194 results about "Pcr analysis" patented technology

This invention relates to plant breeding and the protection of plants from insects. More specifically, this invention includes novel transformation events of cotton plants comprising one or more polynucleotide sequences, as described herein, inserted into specific site(s) within the genome of a cotton cell. In highly preferred embodiments, said polynucleotide sequences encode “stacked” Cry1F and Cry1Ac lepidopteran insect inhibitory proteins. However, the subject invention includes plants having single cry1F or cry1Ac events, as described herein. Additionally, the invention is related to cotton plants derived from that transformation event and to assays for detecting the presence of the event in a sample. More specifically, the present invention provides DNA and related assays for detecting the presence of certain insect-resistance events in cotton. The assays are based on the DNA sequences of recombinant constructs inserted into the cotton genome and of the genomic sequences flanking the insertion sites. These sequences are unique. Based on these insert and border sequences, event-specific primers were generated. PCR analysis demonstrated that these cotton lines can be identified in different cotton genotypes by analysis of the PCR amplicons generated with these event-specific primer sets. Thus, these and other related procedures can be used to uniquely identify these cotton lines. Kits and conditions useful in conducting the assays are also provided. These materials and methods can also be used to assist breeding programs to further develop traits in cotton.

The invention provides a method for establishing a humanized rat drug evaluation animal model. According to the method, a multidrug resistance gene 1 (Abcb1)-knocked-out genetically engineered rat is obtained through a microinjection method by virtue of a CRISPR/Cas9 gene knockout technology and 153kb bacterial artificial chromosome (BAC) fragments containing a humanized Abcb1 promoter and cDNA is simultaneously inoculated into the rat genome through the microinjection method by virtue of a large fragment transgenic technology to obtain a transgenic rat capable of stably expressing human Abcb1 and the genetically engineered rat and the transgenic rat are hybridized to establish the humanized rat drug evaluation animal model. RT-PCR analysis shows that Abcb1 expression profiles of humanized Abcb1 rat are significantly different from those of the rat endogenous Abcb1. The method has the beneficial effects that the humanized rat capable of expressing human Abcb1 is obtained and the rat is used for expressing human Abcb1 genes and has closer expression profiles to those of human so that the model can be well used for the efficacy evaluation of newly developed drugs.

The present invention is directed to detection and measurement of gene transcripts in blood. Specifically provided is a RT-PCR analysis performed on a drop of blood for detecting, diagnosing and monitoring diseases using tissue-specific primers. The present invention also describes methods by which delineation of the sequence and/or quantitation of the expression levels of disease-associated genes allows for an immediate and accurate diagnostic/prognostic test for disease or to assess the effect of a particular treatment regimen.

The present invention relates to methods for the diagnosis of bacterial vaginosis based on an analysis of a patient sample. For example, patient test samples are analyzed for the presence or absence of one or more lactobacilli and two or more pathogenic organisms. The presence or absence of one or more lactobacilli and two or more pathogenic organisms may be detected using PCR analysis of nucleic acid segments corresponding to each target organism. The quantity of the target organisms can then be used to determine a score which is indicative of a diagnosis of bacterial vaginosis.

Systems and methods are provided for analyzing data to determine properties of a PCR process or other process exhibiting amplification or growth. Data representing an amplification can be distinguished from data representing a jump or other error. A modified sigmoid function containing a drift term may be used in determining the properties. A multi-stage functional fit of the amplification data can provide increased accuracy and consistency of one or more of the properties. A baseline of the amplification data can be determined by analyzing an integrated area of a first derivative function of the data. A reference quantitation value can also be determined from locations of maxima of different derivative functions of the amplification data, e.g., a weighted average of the maxima locations for the second and third derivatives may be used.

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 discloses a standard plasmid molecule to check genetic improved soybean strain GTS40-3-2 and constructing method, which comprises the following steps: incorporating strain special fragment of the genetic improved soybean strain GTS40-3-2 and special fragment of soybean internal standard gene Lectin; analyzing exogenesis inserting carrier by-pass ortho gene sequence of genetic improved soybean strain GTS40-3-2; designing strain special PCR primer; augmenting; getting strain special fragment of genetic improved soybean strain GTS40-3-2 and soybean internal standard gene special sequence; constructing into a plasmid molecule with molecular cloning method; getting artificial retooling plasmid molecule pMD-RRS. This standard plasmid molecule can be fit for strain special quantitative PCR analysis and check of genetic improved soybean strain GTS40-3-2 sample.

The invention discloses a capillary bioanalysis system, and an analytical method and applications thereof, and belongs to the field of medical equipment and biological detection technologies. The capillary bioanalysis system comprises a three-dimensional movement sample injecting platform, a temperature control-optical detection module, a magnetic field control module, a fluid control unit and a capillary array. The three-dimensional movement sample injecting platform and the fluid control unit are arranged on the two sides of the capillary array, and the capillary array is provided with the temperature control-optical detection module and the magnetic field control module. According to the capillary bioanalysis system, the droplet technology and the magnetic bead technology are combined, and bioanalysis processes such as loop-mediated isothermal amplification, fluorescence quantitative PCR analysis and immunochemiluminometry are integrated in capillaries. Advantages of the capillary bioanalysis system are that: volume is small, analysis speed is fast, detecting flux is large, and automation degree of operation is high. The capillary bioanalysis system is flexible in application, is suitable for analysis of single sample, batches of samples and on-site rapid detection, is capable of reducing acquisition cost and operation cost of equipment significantly, and possesses excellent economic benefits.

The invention relates to a method for increasing the contents of tanshinone and salvianolic acid in a salvia miltiorrhiza hairy root by using a transgene AtMYC2, belonging to the technical field of gene engineering. The method comprises the steps of constructing a high-efficiency expression vector of a plant by using an arabidopsis transcription factor AtMYC2, and carrying out genetic transformation on salvia miltiorrhiza leaves to obtain a gene AtMYC2 overexpressed transgenetic salvia miltiorrhiza hairy root; analyzing the expression of AtMYC2 in the transgenetic salvia miltiorrhiza hairy root and related genes in biosynthetic pathways of tanshinone and salvianolic acid through qRT-PCR; measuring the contents of tanshinone and salvianolic acid in the transgenetic salvia miltiorrhiza hairy root by using a high-performance liquid chromatography (HPLC); and measuring the antioxidant activity of tanshinone and salvianolic acid in the transgenetic salvia miltiorrhiza hairy root by using a DPPH free radical scavenging method. The invention provides the method for simultaneously increasing the contents of tanshinone and salvianolic acid in salvia miltiorrhiza hairy root and also provides a novel high-quality raw material for producing tanshinone and salvianolic acid with important clinic demands so as to have the positive promoting significance and application value for relieving the problem that the drug resources of tanshinone and salvianolic acid are short.

This invention provides methods of using labeled primers or probes for nucleic acid target detection and to detect the identity or presence of a nucleotide at certain positions in nucleic acid sequences with single molecule sensitivity using nanopore detection, and sets of oligonucleotide primers for use in such methods, as well as methods of quantitative PCR coupled with nanopore detection.

The invention is directed to compositions and methods for rapidly and efficiently extracting nucleic acids and/or targeted nucleic acids sequences from biological samples. The methods of the invention comprise combining the sample with a buffer and magnetic silicon beads and concentrating the beads with a magnet or other electrical field. Liquid may be removed, or not, and an alkaline buffer is added followed by magnetic carboxy beads in a binding buffer so that nucleic acids transfer to the carboxy beads, which can be easily and quickly isolated once again with a magnet. Total nucleic acid extraction is greatly enhanced. Extracted nucleic acids can be analyzed, for example, by PCR wherein the nucleic acids can be identified and characterized. Carboxy beads may also contain a ligand so as to target specific nucleic acid sequences. The invention is also directed to kits comprising the tools and compositions for performing the methods of the invention.

The invention relates to a cotton drought resistant gene GhEXP1 and applications thereof, and belongs to the biology technical field. The related gene GhEXP1 is a cotton expansin gene and is reported for the first time. The gene is cloned from an upland cotton draught resistant species (Ao 3503), and through RT-PCR analysis, people find that the expression of GhEXP1 is up-regulated after a drought treatment. The drought resistant performance of strains containing silenced GhEXP1 gene is obviously reduced. The living rate of GhEXP1 transgene arabidopsis plants is 80% after a 30-day drought treatment; while the living rate of referential wild arabidopsis is 10%; and the result shows that the GhEXP1 gene can prominently improve the drought resistant performance of plants. The GhEXP1 gene is advantageously used for improving the plant drought resistance and has a wide application prospect.

The invention discloses three groups of pumpkin SSR labeled primers applied to multiplex PCR reaction, belongs to the technical field of plant molecular biology DNA marks, and discloses an application of the primers to identification of pumpkin varieties and a pumpkin variety identification method. A batch of pumpkin SSR marks are developed by using pumpkin transcriptome sequencing results; three marks are selected from the marks and are characterized in that the polymorphism is high, the strip pattern is excellent, the size difference of amplification products is about 50bp, and primers cannot be matched with each other for forming hairpin structure SSR loca; by virtue of optimization of a PCR reaction system and an amplification program, a multiplex PCR analysis technical system with three SSR loca is built; the SSR marks are excellent in multiplex PCR amplification strip pattern, high in polymorphism and easily readable in results; the analysis efficiency of SSR is remarkably improved; the SSR marks can be applied to the fields of identification of pumpkin varieties, construction of fingerprint maps and identification of purity of hybrid seeds.

The invention discloses a method for extracting total DNAs of soil microorganisms at high purity. CTAB (Cetyltrimethyl Ammonium Bromide), lysozyme, protease and SDS (Sodium Dodecyl Sulfonate) act together to perform lysis of cells and simultaneously freeze and thaw the cells repeatedly; therefore, an excellent cell lysis effect is achieved; the humic acid is removed by utilizing PVP (Polyvinyl Pyrrolidone), CaC12 and BSA (Bull Serum Albumin); and the DNAs are precipitated by utilizing isopropanol so that the purity of the obtained DNAs is high. The method is simple and convenient; and without being purified, the DNAs obtained by the method can be used for subsequent PCR (Polymerase Chain Reaction) analysis and DGGE (Denaturing Gradient Gel Electrophoresis) analysis. The purity of the extracted DNAs meets the requirement for directly performing subsequent molecular biological research; the extracted DNA segments can be subjected to 16SrDNA amplification and DGGE atlas analysis; glue cutting recovery and basic group sequencing can be performed on specific stripes of the DGGE band; unknown bacteria can be identified through comparison with the conventional sequences in an RDP (Ribosomal Database Project) database or by establishing a new sequence probe; and therefore, the structural composition of the microbial community can be determined, and a foundation can be laid for researching the structural diversity and ecological functions of the soil microorganisms in the rhizosphere of the mulberry in future.

The invention discloses a method for extracting total DNA (deoxyribonucleic acid) of mulberry rhizosphere soil microorganisms by adopting a plurality of measures, which can achieve a good cell lysis effect by utilizing PVP (polyvinyl pyrrolidone) prewashing to remove humic acid, the synergy of CTAB (cetyltrimethyl ammonium bromide), lysozyme, protease and SDS (sodium dodecyl sulfate) to lyse cells and repetitively freezing and thawing the cells at the same time and utilizes PEG8000 to precipitate DNA, so that the purity of the obtained DNA is high. The adopted method is simple and convenient, and the DNA can be used in subsequent PCR (Polymerase Chain Reaction) analysis and DGGE (denaturing gradient gel electrophoresis) analysis without needing to be purified. The extracted DNA is so pure as to meet the requirement of direct subsequent molecular biology research, an extracted DNA fragment can be used in the amplification and DGGE map analysis of 16SrDNA, moreover, the specific band gel extraction and base sequencing of a DGGE band can be carried out, comparison with the sequence existing in an RDP (ribosomal database project) database can be carried out, or a new sequence probe can be established to identify unknown bacteria, so that the composition of a microbial community structure can be determined, and thereby laying a foundation for the future research of the community structure diversity and the ecological functions of the mulberry rhizosphere soil microorganisms.

Described herein are devices and methods for extracting cellular material from living cells and then depositing them into to a receptacle in a nanoliter scale. Using a nanopipette integrated into a scanning ion conductance microscope (SICM), extraction of mitochondrial DNA from human BJ fibroblasts and Green Fluorescent Protein (GFP) transcripts from HeLa/GFP cells was achieved with minimal disruption to the cellular milieu and without chemical treatment prior to obtaining the isolated sample. Success of the extraction was confirmed by fluorescence microscopy and PCR analysis of the extracted material. The method and apparatus may be applied to many different cell types and intracellular targets, allowing not only single cell analysis, but single subcellular compartment analysis of materials extracted in their native state.

The invention belongs to the field of plant gene engineering and transgenic breeding, and relates to a method for improving the stress resistance of cut chrysanthemum through trans-CgHSP70 genes. CgHSP70 established plant expression vectors obtained by cloning from Zhongshan purple cinnamon of the chrysanthemum are transferred into the cut chrysanthemum by using an agrobacterium-mediated method for cultivation so as to initially obtain resistant plants, and positively transferred plants are obtained through the screening of hygromycin resistance; PCR (polymerase chain reaction) and fluorescence quantitative PCR analysis is performed on converted plants to prove that endogenous genes are transferred into genome DNA of transgenic plants and transcription occurs; and the resistance analysis on the offsprings of the transgenic plants proves that the resistance to high temperature, drought and high salt is obviously improved. In the invention, the stress resistance of the cut chrysanthemum is improved through the conversion of the endogenous CgHSP70 genes and normal transcription expression as well as the induction of the expression of the genes at an adverse environment; a novel and practical method is provided for selecting and breeding stress resistance varieties of chrysanthemum by using a gene engineering technology; and the breeding progress of the biotechnology of the chrysanthemum can be effectively accelerated.

The invention relates to a six-color real-time fluorescence quantitative PCR (Polymerase Chain Reaction) analyzer. The six-color real-time fluorescence quantitative PCR analyzer comprises a light source excitation part (A) and a light detector part (C), and is characterized in that the light source excitation part (A) is provided with six independent light source excitation passages, the light detector part (C) is provided with six independent detection passages, and a certain angle is formed between the light source excitation part (A) and the light detector part (C). A light path system adopts a fixed light path, light filters are superposed to realize the selection of wavelength coverage, and the wavelength coverage of each passage can be changed by changing the wavelength of the light filters so as to realize different wavelengths.

The invention belongs to the technical field of molecular biology and particularly relates to a method for extracting DNA from a plant rich in polysaccharide and polyphenol. The provided method for extracting DNA from the plant rich in polysaccharide and polyphenol comprises the following steps: using a specific lysis solution to lyse a cell, extracting the DNA with Tris phenol-chloroform mixed solution, chloroform and a sodium perchlorate combination solution, and purifying the extracted DNA through a self-made adsorption column membrane, thereby obtaining the high-quality genomic DNA. The purity of the DNA extracted through the method for extracting DNA from the plant rich in polysaccharide and polyphenol is high, the impurity is low, the DNA is further prevented from being damaged by asuperoxide anion free radical or active oxygen, the completeness of the extracted genomic DNA is guaranteed, and the method is favorable for the scientific researches like construction of the gene library, PCR (Polymerase Chain Reaction) analysis, and Southern hybridization.

The invention relates to a variable-temperature metal module with precise temperature compensation and a temperature compensating method. Sample holes are arranged on the variable-temperature metal module. The variable-temperature metal module comprises temperature compensating heaters, the arrangement number of the temperature compensating heaters is less than or equal to the arrangement number of the sample holes, the temperature compensating heaters are connected with a control circuit, and each temperature compensating heater is singly arranged around the sample holes. The invention is used for a gene amplifying instrument and a full-automatic medical PCR (Polymerase Chain Reaction) analytic system and can increase the temperature uniformity of the variable-temperature metal module to +/- 0.05 DEG C from +/-0.3 DEG C in the conventional technology.

The invention provides an apparatus for preparing a biological sample for PCR analysis has an inlet (4) for receiving the sample (15) and a sieve (13) through which the sample is forced by screwing down an inlet cap (16). A lysis solution between two rupturable seals (11 and 12) is effective to disrupt the cells of the sample and release nucleic acid. The apparatus is mounted releasably on a PCR machine (2) having a motor (2') releasably coupled with a drive mechanism (3, 36,40) in the apparatus to effect a rotary and a vertical up and down movement of its components (30, 31, 32, 42). The apparatus has a transparent cuvette (5) and a needle (71) extending to the lower end of the cuvette, by which the prepared sample is dispensed to the cuvette for PCR analysis.

The invention discloses a plasmid reference molecule which is suitable for the quantitative determination of nucleic acids from a transgenic soybean GTS40-3-2, comprising a structure-specific sequence and a strain-specific sequence of the transgenic soybean GTS40-3-2 and a specific fragment of a soybean endogenous reference gene Lectin. The invention is characterized by designing primers to obtain the structure-specific sequence and the strain-specific sequence of the transgenic soybean GTS40-3-2 and the specific fragment of the soybean endogenous reference gene Lectin by PCR amplification through the analysis of inserting the exogenous source of the transgenic soybean GTS40-3-2 in the gene sequence; constructing by molecular cloning to form a artificial recombinant plasmid molecule pXL02 in a plasmid molecule; detecting the phosphorus content of the plasmid reference molecule by inductively coupled plasma mass spectrometry (ICP-MS) to calculate the concentration of the plasmid reference molecule. According to the invention, the plasmid reference molecule constructed in the invention can completely replace a transgenic soybean GTS40-3-2 positive standard sample, and the plasmid reference molecule is completely suitable for the quantitative PCR analysis and detection of structure specificity and strain specificity of the transgenic soybean GTS40-3-2 sample.