365 results about "Sanger sequencing" patented technology

Methods of identifying DNase I Hyper-Resistant Sites (DHRS), or in board sense, highly compact chromatin and characterizing the DNA methylation status of DMRs such as CpG islands and CpG island shores are provided. The methods are particularly useful for analysis of genomic DNA from low quantities of cells, for example, less than 1,000 cells, less than 100 cells, less than 10 cells, or even one cell, and can be used to generate chromatin and methylation profiles. The downstream analyses include in parallel massive sequencing, microarray, PCR and Sanger sequencing, hybridization and other platforms. These methods can be used to generate chromatin and DNA methylation profiles in drug development, diagnostics, and therapeutic applications are also provided.

Exemplary methods, procedures, computer-accessible medium, and systems for base-calling, aligning and polymorphism detection and analysis using raw output from a sequencing platform can be provided. A set of raw outputs can be used to detect polymorphisms in an individual by obtaining a plurality of sequence read data from one or more technologies (e.g., using sequencing-by-synthesis, sequencing-by-ligation, sequencing-by-hybridization, Sanger sequencing, etc.). For example, provided herein are exemplary methods, procedures, computer-accessible medium and systems, which can include and/or be configured for obtaining raw output from a sequencing platform configured to be used for reading fragment(s) of genomes, obtaining reference sequences for the genomes obtained independently from the raw output, and generating a base-call interpretation and/or alignment using the raw output and the reference sequences. For example, a score function can be determined based on information associated with the sequencing platform that can be used to analyze polymorphisms based on the base-call interpretation and/or alignment.

The invention discloses a method for breeding rmnd5b (required for meiotic nuclear division 5homolog B) gene deletion type zebra fish through gene knockout and belongs to the field of gene knockout. According to the method, construction of a gRNA expression vector and gRNA in-vitro synthesis are performed through design of a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) gene knockout target site, micro-injection is performed on an embryo of the zebra fish, the effectiveness of the target site is detected, tail cutting identification is performed, TA cloning is performed on a target sequence, plasmids are subjected to Sanger sequencing, an F1 generation of heritable zebra fish mutants is obtained, the same mutant female fish and male fish are picked from mutants of the F1 generation, hybridization is performed, an F2 generation of the zebra fish mutants is obtained, F2 generation homozygote is picked from the F2 generation of the zebra fishmutants, F3 generation pure-line inheritance is performed, and an rmnd5b gene deletion type zebra fish strain is obtained. The method is lower in off-target rate and has good medical research value inresearch of the correlation between rmnd5b gene deletion and development of other organs.

Methods for highly parallel Sanger sequencing are discussed. In particular, provided herein are methods using particles to clonally amplify templates and to introduce the amplified nucleic acids into many parallel channels with a single template per channel. Once in the channels, the nucleic acids are separated by size using electrophoresis to produce long read length sequencing information. Methods involving optical detection of the size-separated nucleic acids and analysis of the resulting electropherograms to yield the sequences are disclosed.

The invention provides an STAG2 gene mutant sequence and a detection method thereof as well as use of STAG2 gene mutation in detecting bladder cancer and belongs to the technical field of the molecular biology gene detection. The detection method comprises the following steps: (1) extracting a tumor tissue DNA and a peripheral blood DNA from a tumor tissue and the peripheral blood of a bladder cancer patient; (2) performing whole genome sequencing and whole exon sequencing on the two DNAs extracted in the step (1), respectively; (3) performing whole exome sequence alignment and somatic mutation detection on the sequencing results of the step (2), thereby obtaining a mutant gene; (4) verifying the somatic replacement, insertion and deletion of the mutant gene obtained in the step (3) by use of Sanger sequencing; and (5) identifying the mutant gene as the remarkable mutant gene. The detection method has the advantage that the mutation of the STAG2 is one of unfavorable indexes of the bladder cancer and also is capable of serving as a new acting target for treating the bladder cancer.

The invention relates to the technical field of gene detection, and provides a method and a primer for detecting mutation sites of all exon sequences of human BRCA1 and BRCA2 genes. The specific primers include forward and backward primers for amplifying all 24 exons covering the BRCA1 gene, and base sequences of the primers are as shown in SEQ ID NO: 001-062, as well as forward and backward primers for amplifying all 27 exons covering the BRCA2 gene, and base sequences of the primers are as shown in SEQ ID NO: 063-142. By virtue of a Sanger sequencing method, the method and the primer disclosed by the invention can be used for detecting the mutation of all exons of the BRCA1 gene and the BRCA2 gene and can be used for extending all exons of the entire BRCA1 and BRCA2 genes, covering all to-be-detected mutation sites. The method and the primer disclosed by the invention are quite high in specificity and accuracy, simple to operate and low in cost, and can be used for greatly enhancing the risk assessment of hereditary breast cancer and effectively reducing the onset risk of the breast cancer.

The invention relates to a human idiopathic basal ganglia calcification pathogenic gene and a detection method thereof. Human idiopathic basal ganglia calcification, namely IBGC is a neurodegenerative genetic disease. The invention provides seven mutation forms of four pathogenic genes including SLC20A2 (Sodium-dependent phosphate transporter 2), PDGFRB (Platelet-derived Growth Factor Receptor Beta), PDGFB (Platelet-derived Growth Factor Subunit B) and XPR1 (Xenotropic and Polytropic Retrovirus Receptor 1) and sequences of the seven mutation forms are shown as SEQ ID NO.1 to SEQ ID NO.7. The form of the pathogenic gene provided by the invention is not reported until now and can provide evidence and lay a foundation for analysis and medicine development of a pathogenic mechanism, pathogenic gene screening and detection, formulation of a therapeutic regimen and the like. Meanwhile, the invention constructs a pathogenic gene detection method; the pathogenic gene detection method comprises the following steps: firstly, capturing a pathogenic gene exon region by utilizing multi-PCR (Polymerase Chain Reaction); carrying out next generation sequencing on the pathogenic gene exon region and carrying out information analysis to find out mutation; finally, identifying the mutation by utilizing Sanger sequencing, wherein a PCR captured primer group comprises amplification primer sequences SEQ ID NO.12 to SEQ ID NO.23, and amplification primer sequences of a Sanger sequencing segment are shown as SEQ ID NO.24 to SEQ ID NO.35. The detection method provided by the invention covers all exons of the four pathogenic genes and can be used for efficiently, comprehensively, rapidly and accurately acquiring mutation information.

The invention provides an SNP (single nucleotide polymorphism) marker combination and identification method of a small Meishan pig and a raw meat product. The method comprises the following steps: extracting a genome DNA of raw pork or a meat product, performing AGE (agarose gel electrophoresis) and Sanger sequencing after performing PCR (polymerase chain reaction) amplification on the genome DNA,and identifying the small Meishan pig and the meat product thereof according to an SNP genotype of a characteristic site of a sequencing result, wherein in the step of Sanger sequencing, peculiar mutation occurs at identified sites: SNP6, SNP12, SNP16, SNP71 and SNP79. According to the method, the problem that an identification method of the small Meishan pig and the meat product thereof does notexist in the prior art is solved.

The invention relates to a quality control method for detecting human BRCA1/2 genovariation based on high-throughput sequencing, a reagent kit, and an application thereof. In the quality control method, a plurality of genome DNAs of a human tumor cell line with positive BRCA1/2 genovariation are extracted, and are determined by means of a Sanger sequencing method, wherein a variation positive locus is used as a positive comparison locus while a wild type locus is a negative comparison locus; the genome DNAs are mixed at a certain ratio to obtain a quality control product that can be used for detecting human BRCA1/2 genovariation through high-throughput sequencing. The reagent kit disclosed by the invention comprises the quality control product for detecting human BRCA1/2 genovariation.

The invention discloses a high-flux detection method of a circulating DNA liver cancer driver gene. According to the method, a mutant gene which is the most common of the existing liver cancer is selected and combined with a second-generation high-flux sequencing technology to more comprehensively detect a gene mutant condition of the liver cancer. Since a blood plasma sample of a patient is adopted, the method can greatly reduce the detection injury of a patient; secondly, the whole-exome detection is performed on 75 liver cancer genes, the method is more comprehensive compared to the detection method based on real-time PCR and first-generation Sanger sequencing technology; and since only 75 genes are detected, the sample can be detected in a greater sequencing depth, so that the detection precision can reach 0.1 percent. Therefore, a reference can be provided for more and better individual precision therapeutic schemes of the patient.

The invention provides a method for taking DNA as an efficient text information storage medium, which is used for storing text information and is beneficial to culture protection. The method for taking DNA as a text information efficient storage medium comprises a storage process and a decoding process. The storage process comprises the following steps: establishing an information sequence, dividing text content into a plurality of storage units, and converting each storage unit into a DNA sequence by utilizing DNA tool software, thereby converting the whole text into a plurality of DNA sequences; adding 20bp protection sequences to the two ends of the information sequence respectively; adding primer binding regions at two ends of the protection sequence. The decoding process comprises thefollowing steps: culturing bacteria, extracting plasmids, carrying out polymerase chain reaction to obtain an amplification product, carrying out agarose gel electrophoresis and imaging identification on the amplification product, sequencing the amplification product by using a Sanger sequencing method, and reducing the DNA sequence obtained by sequencing into a Chinese character text by using DNATools software.

The invention discloses a multi-factor model for predicting a breast cancer onset risk and an establishing method of the multi-factor model. The establishing method of the multi-factor model comprises screening gene detection, wherein the screening comprises the following steps: (1) collecting and treating a peripheral blood sample; (2) establishing library DNA of the sample, and performing computer sequencing on a Hiseq sequencing platform; (3) performing biological information analysis on computer data, and finding pathogenic mutation; (4) sequencing mutation sites by using a golden standard Sanger sequencing technique, thereby obtaining accurate gene mutation information. By adopting the multi-factor model which is disclosed by the invention and is used for predicting the breast cancer onset risk, a part of people can be screened and diagnosed at an early stage, active interference measures can be taken, and the multi-factor model is particularly applicable to prediction on high-risk people suffering from familial breast cancer.

A multiplex polymerase chain reaction assay that targets nine tetranucleotide short tandem repeat (STR) markers in the mouse genome. Unique profiles were obtained from seventy-two mouse samples that were used to determine the allele distribution for each STR marker. Correlations between allele fragment length and repeat number were determined with DNA Sanger sequencing. Genotypes for L929 and NIH3T3 cell lines were shown to be stable with increasing passage numbers as there were no significant differences in fragment length with samples of low passage when compared to high passage samples. In order to detect cell line contaminants, primers for two human STR markers were incorporated into the multiplex assay to facilitate detection of human and African green monkey DNA. This multiplex assay is the first of its kind to provide a unique STR profile for each individual mouse sample and can be used to authenticate mouse cell lines.

The invention discloses primers and a method for detecting myelosis myelodysplastic syndrome and especially detecting DOCK2 gene mutation of a patient with myelosis myelodysplastic syndrome. The primers comprise (i) primers for amplifying 6th, 22nd, 33rd, 37th, 40th and 44th exon sequences of the DOCK2 gene. An Sanger sequencing technique and sequencing primers are adopted. The primers and method can be used for quickly detecting mutation of 6th, 22nd, 33rd, 37th, 40th and 44th exons of the DOCK2 gene in the body of a patient with myelosis myelodysplastic syndrome. The primers and method have the advantage of accurate detection result, can be used for assisted diagnosis of severe combined immunodeficiency disease, and have important reference meanings for early intervention and early therapy.

The invention discloses a primer combination, method and kit for constructing a targeted library of multiple inherited metabolic liver diseases based on high-throughput sequencing. The base sequence of the primer combination is shown as SEQ ID No. 1 to SEQ ID No. 2245, and the primer combination covers total 703 exons and cutting regions of 43 genes such as SERPINA1, G6PC, SLC37A4, AGL and the like, and is capable of detecting 41 sub-type gene variation loci of totally 20 inherited metabolic liver diseases comprising alpha-1-antitrypsin deficiency, glycogen storage disease, citrullinemia, aminosuccinuria, hemochromatosis, porphyrinopathy, cystic fibrosis, bile acid synthesis defect, Gaucher disease, hereditary fructose intolerance, cholesterol ester storage disease, Gilbert syndrome, Dubinsyndrome, Rotor syndrome, progressive familial intrahepatic choleatasia and the like. The primer combination disclosed by the invention is simple in operation, high in accuracy and low in time consumption, and the time cost and manual cost used for detecting genes and fragments one by one during Sanger sequencing can be greatly saved.

The invention relates to an InDel molecular marker related to cashmere traits of goat and an application of the InDel molecular marker. The marker is located between the 95454685bp and the 95455189bpof the No.6 chromosome of the goat; the polymorphism of the InDel molecular marker is -/insertion fragment; and a sequence of the insertion fragment is shown as SEQ ID NO.1. The invention also provides specific primers and a method for detecting the InDel marker; and sequences of the specific primer pair are shown as SEQ ID NO.2-3. Through the specific primer pair provided by the invention, on thebasis of an electrophoretic band obtained through PCR and agarose gel electrophoresis as well as analysis and statistics on Sanger sequencing and in accordance with cashmere goat allele and non-cashmere goat allele types on an InDel site, it can identify cashmere or non-cashmere traits of a goat variety. The InDel molecular marker provided by the invention is low in amount of detected samples inneed, simple and convenient in method and accurate in identifying result, and the InDel molecular marker has a good prospect of application and promotion.

The invention belongs to the technical field of animal husbandry or animal genetic breeding and discloses identification and application methods for an MC1R gene haplotype. The identification method for the MC1R gene haplotype comprises the steps that an MC1R gene is subjected to PCR amplification, and single-ended Sanger sequencing is adopted for obtaining a variation sequence; based on the established haplotype, the genotype and phenotype of the hair color of a breeding group are subjected to association analysis, and the haplotype for controlling a black feather and the black feather homozygous genotype are identified. According to the identification method, the haplotype for controlling the black feather and the black feather homozygous genotype can be accurately identified. According to the identification method, key SNPs loci are simultaneously identified in one sequencing reaction; but in the prior art, two sequencing primers are used for simultaneously identifying the SNPs loci. Compared with a PCR-RFLP identification method in the prior art, through Sanger sequencing, the identification accuracy is further improved.

The invention belongs to the technical field of plant gene engineering and particularly relates to an accurate and efficient editing method of an upland cotton genome. A pRGEB32-GhU6.7-NPT II vector containing a cotton endogenous promoter pGhU6-7 is modified, original Cas9 protein is replaced with LbCpf1 protein, and a vector GhRBE3 with editing capacity in cotton is constructed. GhCLA is selected as a target gene to verify application of GhRBE3 in cotton. One target is designed, a Cpf1 gene editing system is imported into the cotton genome by agrobacterium tumefaciens mediated genetic transformation, Sanger sequencing and high-throughput sequencing are performed on transgenic plants to detect the editing efficiency and the off-target effect of the method in the allotetraploid cotton genome. The method has good editing efficiency and specificity.

The invention provides a reagent and a method for detecting the ninth exon mutation of a CALR gene by using a Sanger sequencing technology. The reagent and the method are high in detection specificity, and the mutation types of insertion and deficiency of the ninth exon of CALR can be specially analyzed, so that the false positive problem is eliminated, and missing inspection of rare mutation can also be avoided. The reagent and the method can be used for clinically screening genes of patients suffering from primary thrombocythemia and primary myelofibrosis.

The invention relates to a detection primer, a detection kit and a detection method of Leber's hereditary optic neuropathy (LHON) mitochondrial DNA gene mutations. The invention, on the basis of Sanger sequencing principle, has the characteristics of being highly sensitive, stable and accurate; four most common pathogenic mutations of LHON mtDNA can be simultaneously detected, so that the problems of an existing LHON mitochondrial DNA mutation detection method, which is low in sensitivity, not strong enough in specificity, capable of causing pollution easily, high in expense and the like, can be solved; therefore, an accurate gene diagnosis method is provided for LHON; and the invention has important implications for genetic counseling and gene therapy of the disease (the LHON).