30 results about "Genetic library" patented technology

The invention relates to a full-length sequence amplification primer, an amplification method and a three-generation sequencing method of an HLA-I antigen gene, and belongs to the field of gene detection. The HLA-I antigen gene is subjected to PCR (Polymerase Chain Reaction) amplification by using 8 pairs of characteristic amplification primers. And each gene can be subjected to high-yield specific amplification on a full-length sequence containing complete intron and exon regions only through one round of amplification and enrichment, and 7.8 Kb long-fragment genes can be captured once at most. The experimental deviation caused by simultaneous amplification of a single gene by using a plurality of pairs of segmented primers and the indirect error caused by gene splicing are effectively avoided. The method comprises the following steps: carrying out quality inspection and purification on amplicon PCR (Polymerase Chain Reaction) products, mixing samples, constructing an HLA-I type antigen gene library, carrying out accurate and complete sequencing on the full-length sequence of the HLA-I type antigen gene by adopting a three-generation PacBio Sequel II platform, and ensuring that the Hifi reads base accuracy can reach 99% or above. The reading of HLA-I type antigen gene full-length sequence variation information and the haplotype analysis at a high resolution level can be realized.

The invention relates to application of a proteasome inhibitor in preparation of a medicine for treating tumors resistant to a tyrosine kinase inhibitor, and in the application, a plurality of strains of liver cancer cells with drug resistance to lenvatinib or regorafenib are constructed by utilizing a plurality of human liver cancer cell lines with different genetic backgrounds; a high-throughput CRISPR (clustered regularly interspaced short palindromic repeats) gene knockout screening technology and a patent medicine gene CRISPR library are utilized, and gene targets of which the gene knockout or inactivation can better kill drug-resistant cells are specifically identified from thousands of patent medicine genes. Wherein the function inactivation of a plurality of genes in the proteasome family shows more obvious cell killing activity in a plurality of drug-resistant cell models compared with non-drug-resistant cells. The invention discovers and verifies that the proteasome inhibitor has a specific killing effect on tyrosine kinase inhibitor drug-resistant liver cancer for the first time.

The present disclosure provides methods, compositions, and systems for generating a library of variant genes comprising nucleic acids.

This invention relates to the field of biomedical technology, specifically to a snake-derived nanobody against KLH and its preparation and application. The invention involves immunizing the black-browed rat snake with KLH, extracting total RNA from its splenic lymphocytes, constructing a phage display VH gene library, and, after multiple rounds of screening, obtaining nanobody sequences that specifically bind to KLH, such as KLH-1 and KLH-2. This nanobody can be efficiently and solublely expressed and purified in *E. coli*, exhibiting characteristics of small molecular weight and high affinity. This invention further provides applications for this nanobody, particularly in the preparation of KLH-targeting detection reagents or drug delivery systems. This nanobody provides a core tool molecule for developing highly sensitive KLH-targeting detection reagents, efficient drug delivery systems, and novel vaccines.

The invention relates to the field of biological information, and discloses a gene library acquisition method for improving single cell amplification genome coverage. Aiming at the problem of insufficient single cell amplification genome coverage, the gene library acquisition method provides a strategy of composite library construction and optimized data integration, and comprises the following steps: constructing at least one original text library and one sub-library with different average fragment lengths for the same single cell sample, and sequencing; performing Barcode resolution and effective single cell identification on the sequencing data through a customizable sequence feature detection method; and further, according to the identification result of the effective single cell, identifying, collecting and merging all sequencing sequence data of the same single cell, generating an integrated sequence data set of the corresponding single cell, and obtaining gene library data of the single cell. The SAG coverage degree of the obtained gene library data is high, and the method has high reliability and application value when being used for genome analysis of the single cell downstream.

The present invention discloses a method for extracting total RNA from a plateau plant, Oxytropis glacialis, and belongs to the field of molecular biology. The method comprises the following steps: (1) grinding the Oxytropis glacialis material with liquid nitrogen, adding a first lysate to mix and lyse, and then centrifuging to obtain a supernatant; then adding a second lysate to the supernatant to lyse, and then centrifuging to obtain a supernatant; (2) adding chloroform to mix, then centrifuging to obtain a supernatant, and then adding isopropanol to the supernatant and centrifuging to obtain a precipitate; (3) adding ethanol for rinsing, and then dissolving in DEPC H2O and storing at low temperature. The present invention can extract RNA samples with higher concentration and purity by grinding the plant material with liquid nitrogen and combining the first lysate and the second lysate with a reasonable ratio, laying an important foundation for subsequent molecular biology analysis such as the Oxytropis glacialis total RNA gene library and transcriptome sequencing.

The invention belongs to the technical field of gene detection, and particularly relates to a primer group for detecting mutation sites of human cytomegalovirus UL54 drug-resistant genes and application of the primer group. The primer group can be used for constructing an HCMV drug-resistant gene library, sequencing HCMV drug-resistant genes and detecting mutation sites of the HCMV drug-resistant genes.

The invention relates to a capture probe group, a kit and a detection method for detecting genetic susceptibility related gene variation of gastrointestinal stromal tumor. The kit comprises a capture probe group for detecting gastrointestinal stromal tumor genetic susceptibility related gene variation conditions and a corresponding reagent based on a next-generation sequencing technology. On the basis of a next-generation sequencing technology, variation conditions of genetic susceptibility related genes NF1, SDHA, SDHB, SDHC and SDHD of the gastrointestinal stromal tumor can be detected at one time by utilizing a hybrid capture method. The capture probe used in the invention has wide coverage and high sequencing depth, and can detect the embryonic line variation of all exon regions of the designed gene. A variant gene library is obtained according to a capture probe, gene variation sites are obtained through sequencing and comparison to a reference genome, all related diseases of gastrointestinal stromal tumor genetic susceptibility related genes can be reported according to databases such as existing literatures and guidelines, pathogenicity of variation and the like, and genetic modes corresponding to the diseases are provided.

The present disclosure provides compositions and methods for prime editing of target DNA molecules (e.g., genomes) that enables incorporation of nucleotide changes and / or targeted mutagenesis. Nucleotide changes can include single nucleotide changes (e.g., any transition or any transversion), one or more nucleotide insertions, or one or more nucleotide deletions. More specifically, the present disclosure provides fusion proteins comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a polymerase (e.g., a reverse transcriptase) that are guided to a specific DNA sequence by a modified guide RNA (termed a PEgRNA). The PEgRNA (relative to a standard guide RNA) has been altered to comprise an extension portion that provides a DNA synthesis template sequence encoding a single-stranded DNA flap that is homologous to a strand of the targeted endogenous DNA sequence to be edited but comprises a desired nucleotide change(s) and is incorporated into the target DNA molecule after synthesis by a polymerase (e.g., a reverse transcriptase). Also disclosed herein are various methods of utilizing prime editing, including treating trinucleotide repeat contraction diseases, installing targeted peptide tags, treating prion diseases by installing protective mutations, manipulating RNA coding genes to install RNA tags for controlling RNA function and expression, using prime editing to construct complex gene libraries, using prime editing to insert immunogenic epitopes into proteins, using prime editing to insert inducible dimerization domains into protein targets, and delivery methods, among others.

The invention belongs to the technical field of gene detection, and particularly relates to a probe set for detecting an ALK fusion gene and application of the probe set. The probe group can be used for constructing an ALK fusion gene library, sequencing ALK fusion genes and detecting the ALK fusion genes.

The invention discloses an RNA (Ribonucleic Acid) element for enhancing in-vivo stability and expression efficiency of mRNA (Messenger Ribonucleic Acid) and a high-throughput screening method, and belongs to the field of molecular biology. In view of the fact that improvement of mRNA stability and expression efficiency is crucial to mRNA vaccine research and development, the prior art mainly focuses on improvement of the stability and expression efficiency by means of synonymous codon replacement and the like on an mRNA coding region, the effects of the methods are limited by cell types and mRNA sequence characteristics, and a large number of computing resources need to be consumed in the implementation process. In order to overcome the limitation, the invention provides a high-throughput RNA functional element screening method based on a reporter gene library system and a group of RNA elements identified by the method. The invention provides powerful technical support for research, development and production of mRNA vaccines, medicines and other mRNA in-vivo protein expression.

The invention relates to the technical field of estimation, and discloses a reverse transcriptase-polymerase chain reaction combination optimization system for malignant tumor lymphatic invasion judgment. Comprising a data acquisition module used for acquiring different gene data in a tumor gene library, performing systematic clustering analysis on omics statistic indexes in the gene data, and combining a gene expression combination with statistical possibility through a systematic clustering method; the linear elimination module is used for arranging corresponding PCR (Polymerase Chain Reaction) primers on different exons of the gene aiming at the gene expression combination so as to eliminate the colinearity of the gene and mRNA (Messenger Ribonucleic Acid); the integral collaboration module is used for respectively optimizing the reverse transcription reaction and the PCR reaction in the two-step reverse transcriptase polymerase chain reaction to realize integral collaboration of the reverse transcription reaction and the PCR reaction; and the invasion judgment module is used for simultaneously analyzing various genes in the lymph by utilizing the optimized reverse transcriptase polymerase chain reaction so as to judge the lymph invasion condition of the malignant tumor.

The invention discloses a drug resistance gene detection system and program for beta-lactam drugs for RA, and relates to the technical field of gene detection. The drug resistance gene detection system comprises: a gene library containing drug resistance markers of beta-lactam drugs for RA; the gene sequencing equipment is used for performing gene detection on whether the to-be-detected sample contains the drug resistance marker or not and the serum type of the to-be-detected sample; and the data processing module is used for giving a drug resistance analysis report based on a detection result of the gene sequencing equipment. According to the invention, drug resistance markers and serum detection are combined, the prediction accuracy of drug resistance is high, and the method has good reference value for predicting pathological changes and prognosis after infection; the method can be applied to the fields of virus detection, risk assessment, drug research and development and the like.

This invention provides a protein engineering method combining single-point saturation mutation scanning and multi-point targeted combined mutation. The method includes: constructing a single-amino acid site saturation mutation library of the target protein, and scanning and analyzing the single-point mutations and their corresponding target characteristics to obtain the correspondence between the mutant sequence and the target characteristics; based on the correspondence between the mutant sequence and the target characteristics, selecting the amino acid sites and amino acid mutations of the dominant mutants of the target characteristics, synthesizing a multi-point targeted combined mutant gene library, further constructing a multi-point targeted combined mutant expression library, transforming it into an expression host to induce the expression of the multi-point combined mutant target protein, and screening for target protein mutants. The dominant mutants obtained by the method of this invention can cover more amino acid sites and mutation directions of the target protein, thus making the selection of modification targets more scientific and rational, and greatly improving the screening efficiency of dominant mutants.

The invention provides an underwater sequencer. The underwater sequencer comprises: a sample extraction device for extracting an underwater microorganism sample; an input port of the library building device is connected with an output port of the sample extraction device, and the library building device is used for building a gene library of the microorganism sample; an input port of the sequencing device is connected with an output port of the library building device, and the sequencing device is used for performing gene sequencing on the gene library. According to related requirements of underwater sequencing, the extraction device, the library building device and the sequencing device are integrated, nucleic acid extraction, library building and sequencing can be automatically carried out without human intervention, operation is convenient, and the integrated underwater sequencer further has the advantages of being small in size, low in power consumption and the like.

The invention discloses a nanopore sequencing pretreatment chip as well as a preparation method and application thereof, and belongs to the technical field of microfluidics. The invention provides a nanopore sequencing pretreatment chip, the chip is composed of a conductive glass layer, a reaction liquid layer and a printed circuit board layer, and the reaction liquid layer comprises a nucleic acid extraction unit, a gene library construction unit and a nucleic acid concentration determination unit. The invention further provides a preparation method based on the chip and application of the chip in sequencing sample preparation. According to the invention, high integration of core steps of sequencing pretreatment is realized, the preparation efficiency is improved, an effective scheme is provided for full automation of sequencing pretreatment, and the method can be widely applied to high-throughput and ultra-sensitive sequencing pretreatment of clinical samples.

The invention discloses a screening method for targeted zinc amino acid chelate metabolism genes based on a Crispr whole genome library. The method comprises the following steps: constructing a zinc transporter gene knockout intestinal epithelial cell single gene knockout cell strain; transfecting the Crispr whole genome knockout library into an intestinal epithelial cell single gene knockout cell strain to obtain an intestinal epithelial cell double gene knockout cell strain; treating the intestinal epithelial cell double-gene knockout cell strain with different zinc sources, dyeing with a fluorescent dye, and sorting zinc transport difference intestinal epithelial cell double-gene knockout cell strains through flow cytometry; the separated cell lines are subjected to gDNA extraction, gRNA sequence amplification and high-throughput sequencing, and key target genes for regulating and controlling metabolism of different amino acid chelated zinc are analyzed and identified through bioinformatics. The invention establishes a method for high-throughput screening of the specific metabolism gene of the amino acid chelated zinc, and a Zn metabolism regulation gene library is also obtained through screening at the same time.

The invention belongs to the technical field of nano antibodies, and particularly relates to a BCMA nano antibody and application thereof. The preparation method comprises the following steps: immunizing alpaca by using BCMA recombinant protein, separating peripheral blood lymphocytes, constructing a gene library, and screening an anti-BCMA nano antibody from the library. The nano antibody provided by the invention has unique complementary determining regions 1, 2 and 3, and the antibody has high specific binding capacity to a BCMA antigen. According to the human FC fusion antibody constructed by the nano antibody, the nano antibody has ideal capability of combining multiple myeloma (MM) cells and inducing T cells to kill the MM cells. The compound can be applied independently or in combination with other MM drugs, and is used for preparing MM therapeutic drugs. According to the technical scheme, the technical problems that in the prior art, a nano antibody aiming at BCMA is lacked and the variety of drugs for treating multiple myeloma is limited can be solved, and the nano antibody has ideal application and popularization prospects.

This invention discloses a Valenciane oxidase mutant and its application in the preparation of naringin. The invention constructs a gene library of the HPO mutant using site-directed mutagenesis. In vitro catalytic experiments using resting cells show that, under the same conditions, the optimal HPO mutant (HPO_G302G-V480A-V482A-A484A, whose amino acid sequence is shown in SEQ ID No. 4 and nucleotide sequence in SEQ ID No. 5) exhibits a catalytic efficiency 2.54 times that of the wild type, thereby increasing substrate utilization efficiency and improving the yield of the target compound, naringin. Furthermore, the bio-preparation method for naringin described in this invention is simple to operate, environmentally friendly, and uses mild reaction conditions, showing great application potential. In addition, this invention is the first report of successfully constructing a beneficial HPO mutant for high-yield naringin production.

The application belongs to the technical field of gene detection, and particularly relates to a construction method and kit of a gene library for detecting new mutations of dominant monogenic diseases before embryo implantation. The application provides a construction method of a gene library for detecting new mutations of dominant monogenic diseases before embryo implantation, which comprises the step of amplifying the gene of the new mutation of dominant monogenic diseases before embryo implantation and related copy number variation fragments in the genomic DNA of specific screening. The application precisely applies NGS sequencing technology to the screening of dominant monogenic diseases in trace cell samples, breaks through the technical bottleneck of high-throughput sequencing under extremely low initial DNA amount in the past, and realizes the screening of new mutations before embryo implantation, thereby providing a brand-new key technical path for blocking genetic birth defects at an earlier stage.

The invention provides a magnetic bead buffer solution, a magnetic bead kit and application of the magnetic bead buffer solution and the magnetic bead kit in gene library construction. The magnetic bead buffer solution comprises ethanol, low-molecular-weight polyethylene glycol, salt ions and a Tris buffer solution, based on the magnetic bead buffer solution, the final concentration of the ethanol is 3-10 vol%, the final concentration of the low molecular weight polyethylene glycol is 28-35 vol%, the final concentration of the salt ions is 1.8-3 M, and the final concentration of the Tris buffer solution is 200-400 mM. When the magnetic bead buffer solution or the magnetic bead kit is applied to gene library construction, enzyme reaction with magnetic beads in the gene library construction process can be realized, the experiment cost and the possibility of sample errors and cross contamination are reduced, and the gene library construction time is shortened.

The invention belongs to the technical field of gene detection, and particularly relates to a primer group for detecting a mutation site of a human cytomegalovirus UL97 drug-resistant gene and application of the primer group. The primer group can be used for constructing an HCMV drug-resistant gene library, sequencing HCMV drug-resistant genes and detecting mutation sites of the HCMV drug-resistant genes.

The invention discloses a method for establishing a porcine viral infectious disease gene library, and relates to the technical field of gene library establishment. By constructing the comprehensive virus gene library, each gene segment of the virus can be covered, the recognition capability on different virus strains is improved, the variety, variation site and epidemic trend of the virus can be more accurately determined by utilizing a high-throughput sequencing technology, and the constructed library is high in adaptability, can cope with new diseases and has a good application prospect. According to the method, viruses which are not completely described can be recognized, particularly new pig viruses or viruses with high variability, high adaptability and flexibility are achieved, and the variability of viral genomes can be effectively overcome through algorithm-assisted fragmentation and genome analysis.

A method for in-planta high throughput evaluation of endogenous genetic regulatory elements of a crop plant, obtaining a nucleic acid encoding a native regulatory element of a trait-related gene, introducing one or more genetic changes in each of a plurality of copies of the native regulatory element, introducing the genetic library into model plants, utilizing high throughput transformation, screening the transformed model plants, and identifying the one or more genetic changes in the altered regulatory element of the selected model plant.

The invention discloses a method suitable for high-throughput amplicon sequencing data analysis of nucleotide sequences with unknown lengths. Constructing a target gene library, and performing high-throughput amplicon sequencing; removing a primer fragment in a double-end sequence from the original data of the sequencing fragment obtained by the first-step sequencing according to a primer sequence; removing a low-quality sequence from the fragment of which the double-end primer is removed, so as to obtain cleanreads; splicing the double-end cleanreads data to obtain Clean Tags, and carrying out statistics on the splicing efficiency and obtaining the length distribution of sequence fragments; clean Tags are rapidly filtered and denoised based on dada2, amplicon sequence variants are obtained according to 100% similarity, and sequence abundance is counted for subsequent molecular element screening statistical analysis. The method is novel, easy to operate, high in efficiency, good in accuracy and wide in application range, can be suitable for short-fragment functional gene amplicon analysis, various pollutant response proteins such as promoters and RNA riboswitches and some molecular elements with undefined functional processes, and has wide application value.

The present invention belongs to the field of biotechnology, and specifically relates to a method for establishing a mitochondrial DNA gene library and a method for obtaining a mitochondrial genome sequence. The method for establishing the library is as follows: (1) after hybridization of the total DNA of the sample with an RNA probe, the target sequence is captured; (2) the target sequence is collected and amplified to obtain the amplified mitochondrial DNA. The amplified DNA is fragmented, blunt-end repaired, connected to a sequencing adapter, and indexed by PCR to construct a library. The obtained gene library is sequenced, and the sequencing results are assembled. The scheme of the present invention enriches the original long-fragment mitochondrial DNA before constructing the sequencing library, so that more mitochondrial DNA data can be obtained, thereby improving the enrichment efficiency, increasing the proportion and coverage of the mitochondrial genome data, and achieving complete assembly; at the same time, the amount of data required for assembly is reduced, and the sequencing cost is greatly reduced. The present invention does not require a reference genome of the corresponding species, and can relatively quickly and economically obtain the complete mitochondrial genome of various vertebrates, providing a feasible technical route for establishing a vertebrate environmental DNA mitochondrial genome database.

The invention relates to a three-generation library building and sequencing method for a full-length sequence of an HLA (Human Leukocyte Antigen)-III antigen gene, and belongs to the field of gene detection. According to the invention, PCR amplification is carried out on HLA-III type antigen genes by using five pairs of characteristic amplification primers. And each gene can be subjected to high-yield specific amplification on a full-length sequence containing complete intron and exon regions only through one round of amplification and enrichment, and 14Kb long-fragment genes can be captured once at most. The experimental deviation caused by simultaneous amplification of a single gene by using a plurality of pairs of segmented primers and the indirect error caused by gene splicing are effectively avoided. The HLA-III type antigen gene library is constructed by carrying out quality inspection and purification on amplicon PCR products and then carrying out sample mixing, the full-length sequence of the HLA-III type antigen gene is accurately and completely sequenced by adopting a three-generation PacBio Sequel II platform, and the Hifi reads base accuracy rate can reach 99% or above. The reading of HLA-III type antigen gene full-length sequence variation information and the haplotype analysis at a high resolution level can be realized.

The invention relates to a full-length sequence amplification primer, an amplification method and a three-generation sequencing method of an HLA-II antigen gene, and belongs to the field of gene detection. According to the invention, PCR amplification is carried out on HLA-II type antigen genes by using 10 pairs of characteristic amplification primers. Each gene can be subjected to high-yield specific amplification on a full-length sequence containing complete intron and exon regions only through one round of amplification and enrichment, and 17Kb long-fragment genes can be captured once at most. The experimental deviation caused by simultaneous amplification of a single gene by using a plurality of pairs of segmented primers and the indirect error caused by gene splicing are effectively avoided. The method comprises the following steps: carrying out quality inspection and purification on amplicon PCR (Polymerase Chain Reaction) products, carrying out sample mixing, constructing an HLA-II antigen gene library, carrying out accurate and complete sequencing on the HLA-II antigen gene full-length sequence by adopting a three-generation PacBio Sequel II platform, and ensuring that the Hifi reads base accuracy can reach 99% or above. The reading of HLA-II type antigen gene full-length sequence variation information and the haplotype analysis at a high resolution level can be realized.

A gene library construction method, the method comprising: providing a first gene fragment; providing a template gene fragment; combining the first gene fragment with the template gene fragment to obtain a target gene sequence having a sequence length greater than that of the first gene fragment; and performing PCR amplification on the target gene sequence to obtain a gene library.