1290results about "Library creation" patented technology

The disclosure provides systems and methods for droplet processing. For example, a method can include providing a first droplet and a second droplet. In some cases, the first droplet may have a first concentration of a reagent and the second droplet may have a second concentration of the reagent. The second droplet may comprise a bead or a biological particle. The method can also include subjecting the first droplet and the second droplet to conditions sufficient to transfer the reagent from the first droplet to the second droplet, thereby decreasing the first concentration in the first droplet and increasing the second concentration in the second droplet.

Strategies, systems, methods, reagents, and kits for phage-assisted continuous evolution are provided herein. These include strategies, systems, methods, reagents, and kits allowing for stringency modulation to evolve weakly active or inactive biomolecule variants, negative selection of undesired properties, and / or positive selection of desired properties.

The invention discloses preparation of a human CD56 immune phage display antibody library, which comprises the following steps: S1, animal immunization: expressing recombinant hCD56 protein in escherichia coli, then immunizing a Balb / c mouse, and detecting immune titer; s2, splenocyte preparation: extracting RNA (Ribonucleic Acid) of immune mouse spleen cells, and performing reverse transcription to obtain cDNA (Complementary Deoxyribonucleic Acid); s3, preparation of an initial bacteriophage display library: amplifying a heavy chain variable region and a light chain variable region by using a PCR technology, and then assembling to form scFv; constructing scFv phagocytids, electrically transforming the scFv phagocytids into TG1 competent cells, and constructing a bacterial library; s4, constructing a phage single-chain library of the hCD56: infecting the bacterial library by using a helper phage M13K07, and constructing a phage single-chain antibody library; and S5, elutriation and screening of a phage library: specifically enriching a phage single-chain antibody library, screening positive clones from the phage single-chain antibody library, and determining a sequence. According to the invention, the human hCD56 antibody scFv sequence fragment can be screened out, and the research and development period is shortened.

The invention discloses a recombinant fusion protein of a human lambda light-chain single-chain antibody, a DNA (deoxyribonucleic acid) molecule, a library building method and application, and the recombinant fusion protein of the human lambda light-chain single-chain antibody is the recombinant fusion protein formed by connecting the human lambda light-chain single-chain antibody, phage Piii protein and luciferase, the amino acid sequence of the recombinant fusion protein of the human lambda light-chain single-chain antibody is shown as SEQ ID NO: 5, the recombinant fusion protein library of the human lambda light-chain single-chain antibody has large capacity, the established single-chain antibody TG1 library is more than 1 * 10 < 9 > cfu / mL, positive clones capable of being combined with ligands can be screened and enriched for multiple rounds, and the recombinant fusion protein library has the characteristics of high sensitivity, high signal-to-noise ratio and the like.

The invention provides a system and a method for screening Cas protein mutants capable of identifying different PAM sequences, and belongs to the technical field of bioengineering. The system comprises a Cas protein mutant screening vector and an sgRNA expression vector, the Cas protein mutant screening vector comprises the following expression elements: a Cas protein coding gene, a plasmid replicon and a first resistance screening tag expression cassette; the sgRNA expression vector comprises the following expression elements: a plasmid replicon, a second resistance screening tag expression cassette, a third resistance screening tag expression cassette and an sgRNA expression cassette. According to the system and the method, aiming at the problem that the efficiency of identifying PAM sequences except NGG by wild type spCas9 protein is low, an active Cas9 mutant capable of identifying PAM sites except NGG is screened out, and the application field and the editing efficiency of a CRISPR gene editing system can be greatly expanded.

The invention provides systems, methods, reagents, apparatuses, vectors, and host cells for the continuous evolution of nucleic acids. For example, a lagoon is provided in which a population of viral vectors comprising a gene of interest replicates in a stream of host cells, wherein the viral vectors lack a gene encoding a protein required for the generation of infectious viral particles, and wherein that gene is expressed in the host cells under the control of a conditional promoter, the activity of which depends on a function of the gene of interest to be evolved. Some aspects of this invention provide evolved products obtained from continuous evolution procedures described herein. Kits containing materials for continuous evolution are also provided.

The invention discloses a liquid-phase hybridization probe pool for joint detection of multiple pathogens and drug-resistant genes. The liquid-phase hybridization probe pool is designed according to target areas of target pathogens and drug-resistant genes; the probe is a probe with unequal length of 30 to 100 nt; comprising a target specific sequence complementarily paired with a target region and a conjugate sequence complementarily paired between adjacent probes; the 5'terminal and the 3 'terminal of the probe are respectively modified with a conjugate sequence A and a conjugate sequence B, so that the adjacent probes can be specifically combined through terminal pairing; the conjugate sequence A is as shown in SEQ ID No. 601, and the conjugate sequence B is as shown in SEQ ID No. 602. According to the method, the constructed library and the specific probe pool are hybridized, so that the capture time is shortened to 30 minutes, and the method is obviously superior to a traditional method. Through optimization, the operation time and complexity are reduced, personal errors are reduced, meanwhile, high sensitivity and specificity are kept, target nucleic acid in a complex sample can be efficiently captured, a high-quality template is provided for subsequent sequencing, and combined detection of multiple respiratory pathogens and drug-resistant genes is supported.

The invention relates to a high-throughput cis-acting element screening carrier and a screening method. Specifically, the invention provides a plasmid vector system containing bar codes, each bar code in the system is in one-to-one correspondence with a candidate cis-acting element, and the activation multiple of the candidate cis-acting element can be obtained by measuring the abundance of the bar codes; in order to eliminate the influence of a bar code on the vector on a detection result, an exogenous intron which can be cut off during transcription is inserted into a coding region in the vector and is used for distinguishing vector DNA and RNA obtained by transcription. The screening method of the biological cis-acting element has high efficiency, wide applicability and high throughput, and has outstanding application value in biological research and breeding.

The invention relates to the technical field of polymerase, and provides a high-fidelity Pfu DNA polymerase mutant and a preparation method and application thereof.The mutant at least comprises one mutation of the following sites on the basis of wild-type Pfu DNA polymerase with the amino acid sequence shown as SEQ ID NO: 1: V82A, G211R, E383K, R594K and W616V. A plurality of mutant Pfu DNA polymerases with good enzyme activity and high fidelity are obtained by performing mutation modification on amino acids at multiple sites (V82A, G211R, E383K, R594K and W616V) of wild Pfu DNA, and mutants with five sites protruding all have the best enzyme activity and the highest fidelity and are suitable for a DNA sequencing technology, an NGS library building technology and the like.

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 belongs to the technical field of biological detection, and relates to a capture probe, a kit comprising the same and application of the capture probe in detection of T cell and B cell immune repertoire under spatial resolution. The capture probe comprises a transcript specific sequence; the transcript specific sequence is derived from the front 40 bp of a reverse complementary sequence of a J region transcript sequence, and the length of the transcript specific sequence is 20-35 bp. The advantages of using the capture probe to detect T cell and B cell immune repertoire under spatial resolution include that not all RNA with poly A tail is reversely transcribed, but only targeted RNA is reversely transcribed, so that the sequencing cost is remarkably reduced, and the detection is not limited by the poly A degradation degree of RNA in a sample.

The invention relates to a method for producing nano-arrays and micro-arrays. The method described herein provides a method of generating an array of spatially separated proteins. The method relies on covalently attaching each of a plurality of proteins to a structured nucleic acid particle (SNAP) and attaching the SNAP to a solid support.

The invention provides a macro virus group sequencing library construction method for removing host ribosome RNA through an enzyme method and a kit, and belongs to the technical field of biology. The method comprises the following steps: hybridizing total RNA (Ribonucleic Acid) of a sample with a DNA (Deoxyribose Nucleic Acid) probe aiming at host ribosome RNA to form a DNA / RNA hybrid chain; rNase H is used for digesting RNA in the hybrid chain, and DNase I is used for digesting the remaining DNA probe in the system; purifying to obtain virus RNA (Ribonucleic Acid); carrying out fragmentation treatment on the virus RNA; carrying out reverse transcription on the fragmented RNA to synthesize a first chain cDNA, then synthesizing a second chain cDNA, and carrying out terminal repair and dA tail addition to obtain double-chain cDNA; connecting the linker with the dT tail with the double-chain cDNA; and carrying out PCR amplification and purification on the connection product to obtain the macro virus group sequencing library. According to the construction method and the kit, the problem that the full-length sequence of the virus cannot be obtained from a low-load sample can be solved.

The invention provides a new bunyavirus neutralizing antibody screening method based on single cell BCR sequencing. The new bunyavirus neutralizing antibody screening method comprises the following steps: S1, collecting peripheral blood B cells of acute stage and recovery stage patients and healthy volunteers of new bunyavirus infected persons; s2, carrying out CD19 < + > CD38 < + > CD138 < + > magnetic bead separation to obtain a single-cell suspension; s3, constructing a single cell transcriptome and a BCR library, separating single cells by using a microfluidic system, and performing high-throughput sequencing; s4, analyzing a B cell subset, a BCR cloning amplification mode and a VJ gene pair enrichment characteristic through bioinformatics analysis; and S5, screening a specific BCR clone type related to the severity of the disease as a neutralizing antibody candidate target. The method provided by the invention provides powerful support for immune monitoring and vaccine design of new bunyavirus infection, and is expected to provide new strategies and methods for clinical treatment and prevention.

The invention provides a construction method of a plant tissue space transcriptome sequencing library, which comprises the following steps: fixing and embedding plant tissues to obtain embedded blocks; performing autofluorescence detection and tissue permeabilization treatment on part of the embedded blocks, and determining an autofluorescence threshold value and target tissue permeabilization time; slicing, pasting and fixing the remaining embedding blocks, and performing microscope fluorescence scanning detection and tissue permeabilization treatment on the tissue-containing chip according to an autofluorescence threshold value and target tissue permeabilization time to obtain a permeabilized tissue chip; and carrying out reverse transcription, tissue removal, cDNA release, recovery and amplification on the permeabilized tissue chip to obtain a cDNA amplification product. The plant tissue transcript constructed by the construction method is not easy to diffuse and high in capture rate, high-quality in-situ capture time-space group data can be obtained, the accuracy and the credibility are high, the sequencing accuracy is ensured, and the application value is high.

The invention discloses a method for annotating a vesicle cell subset based on monovesicle membrane proteomics, and belongs to the technical field of vesicle cell annotation. The method comprises the following steps: 1, carrying out exosome collection on a plasma sample to obtain a sample; 2, coding and labeling the antibody probe, and adding a protein tag and a labeled DNA sequence into the antibody probe to obtain a labeled antibody probe; 3, preparing a corresponding combined product; 4, adding a labeled antibody probe into the sample to obtain an exosome compound; step 5, capturing an exosome conjugate through CTB; 6, adding the binding product into the exosome conjugate to generate a sequence; 7, constructing a sequence library and sequencing to obtain a sequencing sequence; 8, removing a sequencing sequence with low expression quantity to obtain an exosome expression data matrix, and standardizing the exosome expression data matrix; 9, carrying out clustering analysis to find out an exosome core subgroup; and step 10, determining the source of the exosome core subgroup, and annotating the single vesicle subgroup.

The invention provides a simplified genome sequencing library construction method and a kit and related application thereof. The construction method of the simplified genome sequencing library comprises the following steps: (1) respectively carrying out enzyme digestion on a plurality of DNA (Deoxyribonucleic Acid) samples by using restriction endonuclease; wherein restriction endonucleases adopted by all the samples are different from one another, and the tail ends of specific DNA fragments of the samples are generated to serve as sample bar codes of the samples; (2) mixing the enzyme digestion products of the samples, constructing a DNA library, carrying out fragment sorting on the DNA library, and taking the selected library as a simplified genome sequencing library; or carrying out fragment sorting on the enzyme digestion product of each sample, and carrying out DNA library construction by using the selected fragments to obtain the simplified genome sequencing library. According to the method, the experimental operation process is simplified, the library building efficiency is improved, and meanwhile, the feasibility of the restriction endonuclease for recognizing 6 basic groups based on a third-generation long-read-long sequencing platform in the simplified genome sequencing technology is also explored.

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 invention provides a method for constructing a sequencing library, the sequencing library, a sequencing method and a kit for constructing the sequencing library. The method for constructing the sequencing library comprises the following steps: performing digestion treatment on a to-be-detected sample connected with a joint under the action of exonuclease, so as to obtain a target sequencing library.

The invention relates to the technical field of molecular biological detection, and particularly provides a reagent for respiratory tract infection pathogen identification and drug resistance and virulence gene detection. Each primer in the plurality of primers comprises a first sequence as shown in SEQ ID NO: 1-165 and an optional second sequence for distinguishing sample sources. Target spots detected by the super-multiplex primer combination comprehensively cover pathogen directories (totally 353 types) of latest respiratory tract infection diagnosis guidelines at home and abroad, low-cost, high-sensitivity, high-specificity and high-flux detection is achieved, and clinical medication can be effectively and comprehensively guided.

The invention discloses a nano antibody expression vector, which is characterized in that pET28a (+) plasmid is used as a vector skeleton, a nano antibody gene is connected to the downstream of a T7 promoter of the vector skeleton, and a maltose protein gene is connected or not connected between the T7 promoter and the nano antibody gene; the vector expressed by using the nano antibody expression vector can have excellent living activity without purification. The invention further provides a method for displaying the nano-antibody library by using escherichia coli based on the nano-expression vector, and compared with the existing method for displaying the nano-antibody library by using bacteriophage, the method for displaying the nano-antibody library has the advantages that the yield is high; the library diversity is higher, the amino acid loss rate is low, the efficiency is high, the cost is low, the period is short, and the specific nano antibody can be obtained without screening by a flow instrument.

The invention belongs to the technical field of molecular biology, and particularly discloses a specific nano antibody targeting bovine TLR7 protein as well as a screening method and application of the specific nano antibody. The nano antibody can be specifically combined with a B cell antigen epitope of bovine TLR7 protein, and the B cell antigen epitope is selected from SEQ ID NO: 1 and / or SEQ ID NO: 2. The invention discloses a specific nano antibody targeting bovine TLR7 protein as well as a screening method and application of the specific nano antibody. The specific nano antibody targeting bovine TLR7 protein is high in stability and specificity and has super-strong affinity.

The invention is directed to methods for massively parallel template-free enzymatic synthesis of a plurality of different polynucleotides of predetermined sequences. In one aspect, methods of the invention employ large scale arrays of reaction sites each associated with at least one working electrode for controlling deprotection and deblocking steps at predetermined user selected sites. In another aspect, the invention provides template-free enzymatic synthesis with proofreading, wherein completed polynucleotides at predetermined reaction sites are sequenced using a sequencing by synthesis technique, particularly employing electrochemically labile blocking groups.

The invention is directed to methods for massively parallel template-free enzymatic synthesis of a plurality of different polynucleotides of predetermined sequences. In one aspect, methods of the invention employ large scale arrays of reaction sites each associated with at least one working electrode for controlling deprotection and deblocking steps at predetermined user selected sites. In another aspect, the invention provides template-free enzymatic synthesis with proofreading, wherein completed polynucleotides at predetermined reaction sites are sequenced using a sequencing by synthesis technique, particularly employing electrochemically labile blocking groups.

The invention provides an oligonucleotide set and a multiple amplicon library building method, and belongs to the technical field of biology. According to the method provided by the invention, a connection product or an extension product can be generated from a gene-specific multiple amplification primer and a library amplification primer in a system in a connection or complementary extension manner, and then the construction of the amplicon library is completed in one step by taking the connection product or the extension product as a long primer. Compared with a traditional multiple amplicon library building method, the method provided by the invention has the advantages that the uncovering operation step can be reduced, and the risk of cross contamination is avoided.

PURPOSE: To provide a biochip and a manufacturing method for the same.SOLUTION: A detection unit of a biochip includes a substrate, a first insulating layer, a semiconductor layer, a second insulating layer, a first metal layer, a second metal layer, and a protection layer. The semiconductor layer includes a reaction region. The second insulating layer is disposed on the semiconductor layer, and includes a first part, a second part, and a first opening part for exposing the reaction region. The first metal layer is disposed on the second part and includes a source, a drain, a gate, and a first wall structure. The second metal layer includes a second wall structure disposed on the first wall structure. The protection layer is disposed on the first metal layer and the second metal layer. The protection layer includes a second opening part, a third opening part, and a fourth opening part, and includes a flat part that defines the second opening part by surrounding it, a first projection part that defines the third opening part by surrounding it, and a second projection part that defines the fourth opening by surrounding it. In a normal direction of the substrate, the first opening part, the second opening part, the third opening part, and the fourth opening part overlap with each other.SELECTED DRAWING: Figure 4

The invention provides a primer group for high myopia gene detection, application of the primer group, a kit and a high myopia gene detection method, and relates to the technical field of gene detection. Nucleic acid sequences of the primer group for high myopia gene detection are shown as SEQ ID NO.1-296, and every two primers sequentially form a forward primer and a reverse primer according to the sequence numbers of the nucleotide sequences from small to large. The primer group is high in specificity and sensitivity, 89 high-myopia genes and 166 loci can be detected through one-time PCR amplification in the same tube, the detection efficiency of the high-myopia genes is effectively improved, meanwhile, the detection cost is reduced, and convenience is provided for prediction of high-myopia susceptible people.

The invention discloses a method for detecting accessibility of space chromatin and application of the method. The method comprises the following steps: (1) slicing a tissue sample to be detected on a chip with spatial information; (2) fixing, permeabilizing and dyeing a tissue sample to be detected; (3) preparing fragmented genome DNA (deoxyribonucleic acid); (4) performing DNA hybridization based on a transfer mode; (5) carrying out connection and terminal repair reaction on the fragmented genome DNA by using DNA polymerase and DNA ligase; (6) denaturation and melting; and (7) sequencing. According to the method, the transcriptional activity state of chromatin can be reflected through chromatin accessibility sequencing analysis, staining imaging and chromatin accessibility information can be obtained on the same tissue slice at the same time without complex micro-fluidic equipment, experimental operation is easy, repeatability is high, and the analysis result is more remarkable.

Provided is a sequencing method. The sequencing method comprises: providing a solid support, wherein the solid support is provided with a first site, a plurality of nucleic acid molecules are immobilized at the first site, and the plurality of nucleic acid molecules have n nucleic acid templates having different sequences, and n is a natural number ranging from 2 to 4; implementing multiple cycles of base extension on the nucleic acid molecules, acquiring sequencing signals of channels in a base extension process. and in one or more cycles of base extension reaction, sequencing signals at the first site at least including a first sequencing signal having the highest signal intensity and a second sequencing signal having the second-highest signal intensity; and on the basis of the intensity difference of the sequencing signals of the channels, classifying the sequencing signals, and determining sequencing results of the at least two nucleic acid templates. According to the embodiments, by using the sequencing method, sequencing signals can be effectively classified by means of the intensity difference, thereby fully utilizing a multi-molecule signal site, and improving the sequencing throughput.