49 results about "Gene copy" patented technology

The application provides a standard plasmid for detecting the copy number of exogenous genes of a recombinant CVA10 vaccine, and a preparation method and application thereof. The standard plasmid of the application comprises an endogenous gene MOX fragment SEQ ID No. 1, an exogenous gene P1 fragment SEQ ID No. 2 and an exogenous gene 3CD fragment SEQ ID No. 3 which are connected in series in a cloning vector. The application constructs the standard plasmid, adopts Taqman probe-fluorescence quantitative PCR method, and simply, quickly, absolutely quantitatively and accurately analyzes the copy number of inserted exogenous genes, so that the detection time of the determination of the copy number of exogenous genes of the recombinant CVA10 vaccine is effectively shortened, repeated operation is facilitated, and the determination of the copy number of exogenous genes of the recombinant CVA10 vaccine based on the Hansenula platform and the verification of the genetic stability of the exogenous genes of the recombinant CVA10 vaccine have a wide application prospect.

A method and device for predicting a genotype of a target gene, the method comprising: a baseline correction step; a feature extraction step, comprising obtaining a cluster center of a specific region of the target gene according to a corrected depth of each capture site, and taking the cluster center as a feature value of the corresponding region; and a prediction step, comprising predicting a genotype of the target gene in sequencing data of a sample to be tested according to the feature value. The method has the advantages of fast analysis speed, high accuracy, wide application range, no need for reference set correction, avoidance of the influence of sequencing fluctuation on the analysis result, ease of implementation of high-throughput screening typing at a population level, and applicability to individualized medication guidance for more populations and acceleration of large-scale research and development of pharmacogenomics.

The application provides a primer probe composition for detecting genetic stability of cells and application thereof, the primer probe composition comprises primer pairs SEQ ID NO:1-2 and a probe SEQ ID NO:3 of a VEGF light chain gene, and further comprises primer pairs SEQ ID NO:4-5 and a probe SEQ ID NO:6 of a VEGF heavy chain gene. The application quantitatively detects the antibody light and heavy chain gene copy numbers in cells of different culture generations by fluorescence quantitative PCR based on a Taqman probe method, extracts cell genomic DNA and performs PCR amplification, and prepares a target gene standard product from the amplification product, thereby significantly improving the specificity and accuracy of quantitative detection of the gene copy number.

The invention belongs to the technical field of biology, and provides endoglucanase FsCel45B of a GH45 family, a related biological material and a high-efficiency expression method of the endoglucanase FsCel45B. The GH45 family endoglucanase FsCel45B comprises the following A1) or A2): A1) a protein with an amino acid sequence as shown in SEQ ID NO: 1; a2) a protein which is obtained by substitution and / or deletion and / or addition of amino acid residues on the amino acid sequence shown in SEQ ID NO: 1 and has the same function as the protein shown in A1). The invention further provides an expression optimization strategy in pichia pastoris, based on a pichia pastoris expression vector pPICZ alpha A, an AOX1 promoter and an alpha-MF signal peptide sequence are replaced and co-expressed with disulfide bond folding isomerase (PDI) and a vesicle transport related gene Ssa4, the expression quantity is improved by combining with the increase of the gene copy number, and the expression optimization strategy has industrial production potential.

The invention discloses a TaqMan probe qPCR (quantitative polymerase chain reaction) detection primer group for detecting pathogenic vibrio alginolyticus, a kit and application, and belongs to the technical field of molecular biology and pathogenic microorganism detection. The method comprises the following steps: designing a pair of specific primers and probes by taking the vops gene of the vibrio alginolyticus as a new target, connecting the vops gene with plasmids by amplifying the vops gene, constructing a recombinant plasmid standard substance, carrying out qPCR detection on the copy number of the vops gene of the vibrio alginolyticus by utilizing the designed specific primers and probes, and establishing a corresponding relationship between the vibrio alginolyticus and the vops gene. And the vibrio alginolyticus can be quantified through the copy number of the vops gene. On the basis of novel molecular target detection, no amplification signal exists for a detection sample which does not contain vibrio alginolyticus, and the specificity is good; the method is rapid and accurate in detection and high in sensitivity, the lower detection limit is as low as 100-level copies, non-specific amplification can be avoided, and operation is simple and rapid.

A generic cancer prognosis evaluation system based on LAMA5 expression provided by the invention relates to the technical field of generic cancer prognosis evaluation, and comprises the following steps: acquiring multi-source molecular data, establishing a standardized data input path to obtain LAMA5 gene molecular information, and completing sample identification and field standardization by calculating mutation frequency and gene copy number variation of the LAMA5 gene molecular information to obtain a generic cancer prognosis evaluation result. And an LAMA5 generic cancer molecular database is formed. A standardized data input path and a unified field format are formed by establishing an LAMA5 generic cancer molecular database and integrating TCGA, GTEx, a human protein map and other multi-source molecular data, the database achieves generic cancer prognosis evaluation system calculation of LAMA5 mutation frequency and gene copy number variation, the data consistency and statistical reliability of multiple cancer species are improved, and the method is suitable for large-scale popularization and application. And a data basis is provided for pan-cancer level expression and variation analysis.

The invention relates to the technical field of microbial fermentation and genetic engineering, and particularly discloses a yeast engineering bacterium for efficiently producing plastic depolymerases and application of the yeast engineering bacterium. The engineering yeast takes pichia pastoris as an expression host, a molecular chaperone is co-expressed, a vacuole sorting receptor gene is knocked out or knocked down, in addition, a yeast cell factory for efficiently expressing the plastic depolymerases is constructed by further combining regulation and control element optimization and target gene copy number regulation, and the engineering yeast has the advantages that the engineering yeast can be used for efficiently expressing the plastic depolymerases; the regulatory element comprises any one or a combination of more of a strong promoter, a signal peptide and a C-terminal tag. The engineering yeast provided by the invention can realize efficient accumulation and secretion of the plastic depolymerases, obviously improve the enzyme yield and reduce the production cost of unit enzyme activity, and provides a stable and efficient yeast expression system for industrial and low-cost production of the plastic depolymerases; the method has an important application prospect in the fields of biodegradation and resource utilization of waste plastics.

This invention relates to the field of nucleic acid detection technology, specifically disclosing a liquid-phase nucleic acid chip for isothermal amplification of common algal blooms and its application. The method includes: collecting water samples and filtering to enrich algal cells; lysing the algal cells using cell lysis buffer to extract DNA; adding the extracted DNA to the liquid-phase nucleic acid chip for isothermal amplification; detecting the Ct value using a qPCR instrument; and determining the species and cell density of the algal bloom based on a preset standard curve. This invention directly quantifies algal cell density using algal gene copy number, with significantly higher accuracy than microscopic counting, and does not rely on the experience of professional personnel. It can be widely applied in frontline water resource management work.

The application discloses a method for identifying direct discharge of domestic sewage and application thereof. The method comprises the following steps: detecting the copy number concentration of bacteriophage marker genes and Bacteroides marker genes of adjacent two days of a water body to be detected respectively, calculating the ratio of the copy number concentration of the bacteriophage marker genes and the copy number concentration of the Bacteroides marker genes per day, and calculating the ratio between the two days of the water body to be detected, and combining a specific ratio threshold to determine whether the direct discharge of domestic sewage appears in the water body to be detected, and the day when the direct discharge of domestic sewage appears can be accurately determined, so that the direct discharge of domestic sewage can be traced and controlled; wherein the bacteriophage marker gene is CPQ_064, and the Bacteroides marker gene is BacHum, BacH, HuBac and / or HF183.

Provided is a method for preparing a recombinant human collagen. The method comprises: constructing a genetically engineered bacterium which expresses a prolyl hydroxylase and a recombinant human collagen; culturing the genetically engineered bacterium and performing digestion treatment by means of pepsin to obtain a recombinant human collagen type I or type III. The prolyl hydroxylase comprises P4Ha2, P4Hb and vP4H, and by means of controlling the ratio of the copy numbers of the genes thereof, the recombinant human collagen and a natural human collagen are consistent in hydroxylation modification. Also provided is a large-scale production method for the recombinant human collagen. The prepared recombinant human collagen has completely the same sequence composition and length and substantially the same hydroxyproline content as the natural human collagen, and also has a stable full-length triple helix structure and biological activity without immunogenicity and potential risk of viruses.

Disclosed is a method of determining whether a human subject is not a carrier of spinal muscular atrophy (SMA). This method includes the steps of (i) collecting a genomic deoxyribonucleic acid (DNA) sample from a human subject; (ii) screening the genomic DNA sample to determine the human subject's copy number of survival of motor neuron 1 (SMN1) gene and whether one of the copies of the SMN1 gene is positive for a polymorphism associated with non-carriers of SMA having two copies of the SMN1 gene; and (iii) determining the human subject as not a carrier of SMA if the human subject includes two copies of the SMN1 gene with one of those copies being positive for the polymorphism. Also disclosed is a method of determining whether an individual has a decreased risk of being a carrier of spinal muscular atrophy (SMA), where the individual is identified to have a decreased risk of being a carrier of SMA when the individual has two copies of the SMN1 gene with one of those copies being positive for the polymorphism.

The application provides a STRC gene copy number variation detection method based on whole genome sequencing. The STRC gene and the STRCP1 gene are subjected to sequence alignment to find each difference site of the STRC gene and the STRCP1 gene. For each difference site, the sequence of the corresponding STRC position and STRCP1 position in the genome is read from a variation detection file. The total copy number of the true gene and the false gene is calculated by taking the reference site in the genome as a reference. The STRC gene copy proportion on each difference site is calculated. The STRC gene copy number on each difference site is calculated according to the total copy number and the STRC gene copy proportion. The STRC gene copy number on each exon is determined according to the STRC gene copy number on each difference site. The method can realize the detection of the STRC copy number, simplifies the detection process, improves the detection throughput and reduces the cost.

The invention provides a method for improving the yield of lincomycin by modifying a streptomyces lincomycin SLCG3615 gene, which comprises the following steps: knocking out a TetR family transcriptional regulation gene SLCG3615 in the streptomyces lincomycin through a genetic engineering way to obtain a lincomycin high-yield engineering strain, and fermenting the obtained strain to produce the lincomycin. Wherein the nucleotide sequence of the SLCG3615 gene is as shown in SEQ ID NO. 1. The invention also provides an application of the SLCG3615 gene knockout modified streptomyces lincomus. The lincomycin biosynthesis negative regulator SLCG3615 is screened in the research, the SLCG3615 gene copy on the chromosome of the streptomyces lincomycin is knocked out through a genetic engineering way, a lincomycin high-yield strain can be obtained, and a technical support is provided for improving the fermentation yield of the lincomycin in industrial production.

The invention relates to the technical field of synthetic biology, in particular to a saccharomyces cerevisiae recombinant strain with high gene copy integration and high geraniol yield as well as a construction method and application of the saccharomyces cerevisiae recombinant strain. According to the method, the copy number of geraniol synthase genes is increased through a saccharomyces cerevisiae gene multi-copy integration technology, efficient and stable expression of the geraniol synthase genes is achieved, supply of geraniol precursors is improved by further combining the modes of blocking a precursor competitive pathway and the like, a saccharomyces cerevisiae recombinant strain capable of efficiently and stably synthesizing geraniol is constructed, and the method is suitable for large-scale production of geraniol. Restriction factors of synthesis of geraniol by saccharomyces cerevisiae are removed, and the recombinant strain has high genetic stability and good environmental protection property. The recombinant strain constructed by the invention can be used for efficiently synthesizing geraniol and derivatives of geraniol, and is applied to essence, fragrance and food industries.

The present invention addresses the problem of developing a novel drug for treating osteosarcoma. The present invention also addresses the problem of developing a therapeutic marker for studying the therapeutic effect of a drug in the treatment of osteosarcoma. The present invention shows that in order to solve the above-mentioned problem, a compound capable of inhibiting cell proliferation of osteosarcoma is found, and thus a novel pharmaceutical composition for treating osteosarcoma can be provided. It is also shown that osteosarcoma to be treated, which is a novel pharmaceutical composition, is an osteosarcoma in which the number of copies of an MCL1 gene in a cell has been increased or an osteosarcoma in which the production of an Mcl-1 protein has been increased, or an osteosarcoma in which the number of copies of a BCL2L1 gene has been increased or an osteosarcoma in which the production of a Bcl-xL protein has been increased. Therefore, by detecting these, it can be used as a therapeutic marker for studying the therapeutic effect of a novel pharmaceutical composition for the treatment of osteosarcoma.

The invention belongs to the technical field of biological medicine, and particularly relates to a method for measuring the copy number of a CAR gene through double digital PCR. The invention provides the method for determining the copy number of the CAR gene in the cell therapy product based on the digital PCR, and the method does not need a standard curve, has universality, is suitable for identifying the copy number of any CAR gene containing the WPRE virus vector, and has no limitation on the CAR structure. The primer probe pair is good in specificity, high in sensitivity, good in reaction system repeatability, good in stability and good in application prospect.

This invention belongs to the field of biotechnology and provides GH45 family endoglucanase FsCel45B, related biological materials, and efficient expression methods. GH45 family endoglucanase FsCel45B includes the following A1) or A2): A1) a protein with the amino acid sequence shown in SEQ ID NO:1; A2) a protein with the same function as the protein shown in A1) obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence shown in SEQ ID NO:1. This invention also provides an expression optimization strategy in Pichia pastoris, based on the Pichia pastoris expression vector pPICZαA, replacing the AOX1 promoter and α-MF signal peptide sequence, and co-expressing it with disulfide bond folding isomerase (PDI) and the vesicle transport-related gene Ssa4, and combining this with an increase in gene copy number to improve expression levels, possessing industrial production potential.

The invention discloses a primer pair for evaluating large yellow croaker cell senescence based on relative telomere length and a method thereof, and belongs to the field of molecular genetics. The specific method comprises the following steps: 1) taking DNA of a large yellow croaker cell genome; 2) performing qPCR amplification on the DNA obtained in the step S1 by using a first qPCR amplification system containing a telomere universal primer to obtain a telomere copy number Cttel; (3) carrying out qPCR amplification on the obtained DNA by using a second qPCR amplification system containing the forward primer as shown in SEQ ID No.1 and the reverse primer as shown in SEQ ID No.2, so as to obtain the copy number Ctactb2 of the internal reference single copy gene; and 4) calculating the relative telomere length T / S = 2-[delta] Ct, wherein [delta] Ct = Ctel-Ctactb2. The relative telomere length detection actb2 primer provided by the invention can specifically amplify large yellow croaker DNA, and can realize accurate detection by adopting a trace sample.

Described herein are treatments and patient selection methods for cancers such as prostate cancer, breast cancer, liver cancer, bladder cancer, and melanoma. The treatment involves administering a RIPK2 inhibitor to a subject. The patient selection involves selecting a patient who has increased copy numbers or expression of both RIPK2 and MYC.

The invention relates to a real-time quantitative polymerase chain reaction (qPCR) method and a kit, which are used for simultaneously determining classification of specific gene variation and gene copy number. More specifically, the present invention relates to an efficient qPCR method and kit for simultaneously determining the variation and copy number of a target gene by performing real-time quantitative PCR on a reference gene and a target gene with known copy number in the same reaction vessel.

The invention discloses a quantitative analysis method and system for HER2 gene copy number based on a neural network, and relates to the technical field of gene detection, the system comprises a microscopic monitoring module, a laser reflection module, a neural network processing module and a probability adjustment module, the microcosmic monitoring module generates a microcosmic magnified image reflecting a blood sample by adopting receiving and interpretation of an electron microscope, and the laser reflection module adopts characteristics of diffuse reflection laser reflected when artificial laser propagation encounters telomere and centromere of No.17 chromosome; the laser absorption rate change caused by different dyeing intensities in a cell nucleus is measured, the laser absorption rate is evaluated, the neural network processing module combines a processed micro-amplified image with a reflected light analysis result to form an image internal comprehensive analysis result, and the probability adjusting module is used for adjusting the probability according to the reflected light analysis result. The method has the characteristic of high accuracy.

The invention provides a Candida tropicalis ERG11 gene copy number variation digital PCR (polymerase chain reaction) detection method, the total copy number of ERG11 genes is detected through a group of universal primers, a wild type probe of an A395 site of the ERG11 genes in a detection sample and a mutant type probe of the A395 site of the ERG11 genes, and the total copy number of the ERG11 genes is equal to the sum of the copy number of wild target spots and the copy number of mutant target spots; in the PCR detection method, the copy number of the wild target and the copy number of the mutation target are corrected by using the copy number of two reference genes. Compared with an ERG11 gene mutation and copy number variation detection method based on a fluorescent quantitative PCR method, the accuracy of copy number variation detection is greatly improved, and the problem of pain points of tandem sequence copy number recognition and quantitative detection in the candida tropicalis drug resistance detection process can be effectively solved.

The invention provides an L-alanine production strain and a construction method and application thereof.The L-alanine production strain is characterized in that a dns gene, a vnp1 gene and a vnp2 gene in a genome of vibrio natriureticus are sequentially knocked out through gene modification with the vibrio natriureticus as an original strain, a series of key competitive pathway genes pflB, lldh, dldh, pta, ackA and adhE are knocked out in order to block an organic acid fermentation pathway of the vibrio natriureticus, and the vibrio natriureticus is obtained; the production of L-alanine is increased, the accumulation of organic acids is reduced, pXtuf-alaD plasmids are transferred to increase the gene copy number, and by cooperating with the ultrafast growth characteristic of the starting strain vibrio natriureticus, the constructed strain has a clear genetic background, shows efficient transformation ability in the subsequent anaerobic production stage, can efficiently produce L-alanine, and has a wide application prospect. Good industrial application prospects are realized.

This application discloses a primer set and its design method for detecting copy number variations in the SMN1 and SMN2 genes. The primer set includes a first primer for detecting exon 7 of the SMN1 gene, a second primer for detecting exon 8 of the SMN1 gene, a third primer for detecting exon 7 of the SMN2 gene, and a fourth primer for detecting exon 8 of the SMN2 gene. The 3′ ends of each of the first to fourth primers independently meet the following conditions: A1) The first base is absolutely conserved and is specifically complementary to the sequences of exons 7, 8, 7, and 8 of the SMN1, SMN1, and SMN2 genes, respectively; and A2) At least one mismatched base is introduced at positions 2 to 4. Using this primer set to detect copy number variations in the SMN1 and SMN2 genes ensures both specificity and amplification efficiency, as well as minimal dosage.

The invention discloses application of an METTL14 inhibitor in preparation of a medicine for treating malignant tumors carrying DNA outside chromosomes, and belongs to the technical field of medicines. A human colon cancer cell HT29-4 containing ecDNA and an ovarian cancer cell UACC-1598-4 are selected as research objects, an METTL14 stable knock-down and overexpression cell model is constructed through a lentivirus-mediated RNA interference technology, and the knock-down and overexpression efficiency is verified through Western Blot. Kyotype analysis shows that the quantity of ecDNA is reduced by knocking down the METTL14, and the quantity of the ecDNA is increased by overexpressing the METTL14. The copy number of DHFR and EIF5A2 genes carried by ecDNA is detected through qPCR, and the result shows that the copy number is decreased after METTL14 is knocked down, and the copy number of overexpression groups is increased. EdU proliferation experiments and cell scratch experiments show that knock-down of METTL14 inhibits proliferation and migration ability of tumor cells containing ecDNA, and overexpression promotes the phenotypes. The invention provides an effective treatment means for the intractable tumor driven by the ecDNA.

The invention discloses a single-gene hereditary cardiovascular disease related gene detection probe group, a kit and an application of the single-gene hereditary cardiovascular disease related gene detection probe group. The probe group is a specific probe which is selected from exon regions of genes such as ABCC9, ABCG8, ACADM, ACADS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1 or ALMS1 and the like, and 20-30 bp intron regions on the upstream and downstream of the exon regions of the genes such as ABCC9, ABCG8, ACADM, ACADDS, ACADVL, ACTA2, ACTC1, ACTN2, ACVRL1, AGK, AGL, AGXT, AIFM1 or ALMS1. The detection probe and the kit provided by the invention can accurately detect exon regions of 268 pathogenic gene variations related to the single-gene hereditary cardiovascular disease, intron regions of 20-30 bp upstream and downstream of exon and copy number variations of pathogenic genes, under the average 1G data volume, the nuclear genome coverage depth reaches 200 * on average, the 30 * coverage degree reaches 99%, and the detection sensitivity is high. The detection sensitivity of low-frequency mutation in a target area is improved, the detection cost is effectively controlled, an experimental system is stable, and the discrete coefficient of library results among samples is less than 10%.

The application particularly relates to a primer probe, a kit and application for detecting the GJB2 gene copy number of a non-syndromic hearing loss patient by using microdroplet digital PCR. The primer probe combination provided by the application comprises a target gene detection primer pair, a target gene detection probe, an upstream primer of a reference gene, a downstream primer of the reference gene and a reference gene probe, and the sequence information is shown as SEQ ID NO. 6-11; and the application further provides a kit for detecting the GJB2 gene copy number of a non-syndromic hearing loss patient. The primer probe combination and the kit provided by the application are applied to the detection of the GJB2 gene copy number variation of a non-syndromic hearing loss patient, and the accuracy of the detection of genetic hearing loss is significantly improved; according to the determination result, it is determined whether the GJB2 gene expression is abnormal or not, the disease prognosis is evaluated, potential therapeutic drugs are screened, genetic counseling services are provided or individualized medical schemes are formulated, and the application has a wide application prospect.

This invention provides a method for modifying Streptomyces lincosinate. SLCG_3615 A method to increase lincomycin production through gene therapy involves knocking out the TetR family transcriptional regulatory genes in Streptomyces lincomyces via genetic engineering. SLCG_3615 A high-yield engineered strain of lincomycin was obtained, and the obtained strain was used to produce lincomycin through fermentation; among which, SLCG_3615 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention also provides... SLCG_3615 Applications of gene knockout modified *Streptomyces lincomycin*. In this study, the negative regulator of lincomycin biosynthesis, SLCG_3615, was screened and knocked out on the chromosome of *Streptomyces lincomycin* via genetic engineering. SLCG_3615 SLCG_3615 Gene copying can yield high-yield strains of lincomycin, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

This invention provides an SMA detection kit based on digital PCR technology. This study utilizes digital PCR detection technology to simultaneously detect the copy numbers of the SMN1 and SMN2 genes in the same reaction, and also to detect small mutations in the SMN1 gene and other genes simultaneously, improving diagnostic accuracy, simplifying the operation process, and increasing timeliness. It can be applied to SMA carrier screening, clinical diagnosis, and disease subtyping, while also saving on testing costs.

The invention discloses a primer and a method for monitoring chlamys nobilis biomass based on environmental DNA amplification. The sequences of the primers are as follows: MN-F: 5 '-CACCTTTCGTTTGGGCGTTGG-3', and MN-R: 5 '-ACACCAGTAGGGACAGCAATCA-3', and the sequences of the primers are as follows: MN-F: 5 '-CACCTTTCGTTTGGCGTTGG-3'. The method for monitoring the biomass of chlamys nobilis comprises the following steps: 1) constructing a chlamys nobilis target gene copy number-Ct standard curve; 2) collecting each sea area sample and measuring a Ct value; 3) substituting into the standard curve, converting to obtain the copy number of the target genes of the scallops on site, and estimating the biomass according to the copy number. According to the method provided by the invention, the trace DNA generated by the chlamys nobilis can be identified by collecting the bottom water body sample of the sea area, and the biomass of the chlamys nobilis can be evaluated according to the copy number of the target fragment in the eDNA.