541 results about "Ligation" patented technology

The present disclosure features methods, compositions, and kits for the spatial detection of genetic variants in a biological sample by using branched ligation.

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

The present disclosure provides methods and compositions for detecting and spatially locating analyte interactions and gene expression in a biological sample. For example, provided herein are methods of determining a location of at least one analyte in a biological sample using analyte-binding moieties, proximity ligation, and an array including capture probes.

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

Provided herein are methods, compositions, and kits for the spatial detection of analytes using ligation-based capture assays.

The present disclosure provides compositions and methods for generating a plurality of nucleic acid concatemers immobilized to a support by conducting on-support circularization and ligation reactions using a plurality of linear library molecules and a plurality of immobilized splint capture primers. The present disclosure provides methods for seeding and optionally re-seeding the support to increase the density of immobilized nucleic acid concatemers that can be sequenced. In some embodiments, the immobilized concatemers can be used for conducting downstream sequencing workflows including batch sequencing and reiterative sequencing workflows. The present disclosure also provides methods for interrupting an ongoing sequencing run to re-seed the support to generate additional concatemers that can be sequenced.

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 invention provides an anti-beta2-glycoprotein I antibody detection reagent and a chemiluminescence detection kit, and the anti-beta2-glycoprotein I antibody detection reagent comprises a biotin-labeled beta2-glycoprotein I antigen prepared by reacting biotin connected with an LPETG label with the beta2-glycoprotein I antigen under the catalytic action of Sortase A enzyme. The N terminal of the beta2-glycoprotein I antigen is labeled with the biotin with a specific label in a fixed point manner, all amino acid sequence regions (structural domains I-V) of the beta2-glycoprotein I antigen are not influenced, and meanwhile, the purity of a connection product is high, the difference between batches is small, and the method is suitable for large-scale production. The positive detection rate and the detection result accuracy of the anti-beta2-glycoprotein I antibody magnetic particle chemiluminescence detection kit can be improved, and the difference between batches can be reduced.

The invention provides a high-throughput single-cell multi-omics three-dimensional genome detection method and system. The detection method comprises the following steps: enzyme digestion reaction of fixed cells; carrying out proximity ligation treatment on the cells subjected to the enzyme digestion reaction; carrying out analysis treatment including reverse transcription on the treated cells, and respectively constructing a DNA library and an RNA library; and sequencing to obtain three-dimensional genomics information in a single cell. A high-throughput single-cell multi-omics three-dimensional genomics detection technology is designed, chromatin in cells is subjected to opening treatment through different methods, chromatin opening sites are enriched, the throughput of generated data is greatly improved, and the detection accuracy is greatly improved while the chromatin openness is detected in a single cell. The capability of detecting interaction between chromatin open sites and the gene expression capability are improved.

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 disclosure relates in some aspects to methods for analyzing a target nucleic acid in a biological sample (e.g., cell or tissue sample). In some aspects, provided herein are methods and compositions for analyzing a target nucleic acid in a ligation-based assay using one or more probes, and phosphorylation of one or more probes hybridized to a target nucleic acid is performed.

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 present invention provides a method for constructing a plasmid containing a tandem repeat sequence, comprising: providing a first upstream primer and a first downstream primer; the first upstream primer and the first downstream primer respectively have a tandem repeat sequence, and the tandem repeat sequence of the first upstream primer is complementary to at least part of the tandem repeat sequence in the tandem repeat sequence of the first downstream primer; annealing the first upstream primer and the first downstream primer under a gradient temperature condition to obtain a first double-stranded nucleic acid fragment having a tandem repeat sequence, wherein the tandem repeat sequence complementary to each other in the double strand is the first double-stranded tandem repeat sequence; connecting the first double-stranded nucleic acid fragment to a first vector backbone with a sticky end to construct a plasmid containing the first double-stranded tandem repeat sequence. The present invention can significantly improve the success efficiency of tandem repeat sequence connection, obtain more positive clones, ensure the success rate of the experiment, and reduce the construction time.

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 of sequencing comprising click chemistry bioconjugation. In some embodiments, target polynucleotide sequences on the same or different molecules are contacted with and hybridize to probes comprising click functional groups. Probes (e.g., reading probes) hybridizing to adaptor sequences adjacent to different sequences of interest (e.g., barcodes to be sequenced) can be hybridized simultaneously in large pools. In some embodiments, the provided methods achieve multiplexing without requiring separate hybridization of probes (e.g., reading probes) for each sequencing-by-ligation cycle, thereby reducing total hybridization time which is typically a most time-consuming step in in situ technologies. In some aspects, the hybridized probes (e.g., reading probes) are clicked onto detectable probes to analyze a sequence of a target polynucleotide in a sequencing-by-ligation fashion.

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 present disclosure provides methods for molecular neighborhood detection of molecules, such as by iterative proximity ligation or split-and-pool methods for obtaining positional information.

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.

The invention discloses a related product of inorganic pyrophosphatase in synthesis of a nucleotide chain and application, and relates to the technical field of nucleic acid synthesis. The inorganic pyrophosphatase is added in the synthesis process of the nucleotide chain, so that by-products generated in the ligation reaction process can be greatly reduced or eliminated, the reaction speed is increased, the conversion rate and purity of the target product are further improved, and the effect of the target nucleotide chain can be fully exerted; and a new way is provided for high-quality large-scale synthesis of the nucleotide chain.

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.