61 results about "Hybrid capture" patented technology

The invention discloses a construction method and an application of a high-throughout sequencing library. The construction method of the high-throughout sequencing library comprises the following steps: fragmenting genome DNA (deoxyribonucleic acid); repairing terminals of DNA fragments; adding a base A to 3' terminals of the DNA fragments with the repaired terminals; connecting the DNA fragments with sticky ends A to methylation linkers; carrying out hybrid capture on connecting products by using a specificity probe so as to obtain target fragments; carrying out bisulfite treatment on the target fragments so as to convert non-methylated cytosine in the target fragments to uracil; carrying out PCR (polymerase chain reaction) amplification on the converted target fragments; separating and purifying amplification products which form the high-throughout sequencing library. According to the method and application of the high-throughout sequencing library, the high-throughout sequencing library of a genome specific zone of a sample can be conveniently and efficiently constructed.

The invention relates to the field of genetic engineering and biotechnologic detection, and concretely relates to a gene group and a kit for diagnosing lung cancer, and a diagnosis method thereof. The gene group for diagnosing the lung cancer includes ABCB1, AKT1, ALK, APC, ATIC and other 63 genes. The method using the gene group to diagnose the lung cancer comprises the following steps: (1) extracting free DNA and genome DNA from the plasma of a sample to be detected; and (2) breaking the genome DNA into fragments with the length of 150-250 bp, carrying out hybrid capture on the broken genome DNA and the free DNA to construct a DNA library, carrying out online sequencing, and analyzing the obtained sequencing result. Exon and partial intron regions of the 68 genes of the free DNA are enriched at one time by a probe capture technology to realize multi-gene and multi-target parallel deep high-throughput sequencing with high accuracy, optimize the detection flow and improve the detection precision, so liquid biopsy, low-frequency detection and tumor diagnosis become possible.

The invention provides methods for determining the MSI status of a patient by liquid biopsy with sample preparation using hybrid capture and non-unique barcodes. In certain aspects, the invention provides a method of detecting microsatellite instability (MSI). The method includes obtaining cell-free DNA (cfDNA) from a sample of blood or plasma from a patient and sequencing portions of the cfDNA to obtain sequences of a plurality of tracts of nucleotide repeats in the cfDNA. A report is provided describing an MSI status in the patient when a distribution of lengths of the plurality of tracts has peaks that deviate significantly from peaks in a reference distribution.

The invention relates to a nucleic acid detection method combining RNA amplification with a hybrid capture method. The nucleic acid detection method includes the following steps of extracting nucleic acid from a sample or splitting the sample to release the nucleic acid to be detected, conducting transcription and amplification on the nucleic acid to be detected to obtain an RNA product, adding the obtained RNA product together with a DNA probe marked with biotins to a solid phase so that hybridization can be conducted, forming a DNA/RNA heterozygote through hybridization, coating the solid phase with specific antibodies for recognizing the heterozygote or avidin or streptavidin, capturing the heterozygote through the solid phase, and adding avidin or streptavidin or antibodies for recognizing the DNA/RNA heterozygote to the solid phase to achieve obtaining of a nucleic acid signal, wherein the avidin or the streptavidin or the antibodies for recognizing the DNA/RNA heterozygote are marked with enzymes with the secondary amplification capacity. According to the nucleic acid detection method, the inherent pollution problem of the PCR is avoided, RNA molecules can be directly detected, inverse transcription and pre-dissociation of RNA do not need to be conducted, and the operation steps are greatly simplified.

The invention discloses a liquid-phase hybrid capture enriching liquor for a genome DNA sequencing library and a hybrid method adopting the liquid-phase hybrid capture enriching liquor. The hybrid enriching liquor comprises phosphate buffer, sodium citrate buffer (SSC), SDS (lauryl sodium sulfate), EDTA (ethylene diamine tetraacetic acid) and Denhardt liquor. The hybrid liquor and the corresponding hybrid method are low in cost, simple and convenient to implement; the hybrid time can be effectively shortened, the needed DNA fragment can be precisely extracted, influences of a background value on a signal value can be reduced, and the hybrid target enriching effects can be optimized.

The invention discloses a method for fragmenting a trace DNA sample and a method for establishing a DNA library by utilizing the trace DNA sample. The method for fragmenting the trace DNA sample comprises the steps that 1, Carrier RNA is added into the trace DNA sample to obtain a sample to be fragmented; 2, fragmenting processing is conducted on the sample to be fragmented. An inventor discovers that after the exogenetic Carrier RNA is added into the target DNA, that is to say, the target DNA is mixed with the Carrier RNA and then fragmenting is conducted on the mixture, the protective action to the target DNA can be achieved, the destructive action of a fragmenting instrument to the trace DNA is reduced to the maximum extent, and full gene establishing library sequencing and even hybrid capture sequencing can be conducted on DNA of a trace FFPE sample.

The invention discloses a hybrid capture kit for detecting mutation of breast cancer susceptibility genes BRCA1 and BRCA2. The hybrid capture kit comprises a hybridization reagent, a PCR amplification reagent and an enrichment degree detection reagent, wherein the hybridization reagent comprises probe mixtures from SEQ NO.1 to SEQ NO. 173, and the mole ratio is SEQ NO.1: SEQ NO. (2-172): SEQ NO. 173=1: 1: 1. During detection, the use amount of each of the probe mixtures with the concentration of 2nM is 1 [mu]l. The kit disclosed by the invention can be used for high-sensitivity, high-flux, low-cost and easy-operation full-explicit mutation detection for the genes BRCA1 and BRCA2, and can assist a clinical doctor in screening out individuals with the breast cancer susceptibility risk to fulfill the aim of preventing occurrence of breast cancers.

The invention discloses an improved sequence capturing method, which comprises the steps of 1, extracting DNA; breaking the DNA; 2, connecting the two ends of the broken DNA with a connector, whereinthe remote end of a connector sequence at one end of the broken DNA includes a tag sequence with the length being 6 to 8nt; 3, performing Pre-PCR on the DNA of the connecting connector; 4, mixing a probe and a sealing reagent; capturing the Pre-PCR, wherein the sealing reagent includes sealing sequences complementary with sequences except the broken DNA, and the tag sequences are sealed by C3 spacer arms with the same length; 5, after the hybrid capture, performing PCR amplification. A bridge type sealed design strategy is used; corresponding sealing sequences are respectively designed for connector sequences at two ends of inserting fragments; the tag sequences at the middle part are subjected to bridge type connection by the C3 spacer arms; the cost is reduced; the complexity in the library building process is simplified.

The invention discloses a preparation method of a modified DNA (deoxyribonucleic acid) hybridization probe for targeted hybrid capture. The preparation method mainly includes preparing a DNA template prior to PCR (polymerase chain reaction) amplification and purifying a PCR product; subjecting the purified PCR product to USER enzyme digestion and T4 DNA polymerase terminal blunting, wherein one of double-stranded DNA of an Lambda exonuclease hydrolysis product has 5'-phosphorylated single strand, so that bioti-labeled single-stranded DNA is obtained; purifying the bioti-labeled single-stranded DNA by a magnetic beads enrichment method to obtain the modified DNA hybridization probe. The preparation method of the modified DNA hybridization probe for targeted hybrid capture has the advantages that production cost is reduced greatly, and the preparation method is wide in application range including exon regions, intron regions, mitochondrion regions and the like of genomes of any species. The obtained DNA hybridization probe can be applied to construction and sequencing of next generation targeting libraries.

The invention discloses a second-generation sequencing method for dimolecular self-checking library preparation and hybrid capture used for trace DNA ultralow frequency mutation detection. The methodcomprises the following steps: extracting plasma free DNA, performing DNA chemical error repair, preparing a self-checking dimolecular identification code hairpin type joint, repairing plasma free DNA, connecting DNA with the joint, and performing Pre-PCR amplification, excessive hybrid capture, Post-PCR amplification, computer sequencing, data error correction, and mutation analysis and annotation. The method disclosed by the invention can efficiently realize low frequency mutation detection of plasma free DNA. DNA error repair and dual redundancy checking technology can enable the method tohave ultralow false positive rate and high sensitity while detecting trace samples, thus avoiding defects of existing plasma circulating free DNA detection methods, not only realizing cancer mutationdetection and targeted medication guidance, but also realizing early screening of genetic and birth defects of fetus.

The invention relates to a pig 50K liquid phase chip based on targeted capture sequencing, and application thereof. The pig 50K liquid phase chip is composed of pig 50K probe mixed liquor and a hybridization capture reagent which are independently packaged; pig 50K probes are DNA double-stranded probes and are nucleotide sequences designed and synthesized according to screened SNP sites; and the SNP sites are screened by comparing the sequencing results of the whole genome of the pig to a pig reference genome. The pig 50K liquid phase chip prepared by the invention is mixed with a pig DNA high-throughput sequencing library; DNA fragments containing target sites in the pig DNA high-throughput sequencing library are captured for amplification and purification; and, after a product is subjected to high-throughput sequencing, a sequencing result is replied to a pig reference genome for comparison, so that the genotyping of the genome of a to-be-detected pig is obtained. When the 50K liquid phase chip prepared by the invention is used for genotyping pigs; and the problems of high cost, inflexibility and incapability of large-scale use in pig farms in China in the prior art can be solved.

This invention is related to a method for rapidly detecting gene specific methylation through signal amplification using the epi-barcode after hybrid capture of the methylated DNA sequence.

The invention discloses a digital multiple-nucleic-acid detection method on the basis of encoding micro-sphere reactors. The digital multiple-nucleic-acid detection method includes steps of extractingsample nucleic acid; designing primers; labeling the primers; carrying out hybrid capture; generating micro-drop; carrying out amplification reaction; carrying out scanning detection and the like. The digital multiple-nucleic-acid detection method has the advantages that multiple-gene detection merits of hybridization technologies and high-sensitivity quantitative detection merits of digital PCR(polymerase chain reaction) are combined with one another, accordingly, gap in the technical field of multiple-gene quantitative detection can be filled, and the digital multiple-nucleic-acid detection method has profound influence.

The invention belongs to the field of gene detection, in particular to a strategy for direct hybridization of genomic DNA and a method for constructing a DNA library and an application thereof in thefield of nucleic acid detection. The method comprises the following steps: genomic DNA is fragmented and phosphorylated; Fragmented genomic DNA hybridized with capture probes; after hybridization, thecaptured single-stranded DNA was eluted. A ligation product is obtained by linking a single strand DNA with a linker; The ligation product was used as template for PCR amplification to obtain the amplified product, and the amplified product was purified to obtain the sequencing library. The invention provides a method for directly hybridizing fragmented genomic DNA, which is universally applicable to a solid-phase chip capture hybridization system and a liquid-phase capture hybridization system, and greatly improves capture efficiency and balance. At that same time, the invention further provide a DNA library construction method, which has the characteristics of simple method, low cost, wide applicability and the like, and can be use for the construction of a high-throughput sequencing document library of DNA or RNA.

The invention discloses a method for judging benignancy and malignancy of pulmonary nodules by a blood sample. Determination is performed through combination of cfDNA polygene variation and machine learning. The method includes the following steps: performing library construction on peripheral blood cfDNA of a pulmonary nodule patient; performing hybrid capture on the cfDNA by utilizing 18 gene capture probes related to a lung cancer; and performing sequencing by utilizing a sequencer, analyzing the cumulative mutation frequency of each of genes, and judging the benignancy and the malignancy of samples by utilizing a logistic regression model. The method can distinguish the benignancy and the malignancy of the pulmonary nodule patient with high sensitivity and high specificity, avoids experience errors of artificial reading CT imaging, and avoids missing detection of a lung cancer and pain of puncture or surgical sampling biopsy of a benign nodule patient.

The invention belongs to the field of biomedicine, in particular to a kit for detecting integrated viruses of a genome in hybrid capture. The kit comprises the following main components: (1) a solid phase medium fixed with a capture mixture of human genome DNA (Deoxyribonucleic Acid) single-chain molecules, (2) positive and negative quality control materials for identifying the hybrid capture efficiency and specificity, and (3) a primer pair and a molecular probe for carrying out real-time PCR (Polymerase Chain Reaction) identification to the captured mixture of the target DNA molecules, obtained by capture. By utilizing all or partial components in the invention, a complete integrated genome virus detection kit or an integrated genome virus detection kit with the type specificity can be assembled. The obtained virus detection kit can be used for detecting and identifying the integrated state of the genome in a certain common integrated and oncogenic viruses, such as human hepatitis B viruses, human papilloma viruses, Epstein-Barr viruses and human herpes simplex viruses, in tissue or cytology specimens.

Disclosed herein is a system for detecting cancer methylation. The system comprises a sample collection module for collecting a subject sample, a DNA extraction module used for extracting and purifying DNA in the sample, a library building module used for building a DNA library for sequencing for the purified DNA sample, a conversion module used for converting the constructed DNA library by using a bisulfite, a pre-PCR amplification module used for carrying out pre-PCR amplification on the DNA library subjected to bisulfite conversion, a hybrid capture module used for carrying out hybrid capture on the sample subjected to the pre-PCR amplification by utilizing a probe composition, a post-PCR amplification module used for amplifying a product captured by hybridization by utilizing PCR, a sequencing module used for carrying out high-throughput next-generation sequencing on a product subjected to hybridization capture after PCR amplification, a data analysis module used for analyzing the sequencing data and determining the methylation level of the sample, and an interpretation module used for interpreting the illness condition of the patient based on the methylation level of the sample.

The invention relates to a lymphangiomyoma joint detection method and an application thereof, and belongs to the technical field of gene detection. The method comprises carrying out liquid phase capture targeting sequencing, designing Panel for TSC1 and TSC2 gene whole exon coding regions highly related to LAM and mutant genes closely related to solid tumors, constructing a gDNA library, carryingout hybrid capture, sequencing on a machine, carrying out sorting by analyzing the sequencing data through bioinformatics processing, and if the TSC1 and TSC2 genes are negative, carrying out supplementary detection by adopting a chromosome chip analysis method, if the single strike site is detected, performing supplementary detection by adopting multiplex ligation-dependent probe amplification, and if the sites have no clear sources of the somatic mutation and the embryonic system mutation, carrying out supplementary detection by adopting Sanger detection. The method improves the positive variation detection rate of LAM patients.

The invention discloses a low-quality FFPE DNA processing method and device and a storage medium. The processing method comprises the following steps: carrying out fragmentation treatment on FFPE DNA,performing heating to remove a secondary structure, and carrying out enzyme digestion; repairing the tail end after digestion, adding poly A tail, and using an A base connector; then, carrying out PCR amplification, hybrid capture and sequencing to obtain sequencing data; and comparing the sequencing data with a human reference genome, and filtering out sequences which are partially compared to two different places of the same chromosome at the same time and of which the comparison initial position distance is less than 500bp from a comparison result. According to the method disclosed by theinvention, most of single chains are eliminated through the heating and the enzyme digestion, so that the single chains cannot form stable double chains in terminal filling-in after being connected toone another, and false positive of subsequent mutation detection caused by single chain residues is reduced; and through data comparison and filtering, single-chain interference is further removed, and the accuracy of mutation detection results is improved.