127 results about "Upstream and downstream (DNA)" patented technology

An isothermal process for amplifying a nucleic acid target molecule that relies on an upstream primer, a downstream primer, a strand invasion system and an oligonucleotide, wherein the upstream and downstream primers are not substrates for the strand invasion system during the amplification process and do not amplify the target molecule independently of the strand invasion system, wherein the oligonucleotide is a substrate for the strand invasion system.

The present invention discloses methods for identifying and analysing microsatellite-associated polymorphisms between different DNA samples. Different DNA samples, e.g. from different individuals, are analysed using a PCR based on the combination of a RAMP-primer and an AFLP-primer and polymorphisms between the different DNA samples are identified. The polymorphisms thus identified may be isolated and further analysed by e.g. DNA sequence analysis both upstream and downstream from the microsatellite-associated polymorphism. These sequences may subsequently be used to devise and synthesise new means for analysis of the polymorphic locus, such as e.g. PCR-primer pairs or oligonucleotide probes.

The invention relates to a cyclic RNA molecular marker for diagnosis of gastric cancer, the cyclic RNA molecular marker is characterized in that the cyclic RNA is hsa-circ-0001017, the invention also provides a method for detection of the cyclic RNA molecular marker in plasma, and the method comprises the following steps: (1) collecting blood, and extracting total RNA in the plasma; (2) performing reverse transcription of the total RNA into cDNA; (3) performing droplet digital PCR detection of a cDNA solution of the step (2) by use of specific amplification back-to-back primers and amplification upstream and downstream primers of housekeeping gene GAPDH, after the completion of the reaction, detecting fluorescence signal values of all droplets, setting a threshold, and determining whether the droplets include the cyclic RNA or the housekeeping gene GAPDH, wherein the droplets higher than the threshold are positive droplets, and the droplets below the threshold are negative droplets; and (4) counting the number of the positive droplets, and calculating the copy number of the hsa-circ-0001017 and the housekeeping gene GAPDH in the plasma for quantitative detection of the hsa-circ-0001017 and the housekeeping gene GAPDH in the plasma. Compared with the prior art, the advantages are that the hsa-circ-0001017 can be specifically expressed in plasma in patients with gastric cancer, and can be used as a new molecular marker for diagnosis of the gastric cancer.

The invention provides primers, a method and a chip for detecting alpha and/or beta-thalassemia point mutation and deletion mutation based on whole-gene capture sequencing and application of such primers, such method and such chip. The primers, the method, the chip and application thereof have the advantages that through designing of capture probes, relevant genes involved in alpha-thalassemia and beta-thalassemia are enriched and all mutation information including SNP and indel in full-length sequences of genes is detected; through addition of autosome, X-chromosome and Y-chromosome regions as well as upstream and downstream regions of coded genes as references, structure variations such as SNV and CNV are detected; compared with existing various hotspot mutation site detection technologies, the method is capable of detecting hotspot mutation information as well as some rare mutations and undiscovered new mutation types to detect and analyze full-length sequence specificity of target genes, fully covers the mutation types and makes up the defect that a conventional detection method easily causes missing detection of low-frequency mutations and rare mutations greatly.

The invention provides a method and a system for constructing a model for predicting protein-RNA interaction binding sites. Correspondingly, the invention also provides a method and a system for predicting the protein-RNA interaction binding sites by using the method. A deep learning model is trained using sequence characteristics at and upstream and downstream of an RNA and protein binding site and measured RNA structure characteristics, and the model is used to predict a protein-RNA interaction binding site. In the feature extraction process, a motif acquisition module constructed based on aconvolutional neural network and a context semantic acquisition module constructed based on a recurrent neural network are used. Compared with the prior art, the trained model has remarkable progressin the aspects of judgment accuracy, calculation time and universality of an application platform.

The invention provides a detection probe for an SNP (Single Nucleotide Polymorphism) of a human CYP2C19 gene. A detection primer consists of a specific upstream and downstream primer pair of the gene and a specific Taqman double fluorescent probe which are respectively used for correspondingly detecting SNPs at CYP2C19*2, CYP2C19*3 and CYP2C19*17 sites. The detection probe has the characteristics of quick detection and high specificity; furthermore, the detection method is simple; positive contrast and negative contrast which are necessary to conventional fluorescent quantitation PCR are eliminated, so that the operation steps and the experimental cost are reduced; the subsequent clinical treatment strategy can be guided more quickly and better.

The invention relates to a method for constructing a high-throughput sequencing library of an immune group library for screening a cross-reaction between samples. The method is characterized in that: RNA of more than 10 ng is taken as a starting amount, a principle of RACE is used to make the template conversion when the RNA is reversely transcribed into cDNA, a known sequence is added to the 5' ends of all cDNAs, a product with the known sequence is used as a template, PCR amplification is performed by using a double-ended Barcode primer, the resulting product is purified and ligated to a sequencing linker sequence to obtain the final sequencing library, which can be used for samples of different receptor sources, during PCR amplification, PCR amplification can be performed using only one pair of primers to achieve equivalent amplification, and the primer preference problem caused by multiplex primer amplification is solved, and the upstream and downstream primers used in PCR amplification are designed to be labeled with Barcode composed of 4 to 12 different bases, which avoids the problem of cross-reaction between samples when multiple samples are simultaneously constructed.

The invention discloses recombinant escherichia coli for heterologously synthesizing ambrein and a construction method thereof. The construction method comprises the following steps of: (1) fusing saccharomyces-cerevisiae squalene synthase gene ERG9 with truncation of 26 amino acid residues at a C end and homologous arms at the upstream and downstream of a lacZ site of the escherichia coli into adonor DNA; constructing plasmid 1; transforming the donor DNA and the plasmid 1 together into the escherichia coli, eliminating the plasmid 1, obtaining the recombinant escherichia coli for synthesizing squalene, and naming the recombinant escherichia coli as a strain 1; (2) connecting alicyclobacillus-acidocardarius squalene-hopene cyclase gene D377C SHC with mutation from the 377th amino acid residue into cysteine residue and bacillus-megaterium cyclase gene BmeTC into one segment, then inserting the segment into escherichia-coli expression plasmid p5C to obtain plasmid 4; (3) transforming the plasmid 4 into the strain 1 to obtain the recombinant escherichia coli for heterologously synthesizing the ambrein, and naming the recombinant escherichia coli as a strain 4. Proved by experiments,the recombinant escherichia coli for heterologously synthesizing the ambrein is fermented to obtain the ambrein.

The invention discloses a molecular identification method of HMGR gene mRNA in pear stock-scion transfer. The molecular identification method comprises the following steps: (1) by taking pyrus bretschneideri rehd as scion and pyrus betulifolia bunge as stock, carrying out micro grafting of test-tube seedlings; (2) extracting total RNA of pyrus bretschneideri rehd, pyrus betulifolia bunge and grafted stock and scion, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification on the total length of the HMGR gene, and carrying out second-time PCR by adopting the amplification result as a template, thus obtaining respective HMGR gene fragments, wherein the sequences of upstream and downstream primers obtained through two times of PCR are respectively shown as SEQ ID NO.1-4; and (3) carrying out enzyme digestion on the PCR product obtained for the second time by adopting restriction endonuclease, and comparing enzyme digestion spectrograms before and after grafting for identifying the transfer condition. The method provided by the invention is rapid and sensitive, is high in accuracy, is simple and convenient, and can be applied to other pear plants.

The invention discloses a vector for knocking out an L-lactic dehydrogenase 1 gene and a construction method of the vector. The construction method comprises the following steps: completely splicing upstream and downstream DNA fragments of a gene which does not contain an L-lactic dehydrogenase encoding gene by using an overlapping extension process, also cloning the spliced fragments to a sub-vector pMD18-T and transforming the spliced fragments to escherichia coli DH5alpha competent cells, screening constructed sub-cloning vectors by virtue of blue-white spots, performing double enzyme digestion to obtain a target strip, then connecting the target strip to a pG+host9 plasmid, and performing bacterial colony PCR and double enzyme digestion identification to successfully obtain a recombinant vector. According to the vector and the construction method thereof disclosed by the invention, a recombinant plasmid of which the L-lactic dehydrogenase encoding genes are knocked out is constructed by using an overlapping extension PCR process, so that excessive base sequences introduced by enzyme digestion and connection can be avoided; and the recombinant vector disclosed by the invention can be used for producing D-lactic acid with high optical purity.

The invention discloses a construction method of a gene detection library of hereditary hypertrophic cardiomyopathy and a kit and relates to mutation of ACTC1, ACTN2, MYBPC3, MYH7, MYL2, MYL3, TNNI3,TNNT2 and TPM1 genes. Target regions comprise an exon region of coding amino acid of the 9 genes and 20 basic group regions at the upstream and downstream of an exon; in order to ensure that all the target regions are covered, a target region library is obtained through a hybridization probe capturing method after the library is prepared; then an LMPCR (Ligation-Mediated Polymerase Chain Reaction)method is adopted to amplify the library; and after the library is purified, a sample library for sequencing is obtained. The library construction method has simple steps and a rapid speed and the cost of constructing the library is effectively reduced; related genes of the hypertrophic cardiomyopathy are covered; an illumina high-throughput sequencing machine is combined and related gene mutation can be rapidly and accurately obtained; and the construction method has great significance on the hereditary hypertrophic cardiomyopathy.

The invention provides a salmonella detection primer group, a salmonella detection method and a salmonella detection kit based on an RPA-LbCas12a-TTECDS system, and relates to the field of salmonella detection. Based on the combination of a recombinase polymerase amplification technology and a CRISPR gene detection technology, salmonella is detected through an optimized and modified crRNA double-target gene combination, crRNA is screened and optimized aiming at an invA gene and a fimY gene of the salmonella respectively, and upstream and downstream primers for RPA reaction are designed according to nucleotide sequences at two ends of a matched sequence of the crRNA, so that the primer group is obtained. According to the primer group and the crRNA, the RPA-LbCas12a-TTECDS system is designed, and t hesalmonella detection method and kit are established according to the system. The detection method and the kit provided by the invention are used for detecting actual samples, and have the advantages of strong specificity, rapid detection, high sensitivity and high precision.

The invention discloses a reverse transcription-polymerase chain reaction (RT-PCR) detection method and a kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3), and belongs to the field of biomedical detection. The invention adopts the technical scheme that the method comprises acquisition of a sample, extraction of virus RNA, design optimization of a specific primer, RT-PCR amplification, agarose gel electrophoresis and result judgment; the specific primer is designed for a specific fragment of BPIV-3 nucleoprotein (NP) genes, and the upstream and downstream sequences of the specific primer are respectively P1: 5'-GGATGTTTGGGAGTGATCTTGAGTA-3' and P2: 5'-TGTGTTGAAAAATGAAGCAAGACCT-3'; the quick RT-PCR diagnosing kit for detecting the BPIV-3 comprises a sample virus RNA extracting reagent, a reverse transcription reagent, a PCR reagent, a positive reference substance and a negative reference substance; and by verifying, the method is sensitive and specific and has applicationprospect.

The invention belongs to the technical field of HER2 gene amplification detection reagents, and particularly relates to a kit for detecting HER2 gene amplification by digital PCR. The kit comprises aprimer probe premixed solution, the primer probe premixed solution comprises (1) upstream and downstream primers for detecting an HER2 gene and a fluorescent probe for detecting the HER2 gene; (2) upstream and downstream primers for detecting a human FAS gene, and a fluorescent probe for detecting the human FAS gene; and (3) upstream and downstream primers for detecting a human ACTB gene, and a fluorescence probe for detecting the human ACTB gene. The kit for detecting the HER2 gene amplification provided by the invention can conduct absolute quantification compared with real-time fluorescentquantification PCR, and a detection lower limit is obviously lower than the fluorescent quantification PCR. Moreover, a primer probe combination of the kit has high specificity and better detection effect.

The invention relates to a TALEN and pMD18 vector-based site-directed mutagenesis system and its application. The system is composed of nuclease TALEN and a pMD18 vector, the nuclease TALEN is a transcription activator-like effect factor shearing a target gene target sequence, and the TALEN is composed of nuclease TALEN-L identifying the 5'-terminal of the target sequence and nuclease TALEN-R identifying the 3'-terminal of the target sequence; and the pMD18 vector is a pMD18 homologous recombinant template plasmid inserted into human AR site-directed mutagenesis gene, and the pMD18 vector is composed of corresponding upstream and downstream sequence of the target gene, and a puromycin screening marker. The system allows genome to be efficiently and accurately edited by using the homologous sequence provided by the Pmd18 vector during the cut-out of a double strand DNA molecule by the TALEN; and puromycin resistant gene is carried out when the introduced homologous sequence is adopted to edit the DNA sequence, so manual labor in the screening process is substantially reduced.

The invention discloses a CDKN2A gene fragment and application of the primers of CDKN2A gene fragment in diagnosing tumors.By designing an upstream and downstream primer pair, the specific CDKN2A gene fragment is determined, the gene fragment shows remarkable high DNA methylation in tumor patients while showing low DNA methylation in normal people, and statistic differences exist between DNA methylation of tumor patients and that in normal people.Occurrence and development of tumors are tracked with the level changes of DNA methylation of the gene fragment, and thus a novel biomarker is provided for diagnosis, treatment effect monitoring and prognosis improvement judgment of tumors.

The invention mainly discloses a DNA template for modifying primary cells by gene editing and a fixed-point insertion method. The DNA template comprises a plasmid, a target gene which is constructed on the plasmid and needs to be introduced, homologous sequences (homologous arms) at the upstream and downstream of a target sequence, and a DNA sequence recognized by Guide RNA; then, an RNP complex of a gene editing protein and the Guide RNA, and the DNA template are delivered to a cell line or a primary cell by utilizing an electroporation technology, so that a target gene is inserted into a genome of the cell line or the primary cell at a fixed point, and the cell line or the primary cell is modified. One practical application of the DNA template is that a chimeric antigen receptor T (CAR-T) cell inserted at a fixed point can be produced without depending on viruses, and therefore, the DNA template has better safety and effectiveness in the application of immune cells for treating diseases.

The invention discloses a method for specificity up-regulated gene expression through a targeting core promoter utilizing small molecule RNAs (including micro-RNAs and small interfering RNAs) and a series of regulation target genes. According to the invention, when a TATAbox sequence is contained in the promoter, a target site is in the range of expanding 20 basic groups from the upstream and downstream sides respectively by taking the TATAbox sequence as the center; when no TATAbox sequence is contained in the promoter, the target site is a 1-50 sequences on the upstream side of the genetic transcription initiation site. The micro-RNAs hsa-let-7i, hsa-miR-138, hsa-miR-92a, hsa-let-7c and hsa-miR-181d specifically regulate up the expression of interleukin-2, insulin, thyrocalcitonin, histone and c-myc gene respectively. Besides, aiming to the randomly-selected 19 gene promoters, the artificially-synthesized small interfering RNAs (siRNAs) can enhance the transcriptional activities of 78.9% of the genes. The invention aims to provide the method for specificity up-regulated gene expression utilizing micro-RNAs and has higher application values and broad application prospects in the biotechnology and the biomedicine field.

The invention provides a CRISPR/Cas12a one-step nucleic acid detection method. The method comprises the following steps of 1, selecting a target sequence of bacteria to be detected, designing upstream and downstream primers of RPA, performing specific screening, amplifying the target sequence to obtain a primer amplicon sequence, and performing specific verification on the primer amplicon sequence by utilizing NCBI BLAST; 2, designing specific crRNA; 3, designing a single-stranded DNA reporter molecule; 4, mixing the single-stranded DNA reporter molecule, the RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer 2.1, RNase Inhibitor and a target to be detected to obtain a reaction mixture; 5, constructing a one-step detection kit comprising a reactant placing tube and an injector, placing the reaction mixture at the bottom of the reactant placing tube, and preloading crRNA and Cas12a protein into the injector; and 6, after the reaction mixture reacts, pushing a piston of the injector to enable the crRNA and the Cas12a protein to be mixed with the reaction mixture for a reaction, and performing blue light excitation to obtain a detection result.

A method for multicopy integration of a target gene to a saccharomyces cerevisiae genome includes: selecting target sequences, designing homologous arm primers in length of 15-90bp on two sides of each cleavage site in the corresponding target sequences to primer front ends of amplification target gene cassettes to synthesize homologous arm loaded primers, and amplifying the target gene cassettesto obtain donor DNA fragments; subjecting transcribed gRNA vectors in corresponding target sequences and the donor DNA fragments to co-transformation into Cas9 nuclease expressing saccharomyces cerevisiae to realize gene integration; removing Cas9 nuclease expression vectors and the transcribed gRNA vectors by a non-resistant flat plate. The method has advantages that the homologous arm primers ontwo sides of each cleavage site is in sequence length of 15-90bp, and by respective adding of the homologous arm primers to upstream and downstream primers of the target gene cassettes, an integratorgene template can be obtained directly through once PCR, so that multi-copy integration of target genes is realized. Compared with an existing method, the method has advantages of low time consumption and simplicity in operation.

The invention discloses a large genomic rearrangement (LGR) detection method based on capture sequencing. The capture probes of the invention comprise exon probes and single nucleotide polymorphism (SNP) probes. The exon probes comprise at least three probes aiming at each exon of the gene and 60bp regions at two ends of the exon, and can achieve 2X complete coverage for the exon and the 60bp regions at two ends of the exon. The SNP probes comprise SNP loci with the population occurrence frequency of 30%-70% in the gene intron region and the upstream and downstream within the 5000bp ranges ofthe gene. Meanwhile, the invention further provides an algorithm formed by integrating a reads depth algorithm module, an allelic imbalance algorithm module and a discordant sequence algorithm module,and the algorithm is used for carrying out large genomic rearrangement analysis on the NGS sequencing data. By adoption of the method, the LGR can be effectively detected, the requirement for the purity of a nucleic acid sample is lowered, false positive caused by single-base variation is reduced, and high detection performance is achieved for exon amplification.

The invention discloses a PML-RAR alpha fusion gene detection kit and a detection method thereof. The kit comprises a first group of probes for detecting upstream and downstream sequences of a PML gene and a second group of probes for detecting upstream and downstream sequences of a RAR alpha gene. Each one of the probes is labeled with a dye. The dyes labeling the two groups of probes are in different colors. The two groups of probes are amplification products obtained by amplification based on a human genomic DNA as a template and amplification primers. The length of the probes is designed according to a lot of experiments. Through experiment result comparison and statistic analysis, the optimal length is obtained and the optimal balance between the detection specificity and hybridization time is realized. The PML-RAR alpha fusion gene detection kit guarantees result specificity and sensitivity, shortens hybridization time, has detection time of 7-8h and improves detection efficiency.

The invention discloses an RNA-protein binding site prediction method and system based on a self-attention mechanism. According to the method, a deep learning model is trained through employing the sequence features of an RNA and protein binding site and upstream and downstream, and an RNA-protein interaction binding site is predicted through employing the model; in the coding process of the sequence features, a k-mer embedded coding mode is introduced, the context relationship between adjacent nucleotides is coded, and more effective features are provided for the model; in the feature extraction process, a prediction model is constructed by using a self-attention mechanism, RNA sequence features are focused from the global perspective, and key subsequences are endowed with higher weights so that the network can fully learn key features, and then the model prediction accuracy is improved; and finally, the method provided by the invention is evaluated on a reference data set, and the method is superior to the prior art in the aspect of prediction precision.

The invention relates to a detection system and a kit for PDGFRA gene mutation. The kit includes PCR detection solution A, PCR detection solution B and SYBR GreenI mixed solution; PCR detection solution A includes V516D for detecting PDGFRA gene exon 12 The upstream and downstream primer A and peptide nucleic acid A of the site, and the PCR detection solution B include upstream and downstream primer B and peptide nucleic acid B for detecting the D842V site of the 18th exon of the PDGFRA gene. The PDGFRA gene mutation detection system and the kit thereof of the present invention have high sensitivity and specificity, require less samples, and can quickly, conveniently and accurately perform PDGFRA gene mutation detection.