85 results about "Sanger sequencing" patented technology

The invention relates to the technical field of biology, and discloses sgRNA for knocking out a TNIP2 gene, the nucleotide sequence is shown as SEQ ID NO: 1, blocking of the sgRNA to TNIP2 expression is verified through sanger sequencing and Western Blotting experiments, a TNIP2 knockout cell line and a control cell have no obvious morphological and growth difference, and the sgRNA can be used for knockout of the TNIP2 gene. The sgRNA can inhibit the replication ability of the H1 subtype influenza virus by regulating and controlling the expression of a host gene TNIP2, in addition, the invention further discloses application of the sgRNA, a cell strain with the TNIP2 gene knocked out and a construction method of the cell strain.

The invention belongs to the technical field of aquatic product breeding, and particularly relates to a KASP molecular marker related to high temperature resistance character of patinopecten yessoensis and application of the KASP molecular marker in genetic breeding. On the basis of genome re-sequencing of 200 patinopecten yessoensis, a PySNP587919 locus obviously associated with the patinopecten yessoensis high-temperature-resistant character is screened by combining a whole genome SNP genotype and whole genome association analysis of the patinopecten yessoensis high-temperature-resistant character, and after the reliability of the locus is verified through Sanger sequencing, the locus is developed into a KASP marker, and the KASP marker can be used for identifying the high-temperature-resistant character of the patinopecten yessoensis and identifying the high-temperature-resistant character of the patinopecten yessoensis. The screening method can be used for screening the high-temperature-resistant comb shells. Furthermore, the invention also establishes a method for screening the high-temperature-resistant patinopecten yessoensis, and the method is efficient and accurate. Therefore, the technical scheme provided by the invention is suitable for molecular marker-assisted breeding work, rapid and effective screening of parents can be realized by typing the PySNP587919 site, and the breeding efficiency is improved.

The invention discloses a molecular marker, a kit and a genotype detection method for predicting high content of highly unsaturated fatty acid in mirror carp muscle, and relates to the technical field of biomolecular detection, in particular to a molecular marker, a kit and a genotype detection method for predicting high content of highly unsaturated fatty acid in mirror carp muscle. The molecular marker for predicting the high HUFA content of the mirror carp muscle is as shown in SEQ ID NO. 1. The primer pair for predicting the high HUFA content of the mirror carp muscle is hufaf1 and hufar1. The genotype detection method comprises the following steps: 1, extracting DNA; 2, carrying out PCR (Polymerase Chain Reaction) amplification; 3, Sanger sequencing is carried out, and the mirror carp with a C single peak appearing at the 75bp position is selected. By means of the molecular marker, the kit and the genotype detection method, individuals with muscle high HUFA content potential can be rapidly identified based on living body losslessness, and therefore accurate and efficient breeding of the mirror carp high HUFA content character is achieved.

This invention relates to a primer set and method for high-throughput assessment of drug resistance levels in rodent populations. The invention provides a set of universal degenerate primers for amplifying the Vkorc1 gene in multiple rodent species. The primer set includes primers for amplifying exon 1, exon 2, and exon 3 of the Vkorc1 gene, respectively. By designing universal degenerate primers and coupling them with next-generation sequencing technology, this invention can simultaneously amplify the Vkorc1 gene coding region of at least 15 rodent species across genera and families. This significantly improves the species universality and detection throughput of rodent drug resistance monitoring, reduces the cost of large-scale screening, overcomes the limitation of Sanger sequencing in accurately analyzing heterozygotes with insertions and deletions, and enhances the ability to discover new resistance mutations. It provides a standardized and low-cost technical solution for efficient assessment of rodent drug resistance.

Provided is an accurate guide RNA (gRNA) screening method for base editing of an asialoglycoprotein receptor 1 (ASGR1) gene, including the following steps: (1) gRNA design; (2) primer design; (3) in vitro transcription of gRNA; (4) cell transfection; (5) collection of cells, and extraction and polymerase chain reaction (PCR) of a genome; and (6) Sanger sequencing.

The invention discloses a construction method of a COL4A5-K229X point mutation X-linked Alport syndrome mouse model. The construction method comprises the following steps: aiming at c.685Agt of a No.12 exon of a mouse COL4A5 gene; carrying out T point mutation, and designing and preparing Cas9 mRNA, gRNA and a donor vector; the components are mixed and then microinjected into fertilized eggs of a C57BL / 6J mouse to obtain an F0-generation mouse; identifying the genotype through PCR (Polymerase Chain Reaction) amplification and Sanger sequencing, and screening positive mice; mating the positive F0-generation mice with the wild-type mice, and breeding F1-generation and subsequent generations; the phenotype of the model is further verified through qPCR, biochemical analysis, light microscopic examination, transmission electron microscope and immunofluorescence. The model constructed by the invention shows hematuria, proteinuria, azemia, podocyte loss and irregular thickening and layering of glomerular basement membrane, is consistent with phenotypes of human XLAS patients, and provides an animal model tool for analyzing pathogenesis and developing treatment strategies.

The application discloses a molecular marker, a kit and a genotype detection method for predicting high content of highly unsaturated fatty acids in mirror carp muscle, relates to the technical field of biomolecular detection, and specifically relates to a molecular marker, a kit and a genotype detection method for predicting high content of highly unsaturated fatty acids in mirror carp muscle. The molecular marker for predicting high content of highly unsaturated fatty acids in mirror carp muscle is shown as SEQ ID NO. 1. The primer pair for predicting high content of highly unsaturated fatty acids in mirror carp muscle is hufaf1 and hufar1. The genotype detection method comprises the following steps: one, extracting DNA; two, PCR amplification; and three, Sanger sequencing, and the mirror carp with a C single peak at the 75th bp is selected. The application can identify individuals with potential high content of highly unsaturated fatty acids in muscle based on living body non-damage and rapidness through the above-mentioned molecular marker, the kit and the genotype detection method, so that precise and efficient breeding of the high content of highly unsaturated fatty acids in mirror carp is realized.

The application relates to a primer of a MEP1B gene molecular marker related to a pig average daily weight gain trait and application thereof, and belongs to the technical field of biology. The MEP1B gene molecular marker of the application is located at the 116042900th base of chromosome No. 6 of a pig reference genome Sscrofa11.1 version 6, and has C / T polymorphism; the average daily weight gain of a pig with a TT genotype at the site is significantly higher than that of a pig with a CC genotype. The application uses forward and reverse primers to amplify a segment containing the above-mentioned molecular marker site, and Sanger sequencing is performed on the amplification product, so that the genotype of an individual can be quickly and accurately identified; the molecular marker can be used as a genetic marker for pig breeding, and pigs of a proper weight can be selected and bred; the pig average daily weight gain trait can be efficiently and accurately detected, and the pig average daily weight gain trait has important value for pig breeding and production.

The present invention relates to a multiplex Sanger sequencing kit and method. Using the kit and method according to the present invention can facilitate multiple amplification and sequencing in Sanger sequencing for diagnosing genetic diseases and the like, and thus can significantly save costs and time.

This invention relates to a 3'RACE library construction and sequencing method and its application based on third-generation sequencing technology. The method includes: S1, using primers containing anchor sequences and Oligo d(T) to reverse transcribe sample RNA to obtain cDNA, which is then used as a template for specific amplification to obtain the target gene; S2, designing specific primers containing third-generation sequencing adapters and barcodes, using the target gene as a template for amplification, and constructing a high-throughput third-generation sequencing library; S3, performing third-generation sequencing and analysis on the library to obtain the complete 3' end sequence information of the target transcript. This invention abandons traditional vector construction and Sanger sequencing methods, leveraging the high throughput and long read length characteristics of third-generation sequencing to ensure that Race experiments obtain a large amount of long 3' end sequence information in a short period, improving the overall efficiency and data accuracy of the experiment, and laying the foundation for refined mRNA research.

The present application relates to a POMT1 gene molecular marker related to chicken abnormal egg trait and application thereof, and belongs to the technical field of biology.The POMT1 gene molecular marker of the present application is located at the 6831149th base of chromosome 17 of chicken reference genome GRCg7b version 17, and the base mutation is G or T, and the genotype is G / G, G / T and T / T.The abnormal egg rate of the chicken with G / G genotype is lower than that of the individuals with G / T and T / T genotypes, and the abnormal egg rate of the chicken with G / T genotype is lower than that of the individual with T / T genotype.The present application amplifies the fragment containing the above-mentioned molecular marker site by using forward and reverse primers, and performs Sanger sequencing on the amplification product, so that the genotype of the individual can be quickly and accurately identified.The molecular marker can be used as a genetic marker for chicken breeding, and the chicken with low abnormal egg rate can be selected and bred, the chicken abnormal egg trait can be efficiently and accurately detected, the population abnormal egg rate is reduced, and the chicken breeding and production have important value.

The invention provides a microbial pollution source tracing method based on Sanger sequencing overlapping peak analysis, which comprises the following steps: analyzing and enumerating multi-base overlapping peaks in a sequencing peak graph to generate all possible base combination sequences, comparing with a microbial database, and screening effective sequences to identify bacterial strain types. And high-threshold homologous comparison is further carried out on the identified mixed strains and a pre-constructed pollution source standard gene sequence library, so that the pollution source is accurately traced. According to the method, original invalid sequencing data is fully utilized, repeated experiments are avoided, and accurate identification of main strains and systematic tracing of pollution sources are realized.

The invention provides a system, a kit and a detection method for rapidly detecting a drug-resistant gene of clarithromycin of helicobacter pylori by a one-pot method. The system comprises an RPA (recombinase polymerase amplification) primer for detecting point mutation of an H. pylori 23S rRNA (ribosomal Ribonucleic Acid) gene A2143G and a T7 / crDNA (deoxyribonucleic acid) hybrid chain for transcribing and recognizing a crRNA 2143G point mutation of the H. pylori 23S rRNA gene A2143G. Based on the system, the kit disclosed by the invention can specifically recognize the helicobacter pylori clarithromycin drug-resistant gene within 30 minutes, and independent preparation of crRNA is not needed, so that the reagent cost is reduced, the experimental process is simplified, uncovering operation is not needed, and cross contamination is avoided; the detection limit of the kit is 5 copies / [mu] L, no cross reaction exists, and the result of the kit is consistent with that of a Sanger sequencing method in a clinical sample.

The invention relates to a high-sensitivity multi-gene joint detection kit. The kit is used for detecting mutation conditions of human BRAF, KRAS, NRAS, HRAS, RET, TERT, TP53 and PIK3CA genes. The primer probe and the amplification system are unique, the 37 mutation types of the 8 genes can be specifically and highly sensitively detected only through 5 ng DNA, and the detection sensitivity is as low as 1%-5%. Compared with the defects that the traditional Sanger sequencing detection sensitivity is low, the number of detection sites of a PCR method is small, NGS operation is complex, and the price is high, the method has obvious advantages in clinical application.

The invention discloses a ganoderma species specific target sequence based on artificial intelligence screening, a primer pair, a kit, applications thereof and a species identification method. The target sequence is obtained by learning and screening sequence features through an artificial intelligence model based on disclosed genome data of ganoderma lucidum, the species specificity of the target sequence is further verified through two technical means of agarose gel electrophoresis and Sanger sequencing, the target sequence can be used for distinguishing ganoderma lucidum, ganoderma sinensis and related species thereof, and accurate judgment of a sample to be detected on the species level is achieved. The identification method constructed based on the target and primer system has the characteristics of high specificity, good sensitivity, stable result, simple and convenient detection process and the like, is suitable for identification of ganoderma lucidum sporocarp, hypha, spore powder, medicinal materials, decoction pieces and related products, and provides a basis for ensuring clinical medication safety and standardizing the ganoderma lucidum market.

The invention discloses a primer and probe composition for detecting neurofibroma type I NF1 gene variation sites, belongs to the technical field of gene diagnosis, and provides a group of specific primers and probe compositions aiming at 12 specific high-frequency or pathogenicity-clear variation sites of an NF1 gene. The composition comprises a specific primer pair aiming at 12 specific high-frequency or specific pathogenicity variation sites of the neurofibroma I-type NF1 gene and a double-labeled oligonucleotide probe matched with the specific primer pair, and the probe is suitable for melting curve analysis; the sequences of the primer pair and the probe of the 12 variation sites are SEQ ID NO. 1 to SEQ ID NO. 36. The primer and probe composition is high in specificity and sensitivity, the kit is easy and convenient to operate and low in cost, the detection result is consistent with that of a gold standard Sanger sequencing method, and the primer and probe composition is suitable for rapid screening and auxiliary diagnosis of clinical samples and has wide clinical application prospects and market value.

This application relates to the field of image processing technology, specifically to a high-voltage power supply DNA sequencing visual inspection method and system. The method includes: acquiring a four-channel DNA electrophoresis image using Sanger sequencing technology; acquiring contours in the DNA electrophoresis grayscale image; constructing a DNA local density confidence coefficient and local DNA density parameters for each pixel based on the grayscale values ​​of each pixel within a channel and its neighboring pixels; acquiring the density run matrix for each pixel; constructing a DNA density distribution heterogeneity coefficient and channel tailing effect evaluation parameters for each pixel, and based on these, constructing a sequencing segmentation probability coefficient for each pixel; acquiring a decision threshold for each pixel; using the center of each contour as a seed point, and acquiring each DNA fragment based on an adaptive threshold using a region growing method, thereby obtaining the DNA sequence. This application can improve the accuracy of detecting the base sequence of a DNA sequence.

The invention provides a PMFBP1 mutant gene related to azoospermia syndrome and an application of the PMFBP1 mutant gene. The mutant gene is a PMFBP1 gene c.2641Cgt; the protein p.Arg881Ter is subjected to non-sense mutation, so that the encoded protein p.Arg881Ter is truncated, and the function of a sperm head-tail connection device is damaged. The invention also provides a specific Sanger sequencing detection method and a kit of the mutation, which can be used for rapid molecular diagnosis, genetic counseling and assisted reproduction guidance of the disease, and provides a basis for precise medical treatment.

The invention discloses a primer group and a kit for detecting gene polymorphism of Alzheimer's disease, a method for detecting gene polymorphism and application, the primer group comprises forward and reverse PCR amplification primers aiming at each gene, a target gene segment is amplified through specific PCR, then an amplification product is sequenced by adopting a Sanger sequencing method, and the primer group and the kit are used for detecting the gene polymorphism of Alzheimer's disease. And comparing and analyzing a sequencing result through a software sequence to obtain the base polymorphism information of the target gene.

The application discloses a HIV-1 drug-resistant gene detection method based on NGS technology, belongs to the technical field of gene detection, adopts a series of processes such as NGS data quality control and filtration, genome alignment and extraction of effective information, construction of consistent sequences, construction of a pseudo-reference genome, HIVdb drug resistance detection, visualization of NGS sequencing results and HIVdb drug resistance detection results, a traceability evolution tree and final output results to complete HIV-1 drug-resistant gene detection. Compared with traditional first-generation Sanger sequencing technology, the system and method adopt the application, can detect drug-resistant sites with a mutation frequency of less than 10%, have higher detection sensitivity, and have important significance for early screening and detection of HIV-1 virus and doctors to formulate more effective drug use schemes after the onset.

The application relates to a primer of a VPS13C gene molecular marker related to a porcine eye muscle area trait and application thereof, and belongs to the technical field of biology. The VPS13C gene molecular marker of the application is located at the 110033015 base of chromosome No. 1 of a porcine reference genome Sscrofa11.1 version 1, the base mutation is G, and the genotype is G / C and C / C. The eye muscle area degree of the G / C genotype porcine is higher than that of the C / C genotype individual. The application amplifies the fragment containing the above-mentioned molecular marker site by using the forward and reverse primers, and carries out Sanger sequencing on the amplification product, so that the genotype of the individual can be quickly and accurately identified; the molecular marker can be used as a genetic marker for porcine breeding, and pigs with appropriate sizes are selected and bred; the eye muscle area of the porcine can be efficiently and accurately detected, and the application has important values for porcine breeding and production.

The application provides a recognition correction method based on Sanger sequencing ab1 file half-peak condition, the application can read sequence and related peak height information of a first peak and a second peak of the ab1 file, screen out files possibly having the half-peak condition through comparison of the first peak and the second peak, then correct corresponding bases and peak heights, and finally obtain corrected scf files and fasta files for later sequence analysis. The application provides an effective method for solving the ab1 file half-peak phenomenon in Sanger sequencing, improves the accuracy and reliability of sequencing data, and avoids sequencing cost, analysis cost and time cost caused by repeated resequencing.

The invention provides a primer pair for detecting TP53 gene exon region mutation and a kit applying the primer pair. The primer pair comprises a first forward primer and a first reverse primer, the nucleotide sequence of the first forward primer is shown as SEQ ID NO: 1, and the nucleotide sequence of the first reverse primer is shown as SEQ ID NO: 2. The primer pair for detecting TP53 gene exon region mutation provided by the invention can be used for performing PCR (Polymerase Chain Reaction) amplification on cDNA (Complementary Deoxyribose Nucleic Acid) obtained by performing reverse transcription on RNA (Ribonucleic Acid) extracted from fresh EDTA (Ethylene Diamine Tetraacetic Acid) anticoagulant whole blood and identifying through Sanger sequencing to find that a PCR amplification product consistent with a theoretical sequence can be obtained; therefore, the specific amplification and mutation detection of the TP53 gene exon coding region with reliable results can be simply, conveniently and quickly completed at one time.

The invention relates to a KIF26B gene molecular marker primer related to chicken continuous laying traits and application of the KIF26B gene molecular marker primer, and belongs to the technical field of biology. The molecular marker of the KIF26B gene is located at the 34213811th base of chromosome 3 of a chicken reference genome GRCg7b version, the base is mutated into A or G, and the genotypes are A / A, A / G and G / G. The total continuous production length of the G / G genotype chicken is higher than that of the A / G and A / A genotype individuals, and the total continuous production length of the A / G genotype chicken is higher than that of the A / A genotype individuals. According to the present invention, the forward and reverse primer pair is used to amplify the fragment containing the molecular marker site, and the amplification product is subjected to Sanger sequencing so as to rapidly and accurately identify the genotype of the individual; the molecular marker can be used as a genetic marker for chicken breeding, can efficiently and accurately detect the continuous laying character of chicken, improves the total egg laying number of chicken flocks, and has important value for chicken breeding and production.

The invention relates to a molecular marker primer related to duck black matter characters and application of the molecular marker primer, and belongs to the technical field of biology. The KIT gene SNP molecular marker is located at the 51648174 basic group of chromosome 4 of a duck reference genome GCF047663525.1 version, the basic group is mutated into T or C, and the genotypes are T / T, T / C and C / C. The ducks of the C / C and T / C genotype individuals show a ubiquitous phenotype, and the ducks of the T / T genotype individuals show a non-ubiquitous phenotype. According to the present invention, the forward and reverse primer pair is used to amplify the fragment containing the molecular marker site, and the amplification product is subjected to Sanger sequencing and genetic typing so as to rapidly and accurately identify the genotype of the individual; the molecular marker can be used for efficiently and accurately identifying the duck black matter character genotype, the accuracy and efficiency of duck black matter character identification are improved, and the molecular marker has important value for protection and utilization of excellent germplasm resources of black-bone ducks.

The present application relates to a kind of KIF26B gene molecular marker primer associated with chicken laying performance and its application, belong to the field of biotechnology.The KIF26B gene molecular marker of the present application is located at the 34213811 base of the chromosome No.3 of chicken reference genome GRCg7b version, and the base mutation is A or G, and the genotype is A / A, A / G and G / G.The total laying length of G / G genotype chicken is higher than that of A / G and A / A genotype individuals, and the total laying length of A / G genotype chicken is higher than that of A / A genotype individuals.The present application amplifies the fragment containing the above-mentioned molecular marker site using forward and reverse primers, and the amplified product is subjected to Sanger sequencing, so that the genotype of individual can be quickly and accurately identified.The molecular marker can be used as a genetic marker for chicken breeding, and can efficiently and accurately detect chicken laying performance, improve the total number of eggs of chicken population, and has important value for chicken breeding and production.

The invention relates to a primer group, a kit and a method for detecting a Fabry cardiomyopathy genetic susceptibility gene, and belongs to the technical field of gene detection. The genetic susceptibility gene of the Brie cardiomyopathy is a GLA gene, and the mutation site of the genetic susceptibility gene is c.640-801Ggt; a; the primer group comprises a PCR (Polymerase Chain Reaction) amplification primer group, and the amplification primer group comprises a forward PCR amplification primer and a reverse PCR amplification primer; the primer group further comprises a Sanger sequencing primer group, and the sequencing primer group comprises a forward sequencing primer and a reverse sequencing primer. The invention also provides a kit and a detection method based on the primer group. According to the primer group and the kit containing the primer group, the c.640-801Ggt of the Fabry cardiomyopathy GLA gene can be detected on the basis of a first-generation sequencing technology; a site mutation has important value for early screening detection and genetic risk assessment of the disease gene.

The invention discloses application of a reagent for detecting SCN2B and / or SCN4B gene mutation in preparation of a hereditary arrhythmia detection product. According to the present invention, through the dual detection system combining whole exon sequencing with Sanger sequencing verification, the accurate capture of the pathogenic mutation such as SCN2B-R28Q / Y69H / P210L and SCN4B-T211M is achieved, and the problem that only SCN5A is determined as the main pathogenic gene in the clinical diagnosis of J wave syndrome (JWS) at present, and the gene detection of about 70-80% of patients is negative is solved. Wherein the three missense mutations of the SCN2B and the SCN4B-T211M variation are reported in the JWS for the first time, and the SCN4B is clear as the JWS pathogenic gene for the first time, so that the pathogenic gene spectrum of the JWS is obviously expanded. A detection product developed on the basis of the method can realize early accurate diagnosis, provide personalized risk assessment (such as sudden death early warning induced by fever) for patients, guide genetic screening of family members and remarkably improve clinical diagnosis and treatment efficiency.

The application belongs to the field of biological detection, and particularly relates to a Ewing sarcoma detection primer group, a kit and application thereof. The Ewing sarcoma detection primer group comprises primer group A and primer group B, and a total of 17 primer sequences. The primer group is designed, Ewing sarcoma characteristic variant genes are detected through multiplex fluorescence PCR and a high-resolution capillary electrophoresis instrument, 24 variation types of 7 kinds of gene fusions can be detected at one time, the minimum detection limit is 10 copies, and the shortest detection time is 240 minutes. The detection result of the primer group can be verified through Sanger sequencing, and is consistent with morphological diagnosis results, the methodological consistency reaches 100%, and a sensitive and efficient Ewing sarcoma genetic variation detection method is provided.

The invention relates to a UMAD1 gene molecular marker primer related to chicken continuous laying traits and application of the UMAD1 gene molecular marker primer, and belongs to the technical field of biology. The UMAD1 gene molecular marker is located at the 24741087th basic group of a chromosome 2 of a chicken reference genome GRCg7b version, the basic group is mutated into G or T, and the genotypes are G / G, G / T and T / T. The total continuous production length of the T / T genotype chicken is higher than that of the G / T and G / G genotype individuals, and the total continuous production length of the G / T genotype chicken is higher than that of the G / G genotype individuals. According to the present invention, the forward and reverse primer pair is used to amplify the fragment containing the molecular marker site, and the amplification product is subjected to Sanger sequencing so as to rapidly and accurately identify the genotype of the individual; the molecular marker can be used as a genetic marker for chicken breeding, can efficiently and accurately detect the continuous laying character of chicken, improves the total egg laying number of chicken flocks, and has important value for chicken breeding and production.