420 results about "Ligation" patented technology

Provided herein are methods of identifying genetic variants in biological samples. In particular, the present disclosure uses spatial transcriptomic templated ligation methods and compositions to identify the presence or absence, and the location of genetic variants in the transcriptome or genome of a biological sample.

Provided herein are methods, compositions, and kits to spatially detect genetic variants in a target nucleic acid via templated-ligation and reversible terminator nucleotides.

Provided herein are methods of detecting a target nucleic acid of interest for spatial gene expression analysis in a sample using barcoded templated ligation probes.

The invention includes a mutant T7 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T7 DNA ligase. The mutant T7 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary. The preferred mutant T7 DNA ligase has at least one of the following mutations: E63K (SEQ ID NO:4), K73E (SEQ ID NO:6), K137E (SEQ ID NO:7), K174E (SEQ ID NO:9), E182K (SEQ ID NO:11), K210E (SEQ ID NO: 13), E243K (SEQ ID NO:15), D245R (SEQ ID NO: 17), E268K (SEQ ID NO:19), E272K (SEQ ID NO:21), E289K (SEQ ID NO:23), K295E (SEQ ID NO:25) and D336R (SEQ ID NO:27).

The present disclosure relates to a method for in situ analysis of single nucleotide variations in an RNA sample, the method comprising the steps of: (a) contacting the RNA sample with a plurality of chimeric padlock probes comprising a modified base at the 3'end portion; (b) adding an RNA ligase to generate a cyclized padlock oligonucleotide for a padlock probe comprising a modified base complementary to the corresponding position of the RNA sample under conditions and reagents allowing the ligation of the padlock probe with single nucleotide specificity; (c) amplifying the cyclized padlock oligonucleotide under conditions and reagents that allow rolling circle amplification, thereby producing an amplified cyclized padlock oligonucleotide; and (d) detecting the amplified cyclized padlock oligonucleotide so as to obtain a map of the mononucleotide in the RNA sample. The disclosure further relates to a kit for in situ analysis of single nucleotide variations in an RNA sample.

The present invention relates to a method for sequencing small non-coding RNAs on a nanopore platform. In this method, sequence-specific structured adapters are used to specifically capture small non-coding RNAs in a single ligation step reaction, thus enabling direct RNA sequencing.

The invention discloses a drug target binding affinity prediction method based on collaborative attention, and belongs to the technical field of natural language processing, and the method comprises the steps: building a CLAT-DTA prediction model comprising an input data representation module, a feature extraction module, an information fusion module and a prediction module; converting drug molecules into fingerprint representation, and pre-training protein sequence data by using ESM; extracting drug data by using Encoder, and extracting protein data by using Bi-LSTM (Bidirectional Long Short-Term Memory); fusing the drug target data using a collaborative attention mechanism; three-layer full ligation is used to predict drug target binding affinity. According to the method, important information can be better polymerized, and the binding affinity between the drug and the protein can be predicted.

The invention relates to a DNA framework structure addressable enzyme ligation method. Specifically, the invention provides a method for accurately regulating and controlling the specific position of an enzyme on a DNA frame structure through an enzyme modification and base complementary pairing principle, and provides a frame nucleic acid with a capture segment and an enzyme with a connecting chain, and the frame nucleic acid with the capture segment and the enzyme with the connecting chain are subjected to base complementary pairing through the connecting chain and the capture segment. Therefore, the enzyme is connected to a predetermined binding site of the framework nucleic acid, and accurate regulation and control of the connection position, number spacing and the like of the enzyme on the framework nucleic acid are realized. The connection method provided by the invention can realize addressable and accurate enzyme position regulation and control on the DNA nano structure, can widen site-specific modification of the enzyme on the DNA frame structure, and has important significance for developing site-specific and high-throughput enzymatic reaction.

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

SNAIL provides cost-efficient detection of specific nucleic acids in single cells, and may be combined with flow cytometry to simultaneously analyze large numbers of cells for a plurality of nucleic acids, e.g. at least one, to up to 5, up to 10, up to 15, up to 20 or more transcripts can be simultaneously analyzed, at a rate of up to about 50, 100, 250, 500 or more cells / second. The methods require only two primers for amplification, and may further include a detection primer.

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.

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.

This application relates to reagents and methods for analyzing associated nucleic acids. Specifically, reagents and methods are provided for analyzing nucleic acids (e.g., genomic DNA) of circulating particles (i.e., particles derived from blood). The method includes associating at least two target nucleic acid fragments of the circulating particles to produce a group of at least two associated target nucleic acid fragments. In the method, the target nucleic acid fragments can be associated by techniques such as barcoding, partitioning, ligation, and / or individual sequencing. Sequencing of the group of associated fragments provides a group of informationally associated sequence reads corresponding to the sequences of the fragments from a single particle.

Described herein are receptor configurations that allow ligation of multiple targets expressed in cancer cells via two main mechanisms: direct binding via an antibody-derived single chain variable fragment (scFv), and indirect binding through the high-affinity engagement of the Fc component of an antibody. When expressed in human T lymphocytes, these receptors (CARFcR) can trigger powerful CAR-T cell activity as well as antibody- dependent cell cytotoxicity (ADCC), either simultaneously or sequentially. CARFcR T cells are not affected by soluble antibodies, but they can be redirected and activated by antibodies bound to target cells. Therefore, such T cells can be used to redirect adoptive T cell therapy towards another tumor-associated antigen "on the fly" using clinically available antibodies. They also provide a practical way to target multiple antigens simultaneously with a single receptor, a feature that should be useful in cell therapies directed against phenotypically heterogeneous tumors.

The invention relates to a DNA (deoxyribonucleic acid) ligation buffer solution and application thereof, the DNA ligation buffer solution comprises 5-15 mM of divalent metal ions, 0.5-5 mM of DTT (dithiothreitol), 10-15% of polyethylene glycol, 0.5-5 mM of ATP (adenosine triphosphate) and a Tris-HCl buffer solution, and the pH (potential of hydrogen) value of the Tris-HCl buffer solution is 7-9. The DNA ligation buffer solution can significantly improve the ligation reaction efficiency, and increase ligation products in the ligation reaction and the number of molecules capable of being sequenced during library construction.

The invention discloses an ultrasensitive immunodetection method based on adjacent nucleic acid amplification signal amplification in the technical field of medical detection. The ultrasensitive immunodetection method comprises the following steps: step 1, immune binding; step 2, enzyme digestion release: releasing a hairpin loop DNA single strand; step 3, complementation and enzyme ligation: adding T4 ligase to cyclize the circular DNA single chain; step 4, rolling circle amplification; and step 6, detecting, and quantifying and positioning by qPCR (quantitative polymerase chain reaction) and an in-situ oligonucleotide fluorescence labeling detection method. According to the present invention, the hairpin ring structure on the oligonucleotide DNA chain coupled to the antibody protects the complementary sequence so as to avoid the generation of the non-specific circular DNA single chain, and the rolling circle amplification technology is combined to amplify the detection signal, such that the detection background signal intensity is inhibited while the specificity of the detection signal is enhanced so as to further improve the detection sensitivity.

The invention relates to the technical field of high-throughput gene sequencing, and discloses a method for one-tube one-step construction of an amplicon library for detecting gene fusion, and the method for one-tube one-step construction of the amplicon library for detecting gene fusion comprises the following steps: carrying out joint connection on total cDNA of a sample in a connection system to obtain a connection product; the connecting system contains ligase and a short Y joint. According to the one-tube type one-step amplicon library construction method for detecting the gene fusion, in order to comprehensively detect the gene fusion and reduce the omission ratio, the gene fusion detection of known and unknown fusion partners is realized by using multiple single-ended anchoring PCR (Polymerase Chain Reaction) to construct a library and designing a specific primer aiming at a specific gene exon; by means of the method, all gene sequences of main gene exon fusion participating in fusion can be amplified without difference, and the problems that an existing detection method is prone to missing detection, and unknown fusion cannot be detected are effectively solved.

Disclosed herein are methods for improving highly sensitive immunoassays that utilize a capture / release mechanism to reduce non-specific binding.

The invention discloses a probe combination for detecting a mitochondrial respiratory chain super complex based on a proximity ligation assay and a hybridization chain reaction and application, and belongs to the field of probe detection. Two adjacent protein subunits capable of reflecting the structural integrity of the hypercomplex are determined as bases, the antigen-antibody combination principle is utilized to promote the antigen-antibody which recognizes and combines the bases to initiate proximity ligation reaction (PLA) due to nucleic acid labeling, cascade amplification of detection signals is realized according to the hybridization chain reaction (HCR) principle, and the detection sensitivity of the hypercomplex is improved. The activity of the oxidative phosphorylation system is judged through the structural integrity of the super-complex, the defects and limitation of a traditional detection method are overcome, and rapid, high-sensitivity and accurate detection on the structure of the super-complex is achieved. Meanwhile, the cells are fixed on a 96-hole fluorescent plate, and the structural integrity of the mitochondrial hypercomplex is detected by using a multifunctional microplate reader, so that parallel detection on 96 samples at the same time is finally realized, and digitization and high throughput of detection are realized.

The present invention refers to the medical field. Particularly, the present invention refers to a method for the generation of a cDNA library from an RNA sample, which comprises: a) providing an adenylated the RNA, b) hybridizing the adenylated RNA with a first synthetic oligonucleotide that comprises a poly T tail, c) reverse-transcribing the hybridized RNA to cDNA, d) attaching the cDNA strand to a functionalized particle through a chemical bond, e) dehybridizing the RNA to expose the cDNA, characterized in that the steps (b) and / or (c) are performed in solution, and in that the method does not comprise ligation reactions between oligonucleotides. This methodology has multiple applications, including miRNA sequencing and quantification.

The present disclosure relates to methods for spatial analysis using RNA templated ligation. Provided herein are methods of using RNA templated ligation to detect an analyte of interest to interrogate spatial gene expression in a sample.

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 invention discloses a method for synthesizing millions of unique DNA tags through primer combination and application of the method, and relates to the field of molecular biology. According to the method, partial complementary pairing characteristics of F, X and Y primers are utilized, and under the synergistic effect of T4 DNA polymerase and ligase, millions of unique DNA tags are generated through one-time reaction through annealing-filling-ligation three-step reaction. The label structure comprises 5'end functional modification sites (such as amino and biotin) and a customizable sequencing joint, and is suitable for high-throughput labeling scenes such as single-cell multiomics, space proteomics and antibody coupling (AOC). Compared with a traditional one-by-one synthesis method, the method has the advantages that the single-tag synthesis cost is reduced by 80% or above, a reaction system is compatible with automatic operation, the tag combination complexity can reach 5.6 * 10 (based on 384 * 384 * 384 primer combination), and an efficient solution is provided for large-scale molecular marking.

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 invention belongs to the technical field of biology, and particularly relates to a T4 DNA ligase mutant with improved connection fidelity. The T4 DNA ligase mutant is obtained by mutating any one or more amino acid sites of lysine at the 365th site and lysine at the 367th site of wild type T4 DNA ligase. Compared with a wild type T4 DNA ligase, the T4 DNA ligase has higher fidelity, and the fidelity of the T4 DNA ligase is maximally improved to more than 10 times of that of the wild type T4 DNA ligase.

The invention belongs to the technical field of biology, and relates to a zero-background carrier as well as a preparation method and application thereof. The zero-background vector provided by the invention comprises an optimized escherichia coli CcdB gene and a corresponding escherichia coli promoter, and the nucleotide sequence of the zero-background vector is shown as SEQ ID NO.1. The zero background vector is applicable to any plasmid vector; both a cloning vector plasmid and an expression vector plasmid have relatively high connection efficiency and positive rate, and a connection product can be directly converted into a common escherichia coli strain, so that a target gene cloning vector can be effectively prepared; the target plasmid inserted with the zero background carrier has high short fragment insertion efficiency, and can also be used for long fragment insertion at the same time. In the genome assembly process, no matter whether a small fragment or a large gene fragment is inserted, the method can ensure an efficient assembly effect, especially in small fragment gene cloning, the efficiency and the positive rate can be remarkably improved, and meanwhile generation of false positive cloning is reduced.

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.