191 results about "Ligation" patented technology

Provided herein are methods, kits and compositions for detecting an analyte of interest to interrogate spatial gene expression in a sample using templated ligation.

Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample using RNA-templated ligation.

The invention discloses a genome sequencing sample processing method based on a microdissection technology. The method comprises the following steps: (1) preparing a paraffin section or a frozen section; (2) carrying out HE staining on the section obtained in the step (1); (3) carrying out laser microdissection on nucleic acid; (4) fragmenting the DNA sample, and treating a product obtained in the step (3) by utilizing an ultrasonication method to obtain a DNA fragment; a non-contact ultrasonic crusher is adopted, the power is 80%, ultrasonic treatment is carried out for 10 s, stopping is carried out for 20 s, and 15 cycles are carried out. And (5) carrying out on-machine pretreatment on the genome sequencing sample, wherein the treatment comprises tail end repair and joint connection treatment. According to the method disclosed by the invention, accurate cutting of the sample can be realized, a DNA sample is extracted from a very small amount of cells and a sample for sequencing is constructed, the sample can achieve the same or higher sequencing data quality as that of a traditional method, the usage amount of the cells is greatly reduced, and the repeatability of an experiment is improved.

The present disclosure provides an application method in rice callus differentiation based on an Oryza sativa Leafy Cotyledon 1 (OsLEC1) gene, including the steps of: selecting guide ribonucleic acid (gRNA) target sites; cloning tandem fragments including gRNA; ligating each gRNA fragment; performing polymerase chain reaction (PCR) amplification on a ligation product; performing enzyme digestion on the purified product and a target vector; transforming the ligated vector; performing Agrobacterium-mediated genetic transformation of rice; and screening and identifying transgenetic plants. In the present disclosure, the differentiation of callus directly affects the emergence efficiency of transgenic plants. The knockout of OsLEC1 can promote the differentiation of rice callus, suggesting that OsLEC1 can serve as an important target gene for improving the transformation efficiency of rice and even gramineous crops. OsLEC1 can be used as a starting point to construct various molecular tools to enhance transformation efficiency.

The present invention provides a method of stabilizing a phi29 DNA polymerase by contacting the phi29 DNA polymerase with a stabilized oligonucleotide that is free from degradation by a 3'exonuclease. The phi29 polymerase exhibits improved temperature stability in the presence thereof compared to the absence of the stabilized oligonucleotide. The method involves preparing a composition comprising a phi29 DNA polymerase and a stabilized oligonucleotide comprising one or more modified nucleotides. The compositions can be used in methods for performing polymerase reactions, nucleic acid replication, and detection of a target nucleic acid or target analyte in a sample. The compositions are particularly useful in rolling circle amplification reactions in which the targets are produced by proximity ligation assay, in particular cyclized lock probes.

Provided in some aspects are methods for light-controlled in situ surface patterning of a substrate. Compositions such as nucleic acid arrays produced by the methods are also disclosed. In some embodiments, a method disclosed herein comprises using photoresist for photocontrollable hybridization and / or ligation of nucleic acid molecules, wherein photoresist removal allows hybridization and / or ligation of nucleic acid molecules at the exposed area. A large diversity of barcodes can be created in molecules on the substrate via sequential rounds of light exposure, hybridization, and ligation.

This invention belongs to the fields of molecular biology and bioinformatics, specifically relating to a truncated bovine virus-encoded protein phage library, its construction method, and applications. This library covers all 162 known bovine viruses that host bovine subfamily viruses, containing 51,794 non-redundant peptide coding sequences of 56 amino acids each. These peptides are fused and displayed on the surface of the T7 phage capsid, with all coding sequences having a uniform total length of 200 bp. The library construction was achieved through sequence acquisition, truncation and redundancy removal, codon optimization and DNA synthesis, and in vitro ligation and packaging with T7 phage. The resulting library exhibits a positivity rate of 93% and a titer of 2.8 × 10⁻⁶. 10 PFU / mL. This invention features an optimized single-round high-throughput screening process adapted to clinical bovine serum samples, enabling efficient identification of bovine virus B-cell epitopes and providing tools and candidate targets for bovine virus vaccine development and diagnostic reagent development.

Provided herein are compositions and methods for generating libraries for high throughput sequencing without using ligation, and methods of using same.

The present disclosure relates to methods and kits for analyzing a macromolecule. In some embodiments, the present disclosure relates to macromolecule analysis methods which employ barcoding and nucleic acid encoding of molecular recognition events. Also provided herein is a method and related kits for transferring information using a plurality of enzymes, including for performing a ligation, extension, and cleavage reaction with nucleic acid molecules associated with the macromolecule for analysis. In some embodiments, the macromolecule for analysis comprises a peptide, a polypeptide, or a protein.

The application relates to the field of second-generation sequencing, in particular to an ultrahigh-throughput multiplex PCR amplicon capture method for second-generation sequencing. The NGS capture method comprises the following steps: 1) performing multiplex PCR amplification on a sample to be detected by using a digestible composite modified primer pair, to obtain a PCR product; 2) performing an enzyme cutting reaction on the PCR product by using a second enzyme mixture containing a primer digestion enzyme, a mismatch correction enzyme and a terminal repair enzyme, including removing the excess primer part on the amplification product by using the digestion enzyme, and removing the non-specific amplification product by using the mismatch correction enzyme; 3) performing a ligation reaction by using a full-length adapter containing an index, to directly obtain a sequencing library or obtain a sequencing library after amplification. The method significantly improves the amplification efficiency and specificity, and has the advantages of simple steps, low cost, short time, wide application value in the fields of genetic disease detection, tumor companion diagnosis and genetic detection and scientific research.

This invention belongs to the fields of biotechnology and genomics, and relates to a method and kit for capturing plant chromatin conformation. The method includes the following steps: (1) cross-linking and fixing the chromatin of a plant sample; (2) digesting the fixed chromatin using an enzyme digestion combination containing restriction endonuclease AluI and restriction endonuclease HypCh4V; (3) ligating the enzyme fragments generated in step (2) at spatially adjacent locations to form ligation products; (4) enriching the ligation products and constructing sequencing libraries using the enriched ligation products. This invention uses a combination of AluI and HypCh4V enzymes to replace traditional single-enzyme digestion, which significantly improves data resolution while reducing technical bias. It can achieve chromatin interaction mapping at a resolution of 500 bp in plants and has broad application prospects in the study of fine tissue characteristics at the single-gene level in plants.

The invention belongs to the technical field of bioengineering, and discloses a plasma membrane localized green fluorescence overexpression vector as well as a construction method and application thereof. The cytoplasmic membrane localized green fluorescence overexpression vector contains an expression vector skeleton and an AtCBL1 gene connected to the expression vector skeleton, wherein the nucleotide sequence of the AtCBL1 gene is shown as SEQIDNO: 1, and the expression vector skeleton also carries a fluorescent protein gene. The construction method of the vector comprises the following steps: S1, carrying out enzyme digestion and recovery on an AtCBL1 gene sequence with a nucleotide sequence as shown in SEQIDNO: 1 obtained by amplification; s2, performing same enzyme digestion on the expression vector carrying the fluorescent protein gene, and recovering; and S3, connecting the AtCBL1 gene subjected to enzyme digestion with the expression vector to form a recombinant plasmid, converting a connection product into an escherichia coli competent cell, and extracting the recombinant plasmid from positive clone to obtain the plasma membrane localized green fluorescence overexpression vector.

This invention discloses a method for repairing DNA damage in FFPE samples, comprising: S1. subjecting FFPE sample DNA to heat denaturation; S2. allowing the DNA to fully renature by slow cooling or ice bath; S3. using random primers to bind to large nicked regions of the DNA fragment; S4. adding a mixed repair enzyme to repair the DNA damage; S5. ligating the DNA repaired in step S4 to obtain large DNA fragments, which are then purified with magnetic beads to obtain the repaired DNA. By using random primer single-strand extension to assist the enzyme reaction system, the efficiency of DNA damage / deletion repair is improved; by using random ligation to assemble fragmented DNA into large DNA fragments, the problem of ineffective amplification caused by excessive DNA fragmentation is solved, without affecting the microarray detection results. The entire DNA repair reaction is performed continuously, without the need for multiple purification operations. Compared with other DNA repair methods, it is simple to operate and low in cost, significantly improving the quality of FFPE sample DNA and increasing its detection pass rate in Infinium CytoSNP-850K whole genome microarray detection.

The invention provides methods for synthesizing a product DNA molecule of any DNA sequence from a universal library of overlapping oligonucleotides. The method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule. The method also involves annealing the plurality of oligonucleotides, performing a ligation step, and performing an amplification step to thereby synthesize a sub-sequence of the product DNA molecule. The invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme. In one embodiment the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence.

A library preparation device is a chip including (a) a biological sample well, (b) an end repair and elution well, downstream from the biological sample well, (c) an adapter ligation and elution well, downstream from the end repair and elution well, and (d) a final library well downstream from the ligation and elution well. The library preparation device is useful in a method of library preparation on a library preparation device.

This invention relates to a hairpin primer for detecting miRNA and its applications. The invention develops a reassembly ligation detection scheme for miRNA detection, designing hairpin primers and developing a two-step detection process. The first hairpin primer loop structure binds to the target miRNA, opening the stem structure. The opened stem structure attracts the remaining miRNA sequence, reassembling with the second hairpin primer to form a T-shaped double-stranded structure. The two hairpin primers, using the DNA strand as a clamp, can be ligated by DNA ligase to form a long fragment containing both the miRNA's reverse-specific and universal sequences for qPCR detection. The stem-loop design improves ligation specificity, eliminates Splint ligase dependence, reduces cost, and increases efficiency. The ligated long fragment contains both specific and universal sequences, facilitating multiplex qPCR detection.

The invention relates to a molecular peptide mutant, the amino acid sequence of which is as shown in SEQ ID NO: 1. In the invention, SpyCatcher is designed and modified to obtain a molecular peptide SpyCatcher-21 with stimulus response to pH on the basis of not affecting the formation of isopeptide bonds, and Pro is introduced into a key loop of the SpyCatcher-21 through analysis of the crystal structure to reduce the flexibility of the loop and obtain a mutant SpyCatcher-21_A82P, which can raise ligation efficiency with SpyTag. The SpyCatcher-21_A82P can be used to achieve double-enzyme catalysis according to objective needs by changing the pH of the environment to obtain different degrees of coupling, or obtain a three-enzyme coupled catalytic system through electrostatic interaction with a positively charged enzyme.

Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample using RNA-templated ligation.

The present invention relates to providing more efficient methods of conjugating two or more peptides. For example, the invention provides more efficient methods of producing antibody-drug conjugates. More particularly, the methods can be used to efficiently produce ADCs with at least two unique payloads. One approach involves the use of peptidyl asparaginyl ligases (PALs) for two consecutive ligation reactions at the N-terminal and C-terminal ends of a protein and the other approach involves the use of a chemical protein modification reaction and a PAL-mediated enzymatic ligation reaction, both able to introduce two different payloads to an antibody at specific sites.

The invention generally relates to capturing, amplifying, and sequencing nucleic acids. In certain embodiments, copies of the sense and antisense strands of a duplex template nucleic acid are captured using linked capture probes and multiple binding and extension steps to improve specificity over traditional single binding target capture techniques. Methods of seeding sequencing clusters with sense and antisense strands of a target nucleic acid are also disclosed including identifying the strands using sense-specific barcodes and confirming base calls using two sense-specific sequencing reads. Linked adapters may be used to increase adapter ligation selectively or efficiency and yield.

The present disclosure provides methods for characterizing a target DNA present in a sample. The methods involve contacting the sample with a type V CRISPR / Cas effector protein and one or more guide RNAs, where the contacting generates a cleavage product comprising a 5′ overhang; and ligating a double-stranded nucleic acid adapter to the cleavage product, to generate a ligation product. The ligation product includes the target DNA, which can be sequenced. The sample can be subjected to one or more amplification steps prior to the contacting step, with primers that provide for amplification of nucleic acids of, e.g., specific pathogens, categories of pathogens, two or more different pathogens, or two or more different categories of pathogens.

The present invention relates to the field of biomedical engineering. Disclosed are a dual-target receptor agonist fusion protein and a use thereof. The fusion protein is a dual-target receptor agonist fusion protein (GLP-1 and GIP, GLP-1 and GCG) having dual native N-termini and fused with an antibody Fc fragment. The fusion protein can be rapidly prepared by using a more efficient and low-cost bacterial expression system, and has high in vitro agonistic activity and in vivo activity. The fusion protein can be obtained in one step by means of self-ligation of SpyCatcher / SpyTag without the need for purification, and is fused with a human antibody Fc fragment. The fusion protein can be used in the production of a novel drug comprising a polypeptide / protein modified by an antibody Fc fragment and having dual native N-termini.

The invention provides a single-cell mRNA and sRNA co-sequencing method, which comprises the following steps: preparing a single-cell suspension, adding the single-cell suspension into a micropore of a chip of a single-cell operation system, and selecting a micropore of a single living cell to carry out an experiment, and then carrying out cell lysis reaction, sRNA 3'end ligation reaction, mRNA reverse transcription reaction, sRNA free linker removal reaction, sRNA 5 'end ligation reaction, sRNA reverse transcription reaction and first PCR (Polymerase Chain Reaction) reaction, and screening and recovering products and carrying out second PCR reaction, thereby obtaining the single-cell mRNA and sRNA co-sequencing library which can be directly used for on-machine sequencing. The method has the advantages of high accuracy, high sensitivity, good repeatability and the like.

Provided are a method for representing a target biomolecule, a ligation method or a preparation method for a sequencing library, an activation linker, a method for preparing the activation linker, a kit for nanopore sequencing, and a use of the activation linker in nanopore sequencing.

The invention relates to the field of gene vector construction, in particular to construction and application of a gene vector integrating fluorescent screening and self-deletion functions, the gene vector comprises a conditional knock-out (cKO) vector and a gene knock-in (KI) vector which are both integrated with a Dre-Rox mediated self-deletion module and a fluorescent screening module, the construction method comprises the following steps: carrying out HindIII / EcoRI double enzyme digestion on a pUC19 vector, carrying out homologous recombination amplification on a target fragment, carrying out connection transformation and screening verification to obtain a qualified vector; the vector is applied to preparation of cKO / KI gene modified mice, and efficient screening of positive individuals is realized through fluorescence preliminary screening, genotype identification and fluorescence quenching detection. The positive screening workload and cost can be reduced by 70% or above, non-target elements are accurately cut off, interference is avoided, time-space accurate regulation and control are achieved, carrier construction is easy and convenient, repeatability is high, and the method is suitable for gene function research, disease model construction and drug target verification.

Provided are methods and compositions for screening / identifying / characterizing nucleic acid delivery vehicles (e.g., liquid nanoparticle (LNP) formulations). Provided is a library of nucleic acid delivery vehicles with different chemical compositions, where the members of the library are barcoded by virtue of including a barcoded RNA molecule (in some cases a barcoded ligation-ready RNA molecule). The barcoded RNA molecule includes a first ribozyme, a second ribozyme, and a barcode sequence between the first and second ribozymes. Once a barcoded RNA molecule is successfully delivered into a cell, the first and second ribozymes cleave the barcoded RNA molecule to produce a 5'-OH end and a 2',3'-cyclic phosphate end, which ends can be ligated to one another by an intracellular RNA ligase, producing a circular RNA (cRNA) that includes the barcode sequence. Detecting barcode sequences from cRNAs therefore identifies the chemical composition of successful delivery vehicles.

Provided herein is a DNA analysis method for DNA in an adapted library, comprising contacting the DNA with a primer or ligation substrate of a loop precursor primer or a plurality of primers and a plurality of ligation substrates; extending the primer in the direction of the ligation substrate or the plurality of primers in the direction of the plurality of ligation substrates using a polymerase, thereby providing extension products; ligating at least a portion of the extension products to the ligation substrates, thereby providing extension-ligation products; and sequencing at least a portion of the extension-ligation products. The primer or plurality of primers bind V regions of the DNA and the ligation substrate or plurality of ligation substrates bind J regions of the DNA, or the primer or plurality of primers bind J regions of the DNA and the ligation substrate or plurality of ligation substrates bind V regions of the DNA.

This invention discloses a method for improving gene editing efficiency in citrus based on cloning the citrus U6 promoter, relating to the field of biotechnology. The method includes: PCR amplification of the promoter CsU6.1 using genomic DNA from Late Orange leaves as a template; recovery of the amplified fragment and ligation into a linearized pNGerRGEB32 vector to obtain a pNGerRGEB32-CsU6.1 vector containing the CsU6.1 promoter; then, using pNGerRGEB32-CsU6.1 as a template, designing primers containing a target sequence for PCR amplification; recovering the amplified product fragment and ligating it into a linearized pNGerRGEB32-CsU6.1 vector to obtain a pNGerRGEB32-CsU6.1-sgRNA gene editing vector containing the target sequence; and then genetically transforming citrus, achieving upregulation of sgRNA expression and improving gene editing efficiency.

The invention discloses a DNA coding compound library screening method based on an adjacent ligation strategy and application, and belongs to the technical field of biological medicine. According to the method, a DNA coding protein strategy is introduced on the basis of a traditional DNA coding compound library screening technology; when the compound is combined with the protein or induces the two proteins to form a compound, the nucleic acid tags carried by the protein and the compound are close to each other and can be connected by DNA ligase; amplifying the connected nucleic acid tag by using primers which are respectively combined with the protein and the compound nucleic acid tag; and determining the compound and corresponding protein information through a high-throughput sequencing technology. The method not only can be used for simultaneously screening binding agents of multiple proteins, but also can be used for simultaneously screening compounds for inducing protein-protein proximity, and has application prospects in drug research and development, disease treatment mechanism research and biological signal channel regulation and control.

The present disclosure relates to biocatalytic ligation methods for generating oligonucleotides and fusion polypeptides for use in the methods. In particular, the disclosure relates to biocatalytic ligation methods that incorporate ATP regeneration and fusion polypeptides that include a polyphosphate kinase domain and an ATP-dependent nucleic acid ligase domain.