that use engineered transposases having improved performance to improve the transposition reactions during the preparation of nucleic acid libraries.

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 provides a library building method for direct sequencing of single-stranded nucleic acid, the library building method comprises providing a to-be-detected sequence fragment, and a preparation method of the to-be-detected sequence fragment comprises cutting a double-stranded molecular sequence formed by complementary pairing of internal bases of the single-stranded nucleic acid by using transposase so as to form the to-be-detected sequence fragment. Compared with an existing library building method of RNA direct sequencing, the library building method has the advantages that a pairing structure is formed by complementary bases of RNA or single-stranded DNA, and a transposase compound can be used for shearing and connecting the pairing structure. This is an attempt which has not been made before, and the library building efficiency is greatly improved through the library building mode.

The disclosure relates to nanoparticles comprising a surface-exposed immune cell binding moiety, a transposable element comprising a gene sequence flanked by inverted terminal repeats (ITRs), a nucleic acid encoding a transposase with specificity for the ITRs, and an mRNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR). Methods are also described for treating a disease or disorder by administering such nanoparticles to a subject in need thereof.

The invention discloses a recombinant PB transposase mRNA (messenger ribonucleic acid) and PiggyBac vector system and application of the recombinant PB transposase mRNA and PiggyBac vector system. The invention provides a recombinant PB transposase mRNA (messenger ribonucleic acid), the nucleotide sequence of the recombinant PB transposase mRNA is shown as SEQ ID NO: 11, and the 5'end of the recombinant PB transposase mRNA contains a cap structure. The invention provides an mRNA type recombinant PB transposase: PBase-Cap-mRNA, so that the risk that a PB transposase plasmid is inserted into a host cell genome is avoided, and the efficiency of inserting a donor plasmid into the host cell genome is improved. The recombinant PB transposase mRNA is capped, so that the stability of the mRNA is improved, the translation of the mRNA is promoted, and the transposon efficiency is improved. The CHO cells are co-transfected by recombining PB transposase mRNA and donor plasmids, so that the efficiency of transfecting host cells by the donor plasmids is improved, and the construction period of stable cell strains is remarkably shortened.

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.

The present invention relates to a composition comprising-a first single-stranded nucleic acid molecule comprising a region complementary to a target and a transposase recognition site,-a second single-stranded nucleic acid molecule comprising a region complementary to the target and a transposase recognition site, and-a third single-stranded molecule comprising a region complementary to the target and a transposase recognition site, the third single chain molecule comprises a sequence encoding a portion of the interleukin 2 [gamma] receptor (IL2-R [gamma]) or encoding a complete IL2-R [gamma] receptor and a transposase binding site.

The invention provides a retrotransposase, which comprises a target DNA binding structural domain containing a zinc finger binding motif, a reverse transcriptase structural domain and an endonuclease structural domain, and can be used for reversely transcribing RNA (Ribonucleic Acid) into DNA. The amino acid sequence of the retrotransposase is shown as any one of SEQ ID No.1 to 6 or SEQ ID No.32 to 43 or SEQ ID No.68 to 71 or has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% and 99% identity with the amino acid sequence of any one of SEQ ID No.1 to 6 or SEQ ID No.32 to 43 or SEQ ID No.68 to 71. The invention also relates to a system for modifying DNA. The system comprises the retrotransposase provided by the invention or a nucleic acid encoding the retrotransposase provided by the invention; and a donor RNA or a nucleic acid encoding the donor RNA, the donor RNA comprising: a sequence that binds to the retrotransposase and a heterologous sequence.

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 systems and methods that can combine T cell receptor sequencing (TCRseq) and Assay for Transposase Accessible Chromatin using sequencing (ATAC-seq), and / or respective aspects thereof. Further provided are systems and methods that can combine ATAC-seq and perturbation sequencing (Perturb-seq), and / or respective aspects thereof.

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.

In an example of a method, a deoxyribonucleic acid sample is exposed to tagmentation in the presence of a tagmentation buffer including a divalent cation cofactor and a transposase enzyme to generate a tagmented DNA fragment complex. A chelator mixture is added to the tagmented DNA fragment complex. The chelator mixture includes a chelator of the divalent cation cofactor at a weight ratio that is at least 1:1 with the divalent cation cofactor; and has a pH ranging from 8 to 9. The tagmented DNA fragment complex is incubated in the chelator mixture at a temperature of at least 55° C. for at least about 60 seconds, so that the transposase enzyme dissociates from a tagmented DNA fragment of the tagmented DNA fragment complex.

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.

Disclosed is a method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell, comprising: (i) permeabilizing the cell; (ii) contacting the permeabilized cell with a first antibody that specifically binds the chromatin-associated factor of interest and with a second antibody that specifically binds to the first antibody; (iii) contacting the permeabilized cell with a transposome that is linked to a specific binding agent that specifically binds the first and / or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; (iv) activating the transposase, thereby excising a DNA segment comprising at least one chromatin binding site of a chromatin-associated factor of interest and tagging the DNA with the first and second DNA molecule; (v) determining the sequence of the excised and tagged DNA segment; and (vi) based on the determined sequence of the excised and tagged DNA segment, determining the at least one chromatin binding site of the chromatin-associated factor of interest in the cell; wherein the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in presence of a crowding agent.

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