595results about "Library creation" patented technology

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 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 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.

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 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 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.

The invention provides a rabbit-source BCR sequence primer group based on high-throughput single cell V (D) J sequencing and application of the rabbit-source BCR sequence primer group. The primer group comprises a specific nested PCR (Polymerase Chain Reaction) primer pair (the sequence is as shown in SEQ ID NO.1-12) aiming at a rabbit BCR heavy chain (IgA / IgE / IgG / IgM) and a light chain (IgK / IgL). Through microfluid single-cell separation, nested PCR amplification and high-throughput sequencing, the naturally paired rabbit BCR full-length V (D) J sequence can be efficiently obtained. According to the rabbit-derived BCR sequence sequencing primer provided by the invention, B cell receptor library information of different tissue sources can be quickly and efficiently obtained. According to the method, paired antibody sequence information can be specifically obtained, B cell response diversity after rabbit immunization vaccine or pathogen infection can be better known, and important data support is provided for design, optimization, effect evaluation and the like of biological products.

The present disclosure generally relates to devices, compositions and methods for designing and producing nucleic acid molecules and the production of encoded proteins using these nucleic acid molecules. In some aspect, the disclosure relates to automation for the in vitro generation of coding DNA molecules, the in vitro transcription of these DNA molecules to generate protein coding RNA molecules, and the in vitro translation of these protein coding RNA molecules to produce proteins.

The present application provides detection and prognosis of infectious diseases. In particular, the present application provides fragment length profiles of nucleic acid libraries, methods of generating fragment length profiles of nucleic acid libraries, and methods of using the fragment length profiles for diagnosis and / or prognosis. The present application further provides methods, compositions, and kits for determining the stage or site of infection in a subject.

The invention discloses a preparation method of a space chip, the space chip and application of the space chip, the preparation method comprises the following steps: hybridizing: connecting a single-stranded nucleic acid molecule to the surface of a solid-phase substrate, the single-stranded nucleic acid molecule comprises a first fixed sequence, a space bar code sequence, a sequencing primer binding sequence and a second fixed sequence, the surface of the solid-phase substrate of the chip substrate is connected with a first probe, and the first probe comprises a first fixed sequence complementary sequence and a second fixed sequence complementary sequence; carrying out ring closing to enable the single-stranded nucleic acid molecules to form annular nucleic acid molecules; carrying out rolling circle amplification; sequencing to obtain sequence information of the space bar code sequence; synthesizing single-stranded nucleic acid molecules by taking the rolling circle amplification product as a template to obtain a single-stranded nucleic acid molecule library; fixing and clustering: fixing the single-stranded nucleic acid molecule library on the surface of a solid-phase substrate to form a single-stranded nucleic acid molecule cluster. According to the invention, the chip with the nucleic acid probe carrying spatial position coordinate information can be prepared.

Metastasis is a major cause of fatality and expense in cancer. Many metastatic patients express calcified, fibrotic, or deep-seated (lymph / Jung / brain) lesions that are not easily profiled. For such patients, decision-making regarding treatment selection is population-based and heavily reliant on standards of care and are not tailored to the individual's tumor. The present disclosure provides methods of identifying treatment protocols for cancer patients using released tumor cells (RTCs). Method described herein include isolating / enriching RTCs, profiling RTCs and comparing RTCs to reference RTC profile libraries.

The application discloses a method for screening garlic fructan adversity response key enzyme genes and metabolites based on transcriptome and metabolome, and the method can better analyze the relationship between genes and metabolites by jointly analyzing the fructan-related differentially expressed genes measured by the transcriptome and the fructan-related metabolism substances with obvious metabolism changes measured by the metabolome, and constructing a gene-metabolite network interaction diagram.

This invention provides a DNA polymerase comprising: a) the amino acid sequence shown in SEQ ID NO: 2; b) an amino acid sequence having one or more substitutions, deletions, and / or additions of amino acids compared to the amino acid sequence shown in SEQ ID NO: 2, and possessing polymerase function; or c) an amino acid sequence having at least 86% sequence identity compared to the amino acid sequence shown in SEQ ID NO: 2, and possessing polymerase function. The DNA polymerase exhibits good thermostability, 5'→3' polymerase activity, 3'→5' exonuclease activity, and good PCR amplification performance. It has significant application potential and room for modification, and can be further optimized according to different application scenarios, possessing considerable room for improvement and application potential.

This application belongs to the field of molecular biology technology, specifically relating to nucleic acid combo products, kits for mycobacterial identification, and their applications. This application provides a nucleic acid combo product for mycobacterial detection, comprising one or more primer pairs with nucleotide sequences as shown in SEQ ID NO.1 to SEQ ID NO.244. This specific primer combination can simultaneously detect multiple mycobacteria and distinguish them down to the subspecies level. Through highly specific primer design, this nucleic acid combo product can achieve accurate identification of common clinical mycobacteria (including complex groups, species, and subspecies levels), with a detection sensitivity of 50-100 CFU / mL, significantly superior to the detection performance of existing technologies. Furthermore, the nucleic acid combo product of this application can not only identify the species of mycobacteria but also simultaneously detect drug resistance gene mutations, providing important evidence for the selection of clinical treatment regimens.

The invention relates to a method for evaluating and positioning nucleic acid pollution of a nucleic acid extraction kit, which comprises the following steps: 1) taking nucleic acid-free water as a negative sample, and performing nucleic acid extraction on the nucleic acid-free water by using the nucleic acid extraction kit according to nucleic acid extraction steps in the kit, the obtained nucleic acid is presumed as the polluted nucleic acid in the nucleic acid extraction kit; 2) amplifying polluted nucleic acid, purifying a PCR product, enriching double-chain cDNA, and determining the concentration of the double-chain cDNA so as to perform quantification, sample mixing and quality control treatment before library construction; (3) carrying out high-throughput deep sequencing by adopting a platform of the Illumina company, and then carrying out bioinformatics analysis to obtain classification information and gene sequences of polluted nucleic acid; and 4) designing a specific primer based on the gene sequence of the polluted nucleic acid, and carrying out pollution evaluation and positioning verification on each component of the nucleic acid extraction kit through qPCR (Quantitative Polymerase Chain Reaction).

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

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

The invention relates to the technical field of bioinformatics, in particular to a Hi-C high-throughput sequencing database building method for eucheuma. The method comprises the steps of pretreatment, formaldehyde crosslinking, cell lysis and enzyme digestion. The pretreatment comprises the following steps: removing mucus on the surface of the eucheuma in a physical scraping manner; cutting into small blocks, and sequentially cleaning with an interferent cleaning reagent, ethanol and water; the interferent cleaning reagent takes water as a solution and comprises Tris-HCl, EDTA (Ethylene Diamine Tetraacetic Acid) glycerol and PEG (Polyethylene Glycol) 8000. The special Hi-C high-throughput sequencing library building method is provided for the eucheuma, a wax coat on the surface of the eucheuma is treated through a specific pretreatment process, and the level of impurities such as polysaccharides, polyphenols, minerals and various endogenous inhibitors in a sample after subsequent cell lysis can be effectively reduced; and the integrity of the cell nucleus can be maintained as far as possible, so that the eucheuma Hi-C library with extremely high quality (especially high effective data volume) can be constructed.

Flow cells and methods are described that enable control over DNA fragment length bias during amplification. One example flow cell includes a substrate and a polymeric hydrogel positioned over at least a portion of the substrate. A plurality of oligonucleotide primers may be attached to the polymeric hydrogel, wherein the plurality of oligonucleotide primers defines a density gradient across the substrate of the flow cell.

The present disclosure relates to methods of producing and screening libraries of disulfide rich proteins, for instance to identify binding peptides specific for a target molecule. In some embodiments, the binding peptides comprise disulfide rich proteins. Also provided herein are methods of producing and screening libraries comprising disulfide-rich proteins and molecular chaperones. The present disclosure also relates to methods of producing or expressing soluble disulfide rich proteins, for instance using a suitable host cell. Also provided herein are compositions comprising soluble disulfide rich proteins.

The application discloses a method for constructing a tag sequence-containing vector library by short oligonucleotides and application thereof. The method needs two oligonucleotides, i.e. a customized tag primer and a universal primer which can be used for all library constructions. The middle of the tag primer is a tag sequence, and both sides are annealing sequences. The universal primer has sequences complementary to the annealing sequences of the tag primer on both sides, and a modified base which can be recognized as a base damage by a host microorganism in the middle. The tag primer library is annealed with an equal amount of the universal primer to obtain primer dimers. The primer dimers have base-paired DNA double strands on both sides, and are cohesive ends. The middle of the primer dimers is an unpaired omega loop structure, and the double strands of the omega loop contain the tag information carried by the tag primer and the modified base carried by the universal primer respectively. The primer dimers are connected with linearized vectors, and then are transformed into E. coli, so that the region of the modified base of the omega loop is replaced by the tag sequence through the base excision repair mechanism of the endogenous cells.