128 results about "Sanger sequencing" patented technology

The invention belongs to the field of biological detection, and particularly relates to an ewing sarcoma detection primer group, a kit and application thereof. The Ewing sarcoma detection primer group comprises a primer group A and a primer group B, and totally has 17 primer sequences. The primer group is designed, Ewing sarcoma characteristic variation genes are detected through multiple fluorescent PCR and a high-resolution capillary electrophoresis apparatus, 24 variation types fused by 7 types of genes can be detected at a time, the lowest detection limit is 10 copies, and the shortest detection time is 240 minutes. The detection result of the primer group disclosed by the invention can pass Sanger sequencing verification and is completely consistent with the morphological diagnosis result, the methodological consistency reaches 100%, and a sensitive and efficient Ewing sarcoma genetic variation detection method is provided.

The invention relates to an IGF2BP3 gene molecular marker primer related to pigeon weight traits and application of the IGF2BP3 gene molecular marker primer, and belongs to the technical field of biology. According to the invention, genome DNA of a to-be-detected pigeon is taken as a template, a specific primer is adopted to carry out PCR amplification on the genome DNA, then a PCR amplification product is subjected to Sanger sequencing and SNP molecular marker genotyping, and then pigeon body weight character selection is carried out. The weight of G / G genotype pigeons is higher than that of A / G and A / A genotype individuals, and the weight of A / G genotype pigeons is higher than that of A / A genotype individuals. By eliminating A / G and A / A genotype individuals and retaining G / G genotype individuals, the molecular marker has the beneficial effects that the molecular marker is used as a genetic marker for breeding pigeons, and the pigeons with large weight and uniformity are bred; the method can efficiently and quickly identify the weight traits of the pigeons, improves the uniformity of the weight of pigeons, provides a scientific basis for the early breeding of the pigeons, and better serves the breeding of the pigeons.

The invention relates to a primer of an RBP4 gene molecular marker related to pig weak litter number character and application of the primer, and belongs to the technical field of biology. The RBP4 gene molecular marker is located at the 105043789th base of chromosome 14 of a pig reference genome Sscrofa11.1 version, the base is mutated into A or G, and the genotypes are A / A, A / G and G / G. The number of weak piglets of the G / G genotype pigs is lower than that of the A / G and A / A genotype individuals, and the number of weak piglets of the A / G genotype pigs is lower 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 is used as a genetic marker for pig breeding to breed pigs with less weak piglets; the method can efficiently and accurately detect the weak piglet number character, improves the survival rate of healthy piglets of a pig herd, and has an important value for breeding and production of pigs.

The invention relates to a primer of a GAREM1 gene molecular marker related to the average daily gain character of pigs and application of the primer, and belongs to the technical field of biology. The GAREM1 gene molecular marker is located at the 116131455th basic group of chromosome 6 of a pig reference genome Sscrofa11.1 version, the basic group is mutated into A, and the genotypes are A / A and A / T. The average daily gain of A / T genotype pigs is higher than that of 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 pig breeding to breed pigs with proper weight; the method can be used for efficiently and accurately detecting the average daily gain character of the pigs, and has an important value for breeding and production of the pigs.

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 discloses an SNP (Single Nucleotide Polymorphism) molecular marker related to growth traits of litopenaeus vannamei and application of the SNP molecular marker, and belongs to the field of molecular marker-assisted breeding of aquatic animals. Specifically, after a target fragment located on the MEF2 gene of the litopenaeus vannamei is amplified through PCR (polymerase chain reaction), the genotype of the SNP molecular marker located on the MEF2 gene is identified by using a Sanger sequencing method or a PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method, and three genotypes of GG, GC and CC are found. Through correlation analysis of the genotype of the SNP molecular marker and the growth traits of the litopenaeus vannamei, it is found that the growth traits of the litopenaeus vannamei with the genotype of GG are superior to those of GC and CC genotypes. Therefore, the SNP molecular marker can be used as a functional site for screening litopenaeus vannamei with excellent growth traits, and the research provides data for mining litopenaeus vannamei growth traits related molecular markers.

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 an InDel molecular marker related to seedless traits of filial generations of grapes and application of the InDel molecular marker. The nucleotide sequence deletion part of the InDel molecular marker for the seedless type of the filial generation of seedless grape and nucleated grape is shown as SEQ ID NO.3, and is positioned at the upstream 263-693 bp of a VviAGL11 gene transcription start site (TSS) which is closely linked with the seedless character of the grape, and the nucleotide sequence deletion part of the InDel molecular marker is shown as SEQ ID NO.6. The nucleotide sequence deletion part of the InDel molecular marker is shown as SEQ ID NO.7. The nucleotide sequence deletion part is shown as SEQ ID NO.8. According to the invention, PCR (Polymerase Chain Reaction) amplification and Sanger sequencing are carried out on VviAGL11 gene promoters of a seedless grape variety 'Zhongqing 18' and a seedless grape variety 'Musang', so that polymorphic sites with significant difference are obtained, and the difference between the polymorphic sites is 431 bp. The InDel molecular marker is suitable for grape seedless molecular marker-assisted selective breeding, and the invention also discloses a method for carrying out genotype identification through an agarose electrophoresis technology in a seedling stage of hybrid generations of seedless grapes and seedless grapes, so that early selection of seedless characters of the grapes is realized; the seedless grape breeding efficiency is further improved, the breeding cost is reduced, and the breeding process is promoted.

The invention relates to a method for identifying Langya chicken and Non-Langya chicken by using SNP molecular markers, and belongs to the technical fields of animal husbandry and molecular biology. The SNP molecular marker for Langya chicken identification comprises 12 loci. The invention further provides a primer combination which comprises SNP loci for amplifying Langya chicken identification. According to the method, the 12 specific SNP sites of the Langya chicken are utilized, the site information is obtained through PCR amplification and Sanger sequencing, prediction is conducted through machine learning, the Langya chicken variety identification model is established based on the 12 SNP site combination, and the method has the advantages of being low in cost, simple in sequencing method, high in speed and high in accuracy.

The invention relates to a primer of a KLHL14 gene molecular marker related to pig weak litter number character and application of the primer, and belongs to the technical field of biology. The KLHL14 gene molecular marker is located at the 116519104 base of chromosome 6 of a pig reference genome Sscrofa11.1 version, the base is mutated into C or T, and the genotypes are C / C, C / T and T / T. 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 pig breeding to breed pigs with less weak piglets; the method can efficiently and accurately detect the character of the number of the weak piglets of the pigs, improves the survival rate of healthy piglets of a swinery, and has an important value for breeding and production of the pigs.

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 present invention provides a circRNA related to the proliferation of porcine skeletal muscle satellite cells and its application. Based on whole transcriptome sequencing, the present invention first discovers that circular RNA circLMO7 is highly expressed in the muscle tissue of Jinfen white pigs, and its expression level significantly decreases at 1 day, 90 days, and 180 days. The objective existence of circLMO7 is confirmed by PCR, Sanger sequencing, and RNase R digestion experiments; further, real-time fluorescence quantitative PCR is used to confirm that the expression level of circLMO7 continuously decreases at 1 day, 90 days, and 180 days in Jinfen white pigs; the EdU, CCK-8, and real-time fluorescence quantitative PCR experiments are used to further verify that circLMO7 plays a significant upregulating role in the proliferation process of porcine skeletal muscle satellite cells. The present invention proposes that circLMO7 can be used as a biomarker to identify the proliferation of porcine skeletal muscle satellite cells.

The invention discloses application of a methyltransferase mutant and deaminase in DNA methylation single base resolution sequencing, and relates to the technical field of gene sequencing. On the basis of a methyltransferase mutant M.MedI-N377K, cytosine at a CG site in DNA (Deoxyribonucleic Acid) can be specifically recognized, and carboxymethyl of carboxyl-S-adenosine-L-methionine (caSAM) is transferred to a C5 site of C in the CG, so that the 5-carboxymethyl cytosine is generated. 5-methylcytosine can be subjected to deamination by deaminase to form thymine, and the thymine is paired with adenine in subsequent PCR amplification; and 5cam C can resist deamination of A3A and is paired with guanine in subsequent PCR (Polymerase Chain Reaction), and 5mC is read as T and C is read as C in a subsequent Sanger sequencing result of an amplification product, so that C and 5mC in DNA (Deoxyribonucleic Acid) are distinguished. The method disclosed by the invention is high in sensitivity, good in specificity and simple to operate, does not involve hydrosulfite treatment, and can be used for carrying out positioning analysis on 5mC in a biological sample under a single-base resolution ratio.

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 present invention belongs to the technical field of sequencing in gene therapy. Provided is a detection method for a rare-mutation BRAF gene. The sequencing method comprises: during an amplification process of the BRAF gene of a target sample fragment, adding a blocking primer matched with a wild-type template in the target sample fragment, so as to bind the blocking primer to a denatured wild-type template; and performing sequencing to obtain a sequencing result corresponding to the target sample fragment. During the process of making a sequencing template, the present invention uses the blocking primer to inhibit the wild-type template of the BRAF gene during the amplification process, so as to relatively increase the proportion of a mutant product, thereby achieving the purpose of accurately detecting samples with a lower mutation rate. Therefore, the present method is of an important significance for detecting rare mutations by using a Sanger sequencing method.

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 discloses a method for constructing a fetal osteoarthritis model caused by prednisone exposure during pregnancy, an intervention target, and its application. circGtdc1 is a newly identified circRNA that is most significantly altered in dysplastic cartilage caused by prednisone exposure during pregnancy. RNase R, AcD, and Sanger sequencing confirmed its circularization. Prednisolone concentration-dependently reduced circGtdc1 expression in chondrocytes. Silencing circGtdc1 significantly inhibited chondrocyte proliferation and matrix synthesis gene expression, while overexpression of circGtdc1 had the opposite effect and could reverse the effects of prednisolone. Intra-articular injection of AAV‑circGtdc1 to overexpress circGtdc1 can reverse chondrodysplasia caused by prednisone exposure during pregnancy. The intervention target established by the present invention is novel, reliable, and simple, providing a possibility for the early prevention and treatment of fetal osteoarthritis.

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 present invention relates to a molecular marker primer for the IGF2BP3 gene related to the weight trait of pigeons and its application, belonging to the field of biotechnology. The present invention uses the genomic DNA of the pigeon to be tested as a template, adopts specific primers to perform PCR amplification on it, and then performs Sanger sequencing and SNP molecular marker genotyping on the PCR amplification product, thereby selecting the pigeon weight trait. The weight of the pigeon with G / G genotype is higher than that of the individuals with A / G and A / A genotypes, and the weight of the pigeon with A / G genotype is higher than that of the individuals with A / A genotype. By eliminating the individuals with A / G and A / A genotypes and retaining the individuals with G / G genotype, the beneficial effect is that the molecular marker can be used as a genetic marker for pigeon breeding to select pigeons with larger and more uniform weight; the pigeon weight trait can be identified efficiently and quickly, and the uniformity of the weight of the pigeon group can be improved, providing a scientific basis for the early selection of pigeons, and better serving the breeding of pigeons.

Described herein are methods, compositions and kits directed to amplification of nucleic acids suitable for both next generation sequencing (NGS) and a second round of sequencing as validation, such as Sanger sequencing.

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 method for identifying aquilaria agallocha and aquilaria sinensis by using a DLAT gene. Aquilaria agallocha and aquilaria sinensis are identified on the basis that the DLAT genotype of the Aquilaria agallocha is TT and the DLAT genotype of the aquilaria sinensis is CC. A No.1879 basic group of a DLAT gene coding region of agilawood aquilaria sinensis and aquilaria sinensis is taken as a target site, a PCR and Sanger sequencing method is adopted, a PCR amplification product of the DLAT gene is subjected to Sanger sequencing, if the genotype of the DLAT is TT, the product is agilawood aquilaria sinensis, and if the genotype of the DLAT is CC, the product is aquilaria sinensis. According to the method for identifying the agilawood agilawood and the aquilaria sinensis through the DLAT gene, high-throughput sequencing is carried out on the agilawood agilawood and the aquilaria sinensis, genotype detection analysis is carried out in a whole genome range, and it is found that the DLAT gene has significant difference between the agilawood agilawood and the aquilaria sinensis, and the gene function is clear. The method is used for identifying the germplasm of the agilawood agilawood and the aquilaria sinensis by utilizing the genotype difference of the agilawood agilawood and the aquilaria sinensis and adopting PCR amplification and Sanger sequencing methods

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.