948results about "Library creation" patented technology

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 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 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 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 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 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.

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.

The utility model discloses a multi-nozzle flexible ink wiping device and a DNA chip printing synthesis system. The multi-nozzle flexible ink wiping device comprises a first bearing platform and a second bearing platform, the first driving assembly is mounted on the first bearing platform; the sliding block assembly is mounted at the power output end of the first driving assembly; the sliding block assembly has the freedom degree of moving along the longitudinal axis of the first bearing platform. A dirty non-woven fabric tightening assembly, a new non-woven fabric supply assembly and an ink wiping assembly are arranged on the sliding block assembly, a guide shaft is arranged between the dirty non-woven fabric tightening assembly and the ink wiping assembly, and a tensioning shaft is arranged between the ink wiping assembly and the new non-woven fabric supply assembly. The center connecting line of the guide shaft and the tensioning shaft is parallel to the center connecting line of the dirty non-woven fabric tightening assembly and the new non-woven fabric supply assembly, and the center connecting line of the guide shaft and the tensioning shaft is lower than the ink wiping assembly. The ink wiping assembly outwards tightly supports the non-woven fabric to form a flexible ink wiping area matched with the multiple sprayers. The multi-nozzle cleaning device can effectively clean multiple nozzles, and cross contamination does not exist.

Provided are a method for constructing a soluble expression plasmid mutant library, and the use thereof. The method for constructing the soluble expression plasmid mutant library comprises: introducing mutations into a ribosome binding site on a soluble expression plasmid, a sequence composition and length of a region between the ribosome binding site and an initiation codon of a fusion protein to be expressed, and a translation initiation region, so as to obtain a mutant library. By means of the method, plasmid mutants with increased expression levels are obtained, thereby increasing the expression level of a target fusion protein and the final yield of a target polypeptide. The present invention solves the problem of low soluble expression levels of certain polypeptides in Escherichia coli.

The invention relates to the technical field of biology, and particularly discloses a gene Panel sequencing method for intrahepatic cholestasis in a gestation period, which comprises the following steps: collecting saliva, performing DNA extraction and purification on a sample, breaking the purified sample, treating the broken sample to obtain a supernatant A, performing linker connection and purification on the supernatant A to obtain a supernatant B, and sequencing the supernatant B to obtain the gene Panel of intrahepatic cholestasis in a gestation period. The invention relates to a Panel detection technology for detecting 186 cholestasis genes covering bile acid metabolism, which comprises the following steps: preparing a supernatant B, carrying out pre-PCR and purification treatment on the supernatant B to obtain a pre-PCR product, carrying out liquid phase hybridization and capture, configuring a PCR reaction system, carrying out Post-PCR reaction and purification to obtain a supernatant C, quantifying the supernatant C, screening ICP susceptible genes through mixed sample sequencing, collecting DNA (Deoxyribonucleic Acid) through a saliva sampling mode, and detecting the 186 cholestasis genes covering bile acid metabolism. Other hereditary intrahepatic cholestasis diseases can be excluded, and ICP genetics susceptibility genes are defined.

The invention discloses a primer composition and a kit for identifying testudinate species and application of the primer composition and the kit. The primer composition comprises primers (SEQ ID NO.1-6) for targeted amplification of specific marker genes 16S, COI and Cytb of testudinate mitochondrial species and an enrichment universal primer (SEQ ID NO.7). According to the invention, full lengths of three mitochondrial genes 16S, COI and Cytb are taken as targets, a specific primer group added with an enrichment tag and an enrichment primer are designed, and a targeted nanopore sequencing method capable of realizing testudinate species identification is established and optimized, and the method can simultaneously capture testudinate mitochondrial species specific marker gene information of a family species level, and can be used for identifying testudinate species. The problem that the existing DNA bar code is difficult to accurately identify the testudinate species due to the lack of database information is effectively solved; and each marker gene can obtain a complete coding sequence, so that genetic information is richer, and the method can be used for geographical traceability analysis of testudinate species.

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 invention relates to a construction method of a nanometer antibody coding DNA library. The method comprises the following steps: screening animal peripheral blood mononuclear cells (PBMC) by using immunogen to obtain positive cells specifically bound with the immunogen; and amplifying a nucleic acid sample derived from the positive cells so as to obtain a coding DNA library of the nano-antibody, wherein the nano antibody is a nano antibody specifically binding to the immunogen, and wherein the amplification comprises the use of one or more groups of primer pairs, each group of primer pairs of the one or more groups of primer pairs comprising one or more forward primers and one or more reverse primers. A large number of diversified DNA sequences covering a large number of low-abundance sequences are obtained. According to the technical route for rapidly preparing the VHH antibody based on high-throughput sequencing, the high-affinity and high-specificity VHH antibody can be efficiently and rapidly obtained, and the raw material requirement of a high-quality diagnostic reagent is met.

The invention discloses a method and a device suitable for CUAMP. The invention discloses a method for extracting liriodendron tulipifera cell nucleuses by a Tag technology and application of the liriodendron tulipifera cell nucleuses. Hybridized liriodendron tulipifera calluses are used as materials, and protoplasts are released through enzymolysis solution vacuumizing and mild enzymolysis for 1.5 h; the method comprises the following steps: sequentially purifying by using a W5 solution and mannitol, detecting the activity by FDA, cracking by using an NE buffer solution, and washing by using a Wash buffer solution in two steps, thereby obtaining 2 * 10 < 6 >-3 * 10 < 6 > high-purity and high-integrity cell nucleuses. The obtained core is clean in background and complete in membrane structure, and can be directly used for CUTamp; tag is used for building a library, so that the magnetic bead capturing efficiency is improved by 46%, and protein-DNA interaction high-throughput sequencing under the condition of low sample size is realized. The method disclosed by the invention is simple and convenient to operate and good in repeatability, and provides key technical support for epigenetic research of rare tree species such as liriodendron tulipifera.

The present invention relates to the technical field of biology, and specifically relates to a primer set and the use thereof in the preparation of a thalassemia detection kit. The primer set comprises primers designed on the basis of at least one of sequences 1)-13): 1) chr16:219267-219290; 2) chr16:228804-228829; 3) chr16:215255-215276; 4) chr16:235945-235971; 5) chr16:196488-196508; 6) chr11:5276986-5277008; 7) chr11:5257290-5257314; 8) chr11:5249915-5249939; 9) chr11:5258978-5259000; 10) chr11:5221888-5221912; 11) chr11:5189975-5189996; 12) chr11:5152341-5152365; 13) chr11:5133905-5133925, wherein the primers are at least partially complementary to sequences 1)-13).

The application belongs to the field of nucleic acid sequencing, and specifically discloses a method for constructing an RNA library. The method comprises the following steps: screening mRNA with a 5' end cap, connecting the mRNA with a 5' end adaptor to obtain a connection product, and then using a reverse transcription primer with a single molecule tag sequence to perform reverse transcription on the connection product, and further designing a suitable amplification primer to perform PCR amplification on the adaptor connection product, so as to obtain the RNA library. Compared with the prior art, in the method, the single adaptor is connected to the 5' end of the complete and intact RNA, the specific first-strand cDNA is synthesized through reverse transcription, the synthesis of the second-strand cDNA is not required, other breaking and digestion processes are not required, and the chain specificity is directly achieved, so that the operation is simple, and a large amount of working hours is saved. The method can reduce the requirement for the initial amount of the sample, reduce the cost of library construction, simplify the detection process, and save the operation time.

The invention provides a DNA library screening method and application of an intracellular mRNA sensor switch. The DNA library screening method comprises the following steps: designing a DNA library containing a switch sequence of a degenerate basic group; preparing a circular expression vector library; preparing a minimum linear expression vector library; performing seamless cloning; carrying out RCA rolling circle amplification; carrying out telomerase digestion; carrying out cell transfection; extracting RNA (Ribonucleic Acid) and carrying out reverse transcription; carrying out library building of next-generation sequencing by taking the cDNA as a template; carrying out next-generation sequencing; analyzing data and comparing to obtain the DNA library of the mRNA sensor switch. According to the DNA library screening method provided by the invention, more sensor switch sequences can be provided for a single site on mRNA in cells, and more selection possibilities are further provided for subsequent targeted therapy.

The invention provides a capture primer group and a kit for targeted Salivirus genome sequencing and application of the capture primer group and the kit, and belongs to the technical field of genome sequencing. The targeted capture primer group of the Salivirus virus genome comprises a primer 1 and a primer 2, the nucleotide sequence of the primer 1 is as shown in SEQ ID NO. 1, and the nucleotide sequence of the primer 2 is as shown in SEQ ID NO. 2. According to the invention, a universal reverse transcription oligo-T primer and the Salivirus specific primer group provided by the invention are put into the reverse transcription step at the same time, so that not only is a macro transcription effect achieved, but also a Salivirus sequence in a sample is synchronously identified for specific capture. According to the method, the complete Salivirus genome sequence can be efficiently obtained, and the method has good coverage and sequencing adaptability to the virus genome.

Processes and materials to detect neoplasms from a biopsy are described. Processes and materials to build a sequencing library are described. Processes and material to perform targeted sequencing are described. Processes and materials to mitigate confounding sources are described. Cell-free nucleic acids can be sequenced and the sequencing result can be utilized to detect sequences derived from a neoplasm.

The invention relates to a capture probe group, a kit and a sequencing library for detecting polymorphism of related genes of solid tumor chemotherapy drugs and a construction method of the sequencing library, and belongs to the technical field of high-throughput detection. The capture probe group for detecting the polymorphism of the related genes of the solid tumor chemotherapy drugs comprises nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.12; the related genes of the solid tumor chemotherapeutic drugs comprise MTHFR (Methylene Tetrahydrofolate Reductase), TP53, TPMT (Trimethylparaben Methyl Methyl Thiazolyl Tetrazolium), XRCC1 (XRCC1), DPYD ( According to the capture probe group, genotypes of genes related to solid tumor chemotherapy can be rapidly obtained through one-time detection, a sequencing library is constructed on the basis of the genotypes, and related information such as medication prompt, drug curative effect, toxicity and metabolic capacity corresponding to a clinical sample is obtained according to the genotypes of the sequencing library; the kit is simple and convenient to operate, consumes less time and is extremely easy to realize automatic detection.

Embodiments of systems, methods, and compositions provided herein relate to assays for selectively controlling enzymatic reactions. Some embodiments relate to methods of inhibiting, reducing, or eliminating secondary DNA (such as mitochondrial DNA) sequencing reads from open chromatic sequencing, whole genome sequencing, or targeted sequencing.

The application belongs to the technical field of forensic medicine, and particularly relates to a multiple SNP combined microhaplotype amplification system and construction method, product and application based on one-step library construction. The application is based on RC-PCR technology, and constructs a one-step library construction composite amplification system. The amplification system comprises RC-Primer with a sequence as shown in SEQ ID No. 1-SEQ ID No. 210, I5-Primer with a sequence as shown in SEQ ID No. 211-SEQ ID No. 214, and I7-Primer with a sequence as shown in SEQ ID No. 215-SEQ ID No. 222. The method provided by the application realizes one-time PCR reaction, simultaneously completes targeted amplification and library construction, greatly shortens experimental time and simplifies experimental steps, and the advantage is more significant when a large number of samples are simultaneously detected.

This invention relates to a method for designing and utilizing a broad RNA modification library that includes all surrounding sequences of the RNA modification. By introducing multiple types of RNA modifications, this invention enables the construction of RNA modification libraries that encompass all possible combinations of surrounding sequences flanking the modified RNA. Therefore, it differs from existing reported methods for constructing RNA modification libraries. Furthermore, using the RNA modification library of this invention for single-molecule RNA sequencing generates a high-quality reference dataset containing all possible combinatorial motifs. If this dataset is used as training data for deep learning (or machine learning), it overcomes the limitations of existing software that is limited to specific RNA modifications or has low prediction accuracy for other modifications. This allows for the development of novel, more accurate RNA modification detection software and the simultaneous detection of multiple RNA modifications.

The invention discloses a primer group and method for human metapneumovirus whole genome sequencing and application. Two groups of specific primer pools are designed for the metapneumovirus, totally comprise 21 primers, can effectively amplify the metapneumovirus type A and the metapneumovirus type B, are high in sensitivity and suitable for multi-platform detection, and provide a new direction for detection, typing, traceability and evolutionary analysis of the metapneumovirus.