86 results about "Target enrichment" patented technology

The invention discloses a method for targeted enrichment of polymorphic sites by introducing thio-modified nucleic acid into a single base. According to the invention, the selector contains a segment of primer extension binding region with more than four basic groups, when the selector is extended by taking a target DNA / RNA as a template, single thio-modified dNTP and another three terminated dNTP / ddNTP, denaturation and renaturation are carried out, a hairpin structure is formed by itself, the selector without thio-modification is digested under the action of T4DNA polymerase, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA. And complementing the sequence at the incision to form an intermediate product with a hairpin structure, so as to fulfill the aim of enriching rare mutation sites. The method is suitable for DNA and RNA templates, and the obtained hairpin ring structure intermediate can be subjected to signal amplification in two ways: enzyme-mediated nucleic acid amplification or PCR amplification after linearization of a ring structure.

The invention discloses a detection system and method for identifying mycobacteria and drug resistance genes of the mycobacteria based on nanopore sequencing, and relates to the field of biological medicine, the detection system comprises a specific targeted enrichment module, a nanopore sequencing module and a biological information analysis module; the specific targeted enrichment module comprises a probe combination, and the probe combination covers a mycobacterium tuberculosis complex conservative identification gene, species-specific genes of common nontuberculous mycobacteria and mycobacterium leprosy, and full-length or partial sequences of drug resistance related genes in a targeted manner; by utilizing the characteristics of nanopore length reading length and real-time sequencing and a tuberculosis specific targeted enrichment strategy, accurate identification of a mycobacterium tuberculosis complex group, synchronous typing of 42 mycobacteria, analysis of 24 drug-resistant genes, and efficient detection of structural variation and low-abundance heterogeneity drug-resistant mutation are realized, the detection period is shortened, the detection cost is reduced, and the detection efficiency is improved. And a comprehensive and reliable technical basis is provided for accurate diagnosis and treatment of mycobacterium infection.

The invention discloses a sequencing method of an immune repertoire, which comprises the following steps: 1) constructing a cDNA library of a sample, the cDNA in the cDNA library being connected with the position information of the cDNA on the sample; 2) enriching cDNA of TCR and / or BCR genes in the cDNA library in a targeted manner to obtain an immune repertoire of the sample; and (3) sequencing the immune repertoire. According to the method disclosed by the invention, targeted enrichment and high-throughput sequencing of TCR and / or BCR sequences are innovatively realized, and gene expression information and spatial position information of each cell in a space are detected at the same time.

The invention relates to a drug-resistant gene targeted enrichment method using dCas9 and application of the drug-resistant gene targeted enrichment method, and belongs to the technical field of nucleic acid enrichment. The drug-resistant gene targeted enrichment method comprises a CRISPR-dCas9 system for targeting a target gene and a target capture magnetic bead matched with the CRISPR-dCas9 system; the CRISPR-dCas9 system comprises a target DNA (Deoxyribose Nucleic Acid), a dCas9 protein and a target nucleic acid targeted sgRNA (Single Guide Ribonucleic Acid), and the targeted capture magnetic bead is a His tag protein purified agarose magnetic bead. According to the method, a CRISPR-dCas9 system and His tag protein are integrated to purify agarose magnetic beads, and dCas9 protein with a His tag is combined with the magnetic beads to construct a novel enrichment scheme; his magnetic beads are combined with dCas9 with a His tag under the condition that protein denaturation is not achieved, and drug-resistant gene targeted enrichment is achieved; the whole scheme is easy to operate, mild in condition, low in cost and suitable for common laboratories and on-site rapid detection to improve sensitivity. According to the method, the target gene is highly specifically recognized through sgRNA, magnetic bead capture is combined, and the method has the advantages of being efficient, specific and easy to operate and has application value in the fields of drug-resistant gene screening and diagnosis, third-generation sequencing and the like.

The invention provides methods for simultaneously enriching multiple target regions of interest in one reaction volume, from cell-free DNA isolated from a blood or plasma sample, followed by high-thought sequencing and sequence read analysis. The invention also provides library of target-specific oligonucleotide primers or probes for the multiplexed target enrichment.

This invention relates to the field of bioinformatics processing and discloses a method and system for mining the structure-activity relationship (SMR) of porcine neurotrophic peptides based on sequence features. The method includes constructing an original sample index table and fusing multi-source production data, extracting peptide sequence features to generate a sequence feature matrix, constructing a sequence-process joint graph containing peptide nodes and process state nodes, training a structure-activity relationship graph neural network to mine SMR relationships, and deriving a process control decision table based on a process response sample set generated by the network, thereby achieving online optimization of the porcine neurotrophic peptide preparation process. This invention solves the problem of SMR mining caused by the separation of process parameters, sequence information, and activity data, achieving accurate characterization of the synergistic effect of sequence and process and reverse optimization of process parameters, thus improving the targeted enrichment efficiency and bioactivity retention level of target neurotrophic peptides.

The present disclosure provides, among other things, a way to amplify and sequence target sequences in a low-input sample. In some embodiments, the method comprises ligating a double-stranded adaptor onto a population of fragments to produce tagged fragments, and linearly amplifying the tagged fragments.

Transcript Enrichment and Quantification Utilizing Isothermally Linear-Amplified Sequencing (TEQUILA-seq) is a versatile, easy-to-implement, and highly cost-effective method utilizing isothermally linear-amplified capture oligos for targeted sequencing. TEQUILA-seq reduces the per-reaction cost of targeted capture by 2-3 orders of magnitude, as compared to a standard commercial solution. When performed on the Oxford nanopore platform for long-read RNA-seq with multiple gene panels of varying sizes, TEQUILA-seq consistently and substantially enriched transcript coverage while preserving transcript quantification. Profiling of full-length transcript isoforms of 468 actionable cancer genes across 40 breast cancer cell lines representing distinct intrinsic subtypes identified transcript isoforms enriched in specific subtypes and discovered novel transcript isoforms in extensively studied cancer genes such as TP53. Among cancer genes, tumor-suppressor genes were significantly enriched for aberrant transcript isoforms targeted for degradation via mRNA nonsense-mediated decay, revealing a common RNA-associated mechanism for gene inactivation. TEQUILA-seq can be broadly used for targeted sequencing of DNA and RNA in diverse biomedical research settings.

The invention discloses a targeted enrichment method for proteome. According to the cell nucleus targeted RNA binding protein enrichment reagent, one end is provided with a furocoumarin group, the other end is provided with a cell nucleus targeted group, and the other end is provided with biotin; the enrichment or identification of the RNA binding protein in the cell nucleus can be carried out. The method for enriching the nucleus-targeted RNPs by adopting the nucleus-targeted RNA binding protein enrichment reagent comprises the following steps: firstly, adding the nucleus-targeted RNA binding protein enrichment reagent into cells for co-incubation, then, irradiating a cell incubation system by adopting ultraviolet light with the wavelength of 254nm, and then, irradiating the cell incubation system by adopting ultraviolet light with the wavelength of 365nm, then carrying out cracking to obtain a cracking solution; incubating the lysis solution and streptavidin magnetic beads, and finally collecting the magnetic beads enriched with the cell nucleus RNPs, so that the cell nucleus targeted RNPs are enriched. According to the method, the cell nucleus positioning of the reagent can be efficiently realized, the RBPs enrichment selectivity and the negative electrode efficiency are improved, and the varieties of RNPs obtained through enrichment are more comprehensive.

A targeted high-throughput sequencing method for detecting splice isoforms, including a method for establishing a sequencing library for high-throughput sequencing, comprising the following steps: 1) reverse transcription of sample RNA using a reverse transcription primer, adding common dNTPs and 3'-modified dNTPs to generate a first-strand cDNA; 2) ligating an oligonucleotide fragment with an alkyne modification at the 5' end to the cDNA fragment obtained in step 1) via a click chemistry reaction; 3) performing PCR amplification using the reaction product of step 2) as a template; and 4) obtaining a sequencing library. Targeted enrichment primers can be introduced during the reverse transcription or PCR amplification steps. The enrichment step reaction system contains multiple gene-specific primers, each designed based on the downstream exon segment of the alternative splicing event in the transcript, ensuring that the random-length fragments generated by reverse transcription cover the splice site.

The invention discloses a method for generating a hairpin ring for enriching mutation or allele information. According to the invention, a primer extension binding region containing more than 8 basic groups is arranged on a selector; when the selector extends by taking the target DNA as a template and is denatured and renatured, a 3 '-terminal suspended hairpin structure is formed by itself, a 3'-terminal suspended sequence is digested under the action of T4DNA polymerase, a sequence at a gap is supplemented, a hairpin structure intermediate product is formed, and the hairpin structure is used for synthesizing a DNA sequence by taking a genome as a template. And carrying out targeted enrichment on polymorphic site information (wild type and mutant type), and then carrying out information decoding through next-generation sequencing. The method is used for enriching information of rare mutation sites, only common dNTP is needed for single primer amplification, then T4DNA polymerase is added for digesting mismatched sequences in a stem region, completely paired sequences are reserved, and the purpose of enriching the rare mutation sites is achieved. And more than 1000 probes can be subjected to multiple analysis in one PCR tube by using standard laboratory equipment.

This invention discloses a mitochondrial-targeted nanomaterial for senescent cells and its application in anti-aging. The material is a nucleic acid aptamer-tetrahedral DNA, mainly assembled from four single-stranded DNA sequences, SEQ ID Nos. 1-4. Preparation involves first hybridizing the four single-stranded DNA sequences, then concentrating them to obtain aptamer-modified tetrahedra, which are then coupled with tannic acid (TA) via a Mann reaction in the presence of formaldehyde to obtain the Apt-tFNAs-TA complex. Finally, it is incubated with excess catechins to obtain the final delivery material, Apt-tFNAs@catechin-TA. This invention's material exhibits good biocompatibility and skin tissue compatibility, significantly improving uptake efficiency and mitochondrial localization ability in senescent cells. It can effectively deliver polyphenolic compounds such as catechins and protocatechuic acid, improving mitochondrial function and addressing the technical problems of low delivery efficiency of active ingredients and insufficient targeted enrichment in senescent cells.

Provided are a humanized / fully human HER2 antibody or an antigen-binding fragment thereof, and an antibody-radionuclide conjugate using same as a targeting group for radioactive labeling, showing a tumor-targeting enrichment ability.

The application discloses a kind of enrofloxacin rapid detection methods based on fluorescent covalent organic framework magnetic beads, belong to food safety detection field.The method uses the fluorescent covalent organic framework magnetic bead of coupling enrofloxacin monoclonal antibody, realizes detection in combination with immunochromatography test strip.Fluorescent covalent organic framework magnetic bead realizes target enrichment separation with inner core, shell fluorescent covalent organic framework material high-efficiency coupling antibody and exports fluorescence signal;Test strip is equipped with detection line (coated enrofloxacin artificial antigen) and quality control line (coated secondary antibody).By comparing the fluorescence intensity of detection line and quality control line, trace enrofloxacin in complex matrix can be qualitatively and quantitatively analyzed.The method is simple, rapid and sensitive, without large instrument, can be detected on site.

The invention belongs to the technical field of microbiological detection and molecular diagnosis, and discloses a primer group, a kit and a detection method for detecting salmonella enteritidis, the kit comprises an immunocapture component, an isothermal amplification component, a CRISPR / Cas12a detection component and auxiliary components; the isothermal amplification component is any one of an RPA (recombinase polymerase amplification) component or an LAMP (loop-mediated isothermal amplification) component, the RPA component comprises a specific primer group targeting 86th to 212th nucleotides in a salmonella enteritidis hilA gene coding region, and the LAMP component comprises a specific primer group targeting 58th to 250th nucleotides in the salmonella enteritidis hilA gene coding region. The primer group, the kit and the detection method for detecting the salmonella enteritidis successfully integrate three key technologies of RAP and LAMP rapid isothermal amplification, CRISPR / Cas12a high-specificity recognition and trans-cleavage and immunomagnetic bead targeted enrichment, and creatively provide the primer group, the kit and the detection method for detecting the salmonella enteritidis.

The invention discloses a multi-target enriched library construction method for simultaneously detecting DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) type pathogens, which is characterized in that after total nucleic acid of a sample is extracted, DNA and RNA libraries are simultaneously constructed in a single-tube reaction, and the proportion of the pathogens in the library is enriched by capturing a probe, so that the sensitivity of pathogen detection is improved, the sequencing data volume is reduced, the library construction steps are reduced, and the detection efficiency is improved. The method has the advantages that the method is simple and convenient to operate, the condition that samples are misplaced possibly due to the fact that RNA is firstly processed into two-chain cDNA and then mixed into DNA to form cDNA / DNA compounds is avoided, only one-tube extraction can be carried out, and the method has high application value on samples such as neonatal blood and child cerebrospinal fluid under the condition that the sample size is small.

The present disclosure provides for methods of preparing nucleic acid libraries for sequencing. In particular, the present disclosure is directed to a workflow which employs a first round of amplification utilizing a universal polymerase chain reaction approach instead of a platform specific indexed polymerase chain reaction approach. Applicant has unexpectedly discovered that a workflow employing a first round of amplification which utilizes a universal polymerase chain reaction approach increases duplex recovery and / or genomic equivalent recovery as compared with a workflow employing a first round of amplification utilizing a platform specific indexed polymerase chain reaction.

Disclosed are methods for preparing a non-naturally occurring composition, comprising: extracting cell-free DNA from a plasma fraction of a blood sample of a pregnant person, wherein the pregnant person is a heterozygous carrier of at least one pathogenic variant in at least one target gene associated with autosomal recessive disorders, wherein the extracted cell-free DNA comprises a mixture of maternal cell-free DNA and fetal cell-free DNA; performing targeted enrichment on the extracted cell-free DNA or DNA derived therefrom to enrich a plurality of target variant loci in a plurality of target genes and generating enriched DNA, wherein the target variant loci comprise the at least one pathogenic variant; performing high-throughput sequencing on the enriched DNA or DNA derived thereof and generating sequence reads, and determining fetal genotypes of one or more of the target genes from the sequence reads.

The invention provides a construction method of a nucleic acid targeted enrichment system based on thermal stability Cas9. The construction method comprises the following steps: providing a sample of nucleic acid molecules containing a sequence of interest; cleaving the nucleic acid molecule with the first and second gRNA-CAS complexes to produce a target nucleic acid fragment and a non-target nucleic acid fragment comprising a sequence of interest; contacting the cleaved nucleic acid molecule with an exonuclease allowing the exonuclease to digest the non-target nucleic acid fragment; the resulting digest is purified comprising the target nucleic acid fragment. In one embodiment, a digest is purified by providing a nucleic acid molecule comprising a sequence of interest, cleaving the nucleic acid molecule with first and second gRNA-CAS complexes to produce a target nucleic acid fragment and a non-target nucleic acid fragment of the sequence of interest, contacting the cleaved nucleic acid molecule with an exonuclease to purify the target nucleic acid fragment comprising the sequence of interest. By means of the nucleic acid targeted enrichment system construction method based on thermal stability Cas9, the purpose of accurately reducing the complexity of nucleic acid can be achieved.

The invention discloses a multiple myeloma sequencing method based on a targeted capture three-generation sequencing technology, a kit and application, and relates to the technical field of gene detection, and the method comprises the steps of sample treatment, targeted probe group design and targeted enrichment capture, three-generation sequencing library preparation, bioinformatics analysis and the like to identify and analyze three types of variations. According to the method, the defects that an existing layered system lacks an accurate genomics basis, and FISH and NGS technologies and combination strategies are low in flux, incomplete in coverage, incapable of synchronously detecting various variations, high in cost and complex in process are overcome, comprehensive information of three key variation types of multiple myeloma SNV, SV and CNV can be obtained at the same time through a single experiment and one-time sequencing, and the method is suitable for popularization and application. The detection efficiency is improved, and sample consumption, operation time and cost are reduced; all hot spot areas and translocation breakpoint areas of the most important clinical related genes of the MM are covered, and a gene variation map which is more comprehensive than that of a standard FISH Panel is provided.

This invention discloses a targeted sequencing method for multiple pathogenic microorganisms, belonging to the field of biotechnology. The targeted sequencing method for multiple pathogenic microorganisms includes the following steps: sample pretreatment; targeted enrichment; PCR amplification; high-throughput sequencing; and bioinformatics analysis. This invention solves the problems of existing technologies, such as inability to accurately type pathogens, detect drug resistance genes, and analyze virulence genes, as well as poor targeting and slow analysis speed. This invention provides high-purity target nucleic acids for PCR amplification through a triple targeted enrichment system; the anti-inhibition multiplex PCR system ensures efficient amplification of target nucleic acids in complex samples; high-throughput sequencing enables rapid sequencing of target nucleic acids; and the bioinformatics analysis module enables simultaneous completion of pathogen typing, drug resistance gene detection, and virulence gene analysis, significantly improving overall detection efficiency and meeting the clinical needs for rapid, accurate, and comprehensive pathogen detection.

The invention provides a mitochondrial specific lipid tracking and monitoring method based on light-controlled release and application of the mitochondrial specific lipid tracking and monitoring method. The invention provides a mitochondrial specific probe (fluorescent probe) molecule which can be used for mitochondrial specific lipid research and can realize light-controlled release and quantitative analysis on the basis of imaging. The probe molecule is high in targeted enrichment performance and very high in release efficiency, and the release efficiency can reach 100% within a short time. The probe molecule has the advantages of simplicity in operation, high sensitivity, high specificity, low biotoxicity and the like.

Improved methods and compositions are provided herein for primer extension target enrichment of target polynucleotides.

The invention belongs to the technical field of biology, and particularly relates to a DNA aptamer for improving CRISPR / Cas9 mediated exogenous gene integration efficiency based on CtIP protein and application of the DNA aptamer, and the nucleotide sequence of the DNA aptamer is as shown in at least one of SEQ ID NO.1 and SEQ ID NO.10. The invention further discloses a preparation method of the DNA aptamer for improving CRISPR / Cas9 mediated exogenous gene integration efficiency based on CtIP protein. According to the invention, the key factor CtIP protein in DNA double-strand breakage repair is taken as a target for the first time, two aptamers are screened, and the donor vector of a CRISPR / Cas9 system is optimized by utilizing the aptamers, so that the integration efficiency of exogenous genes in HEK-293T cells and Hela cells is improved. The affinity of the CtIP protein specific DNA aptamer obtained through screening reaches the nanomole level, and the Kd value ranges from 42.56 nM to 407.29 nM. An aptamer is inserted into the tail end of a homologous arm of a donor carrier, and directional enrichment of a repair template is realized by utilizing an HMEJ mechanism. The aptamer A6913 and / or A13-32 significantly improves the integration efficiency of the exogenous gene, and the integration efficiency is improved by more than 3 times compared with a control group.

The application discloses a target gene for detecting pea foot rot, a primer set and a detection kit, and belongs to the technical field of plant quarantine. The application innovatively develops a rapid detection technology system based on specific molecular markers in view of the core pain points of long detection period (traditional method >= 20 days) and difficult identification of close species (such as D. pinodes and A. pisi) in the quarantine identification of Didymella pinodella. By screening a single-copy gene sequence unique to D. pinodella and combining PCR technology, a whole-process system of "direct sample detection-target enrichment-signal amplification" is established, and the pathogen detection can be completed within 8 hours, the efficiency is improved by 96% compared with the traditional method, the efficiency is greatly improved, and the close species can be effectively distinguished, the customs clearance efficiency is greatly improved, and technical support is provided for the detection of pea foot rot and the breeding of pea disease resistance.

The invention discloses a kit for detecting circulating tumor DNA (deoxyribonucleic acid) of portal vein and branch vein blood of portal vein. The kit comprises: a nucleic acid extraction assembly comprising an optimized lysate and selectively bound magnetic beads; the library construction component comprises a terminal repair / tailing fusion enzyme and a Y-shaped linker with a molecular identifier; the targeted enrichment assembly comprises a specific primer group and a probe group and is used for enriching related gene targets of the abdominal organ tumors; and necessary reaction components. Through an optimized nucleic acid extraction method, an efficient library construction system and a precise targeted enrichment technology, high-sensitivity detection of low-abundance ctDNA in portal venous blood is achieved, and the lower detection limit reaches 0.1% of mutation allele frequency. The kit is particularly suitable for early diagnosis, curative effect monitoring and prognosis evaluation of abdominal organ tumors such as intestinal cancer, liver cancer and pancreatic cancer.

The invention provides a genetic variation and chimera detection method, system and kit. The method comprises the following steps: (a) extracting high molecular weight DNA from a biological sample; (b) labeling the high molecular weight DNA by using a linker containing a UMI sequence to form a UMI labeled DNA library; (c) performing targeted enrichment on the DNA library marked by the UMI by using a probe aiming at a target gene locus to obtain an enriched long-fragment DNA library; (d) performing long read length sequencing on the enriched long fragment DNA library to obtain sequencing data; and (e) carrying out bioinformatics analysis on the sequencing data, wherein the bioinformatics analysis comprises error correction and variation detection based on a UMI sequence. The method is used for simultaneously detecting structural variation and low-frequency chimeric variation in a target genome region.

To provide a method for enrichment of at least one target nucleic acid in a library of nucleic acids.SOLUTION: A first oligonucleotide is hybridized to a target nucleic acid in library of nucleic acids having first and second adapters. The hybridized first oligonucleotide is extended with a first polymerase, thereby producing a first primer extension complex including the target nucleic acid and the extended first oligonucleotide. The first primer extension complex is captured, enriched relative to the library of nucleic acids, and a second oligonucleotide is hybridized to the target nucleic acid. The hybridized second oligonucleotide is extended with a second polymerase, thereby producing a second primer extension complex including the target nucleic acid and the extended second oligonucleotide, and further liberating the extended first oligonucleotide from the first primer extension complex.SELECTED DRAWING: Figure 1

The application provides a circulating tumor DNA-based mutant gene detection method and application thereof, and relates to the technical field of biological detection.The application provides a circulating tumor DNA-based mutant gene detection method, adopts a customized panel technical route, adopts a single-stranded library construction method, preliminarily amplifies free DNA, and then performs target enrichment based on UMI multiplex PCR, so that the detection sensitivity and specificity are effectively improved.It is proved through tests that the method can simultaneously analyze multiple gene mutation results of the same patient, has a sensitivity of more than 70% and a specificity of more than 90% on the mutation allele ratio (AF), and has a good application prospect for MRD monitoring.

The application discloses a PfAgo-based geminivirus double detection test strip and a preparation method and application thereof, and belongs to the technical field of virus detection.The application provides, on one hand, a PfAgo-based geminivirus double detection test strip and a construction method thereof, and on the other hand, application of the test strip in TYLCCNV and PaLCuCNV virus detection.The application uses the ability of PfAgo enzyme to recognize and cut uracil-containing DNA to innovatively develop a ULPD anti-pollution detection system.The system combines dUTP-LAMP technology for target enrichment, and realizes rapid diagnosis through designed gDNA and immunocolloidal gold test strips.