90 results about "Dna amplification" patented technology

The invention provides an intratumoral bacterium detection and analysis method based on circular DNA enrichment sequencing and related model construction, and belongs to the field of molecular biology. The intratumoral bacterium detection method provided by the invention comprises the following steps: extracting total DNA of cancer tissue or para-carcinoma tissue, carrying out linear digestion, purifying a linear digestion product, and enriching to obtain circular DNA; the method comprises the following steps: amplifying circular DNA, and breaking a DNA amplification product to 300-500bp; purifying DNA fragments, constructing a library, and sequencing; and performing genome comparison according to a sequencing result to obtain information of the intracellular bacteria. According to the method, complete bacterial genome and plasmid genome information can be captured, and the detection sensitivity of the intracellular bacteria and the richness of the bacterial genome information are improved. The invention further provides an intratumoral bacterium analysis method which can be used for screening generic cancer bacterial markers, and provides a cancer risk assessment machine learning model based on the intratumoral bacterium, so that the clinical application possibility of the intratumoral bacterium is improved.

The present disclosure relates to Taq DNA Polymerase mutants comprising at least three mutations which are capable of high levels of DNA amplification in the presence of several PCR inhibitors and compositions comprising the same. Also disclosed are methods of performing mutagenesis on wild-type Taq DNA polymerase to obtain the disclosed Taq DNA polymerase mutants, DNA amplification using the same, and cDNA amplification using the same.

The present document describes methods and systems for amplifying and quantifying amplification of a nucleic acid molecule, with a polymerase chain reaction (PCR) or a loop-mediated isothermal amplification (LAMP), by irradiating, with a heating activation light beam from a continuous wave laser a biological enzymatic reaction mixture in solution comprising a nucleic acid template, a polymerase enzyme, and chemically modified nanoparticles. Quantification of the amplification is achieved by irradiating the biological enzymatic reaction mixture during an annealing and / or elongation steps with an ultraviolet (UV) light source and measuring with a photodetector a transmission change in UV light transmission.

The invention discloses a composition, a detection method and a kit for SpaceAS variable splicing space in-situ detection of nucleic acid. The composition comprises a pi-type probe, a U-type probe, an O-type probe, an S-type probe and a signal probe, and the O-type probe adopts an in-vitro cyclization strategy, so that the problem of low hybridization efficiency caused by dependence on enzymatic cyclization reaction in a traditional hybridization method is effectively solved; meanwhile, an efficient probe hybridization system, a high-sensitivity signal amplification mechanism and a multi-round iterative imaging process are combined, so that the detection sensitivity and specificity are remarkably improved. The detection method has the advantages of high efficiency, high sensitivity, high specificity, good repeatability, strong expandability and the like through the synergistic effect of five-step single-stranded DNA probe hybridization reaction and one-step DNA amplification reaction, and can be widely applied to space in-situ detection and analysis of variable splicing events.

The invention belongs to the technical field of DNA amplification, and relates to a mixed enzyme system capable of amplifying circular DNA as well as application and a product of the mixed enzyme system. The invention provides a mixed enzyme system capable of amplifying circular DNA (deoxyribonucleic acid). The mixed enzyme system comprises a polymerase III tau clip loader and a catalytic core compound. The mixed enzyme system provided by the invention is remarkably superior to a traditional method in the aspects of efficiency, cost, safety, applicability, sensitivity and the like, provides a brand new solution for efficient preparation of the circular DNA, and has a wide application prospect in the field of biotechnology.

The present invention discloses a double-stage amplified DNA cascade circulation system and its application, which constructs a precise imaging system through CAR+CHA+HCR. The target is calculated by the "CAR" combination chain to determine whether to release miR-21, thereby achieving accurate detection of cancer cells. The amplification system consists of a "CHA+HCR" double cycle, where CHA consists of hairpin probes H1 and H2. When miR-21 is present, the CHA cycle is opened, and the formed H1 and H2 duplexes open the HCR cycle, thereby displaying a fluorescent signal. The HCR cycle consists of H3, H 3free The two cycles each perform their own functions and complement each other, ultimately forming a large CHA+HCR system, which further enhances the fluorescence signal.

The invention discloses a preparation method and application of carassius auratus rahn aquatic OmpA gene cloning and prokaryotic expression as well as a polyclonal antibody. The preparation method comprises the following steps: culture and DNA amplification of rahn aquatic, design of an OmpA primer, gene cloning and prokaryotic expression, back multi-point subcutaneous injection of an immune rabbit, heart blood sampling and preparation of the polyclonal antibody. The invention further provides OmpA protein prokaryotic expression and protein purification, preparation of OmpA polyclonal antiserum and application of fish bodies. According to the invention, a rahnella aquatilis OmpA gene is cloned, a specific Hind III and EcoR1 double-enzyme digestion primer sequence OmpA-1F / OmpA-1R is designed, a prokaryotic expression vector pET32a-OmpA is constructed, high-immunogenicity OmpA protein is obtained through purification, OmpA specific multi-antibody serum is prepared, and the OmpA specific multi-antibody serum can be used for preparing the multi-antibody serum. The OmpA polyclonal antibody can be used for detecting and positioning the expression application of rahnella aquatica OmpA protein in crucian carp bodies through bidirectional agar diffusion, an immunoblotting method and an immunohistochemical method, and provides a reference basis for research and development and application of rahnella aquatica subunit vaccines and a molecular diagnosis technology as well as immune prevention and control of infectious diseases of the rahnella aquatica subunit vaccines and the molecular diagnosis technology.

The present application relates to a primer combination for amplifying whole genome DNA of sheep embryo cells and application thereof, and the primer combination is composed of 20 or 30 primers. The present application designs random primers based on sheep reference genome sequence, optimizes the reaction system based on MDA whole genome amplification technology, so that the whole genome DNA amplification product with high coverage and low mismatch rate can be generated by using 10 sheep embryo cells or as low as pg level genomic DNA, which can be directly adapted to downstream applications such as second-generation sequencing and targeted capture sequencing, and the detection cost is reduced.

The invention belongs to the field of detection, and discloses a method for detecting staphylococcus aureus in bulk ready-to-eat food through semi-quantitative developing, which comprises the following steps: designing a specific primer aiming at a tuf gene of staphylococcus aureus; dNA is extracted from a bulk instant food sample to be detected to serve as a template; s2, template DNA is added into an isothermal amplification reaction system for a recombinase-mediated isothermal nucleic acid amplification reaction, wherein the isothermal amplification reaction system comprises the specific primer in the step S1 and SYBR Green I fluorescent dye; after the amplification reaction is completed, a color change judgment result of a reaction system is observed: yellow green fluorescence is judged to be positive under an ultraviolet lamp, orange is judged to be negative under the ultraviolet lamp, and DNA amplification only needs 15 minutes; amplification and detection are completed in one tube and one step at the same time, and the detection result can be accurately, simply, conveniently and rapidly judged according to the presence or absence of an S-shaped curve and the change of the color of a reaction system.

The invention discloses a reagent and a method for improving the tolerance of a PCR inhibitor and application, and belongs to the technical field of molecular biology. According to the invention, the thermosensitive uracil DNA glycosidase and the deoxyuridine triphosphate are combined and used as the PCR antiinhibitor to be applied to DNA amplification of a whole blood sample, and experiments prove that the composition can improve the endurance capacity of a PCR reaction system to a whole blood inhibitor, reduce the false negative rate and improve the accuracy and reliability of detection. Meanwhile, the method for carrying out PCR reaction by using the composition is simple to operate, low in cost and high in nucleic acid detection sensitivity. Therefore, the reagent and the method have a good application prospect in detection of nucleic acid extracted from a whole blood sample.

The invention discloses a Phi29 DNA polymerase mutant with improved thermal stability, a construction method of the Phi29 DNA polymerase mutant and a genetically engineered bacterium. The mutant is obtained by performing single-point mutation on serine at the 527th site on an amino acid sequence shown as SEQ ID NO.2. The invention further discloses a construction method of the Phi29 DNA polymerase mutant. The mutant is realized through a single-point mutation design, and compared with a wild type Phi29 DNA polymerase, the mutant shows better thermal stability in a temperature interval of 40-70 DEG C. The Phi29 DNA polymerase mutant obtained by means of the construction method provided by the invention has better thermal stability. When being used for DNA amplification operation at a relatively high temperature, the fluorescent probe can present relatively high thermal stability, so that the fluorescent probe has relatively high application value and development potential in multiple fields of molecular biology research, clinical diagnosis and the like.

The present invention discloses a method for detecting 5-hydroxymethylcytosine in the whole genome and its application, which relates to the technical field of biochemical detection. The method comprises the following steps: 1: Blocking the 3'-OH end of the DNA to be detected; 2: Preparation of glycosylated 5hmC DNA; 3: TdT-mediated DNA amplification; 4: Fluorescence measurement. The present invention constructs a homogeneous, template-independent signal amplification method for detecting 5-hydroxymethylcytosine in the whole genome. This method is based on specific enzyme-catalyzed reactions and multiple signal amplification strategies, and has the characteristics of high sensitivity, good specificity, no need for separation steps, sequence-independent amplification, and the ability to detect 5hmC at any position in the whole genome. Moreover, the present invention can achieve accurate and sensitive detection of actual clinical samples.

The present invention provides an in vitro method for detecting cell-cell interactions and related kits and uses, wherein the method comprises: (a) at least two primary binding agents, wherein a first primary binding agent binds to a first molecule on a first cell and a second primary binding agent binds to a second molecule on a second cell, and wherein the first primary binding agent and the second primary binding agent are immunologically different; (b) at least two secondary binding agents, wherein a first secondary binding agent binds to the first primary binding agent and a second secondary binding agent binds to the second primary binding agent, wherein the first secondary binding agent does not bind to the second primary binding agent and the second secondary binding agent does not bind to the first primary binding agent; and wherein: (i) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; (ii) the first secondary binding agent is labeled with a FRET donor and the second secondary binding agent is labeled with a FRET acceptor; The first and second secondary binders are conjugated or fused to DNA sequences, wherein the DNA sequences are different and connected to form a circle and are amplified by rolling circle DNA amplification and bound by an external fluorescently labeled DNA probe that is complementary to the amplified DNA sequence; or (iii) the first secondary binder is labeled with a FRET donor and the second secondary binder is fused to an enzyme, which reacts with a conjugate comprising a FRET acceptor and a substrate-specific enzyme to form an activated conjugate that binds to an electron-rich portion on the surface of a molecule adjacent to the enzyme; wherein the method comprises: i. contacting an isolated sample containing cells with the at least two primary binders; ii. contacting the sample with the at least two secondary binders; iii. performing a washing step; iv. detecting the interaction between the secondary binders.

The application belongs to the technical field of DNA amplification, and has a mixed enzyme system capable of amplifying circular DNA, application and product thereof. The application provides a mixed enzyme system capable of amplifying circular DNA, wherein the mixed enzyme system comprises a polymerase III tau clamp loader and a catalytic core complex. The mixed enzyme system provided by the application is significantly superior to traditional methods in terms of efficiency, cost, safety, applicability and sensitivity, and provides a brand-new solution for efficient preparation of circular DNA, and has a wide application prospect in the field of biotechnology.

The invention belongs to the technical field of microorganisms, and particularly relates to a specific detection method for survival of fusarium pseudograminearum in soil. The method comprises the following steps: adding PMA into a soil sample, carrying out mixed incubation and exposure treatment, extracting DNA, carrying out qPCR detection, preventing DNA amplification of dead cells in total DNA, and determining the number of viable bacteria of fusarium pseudograminearum in the soil. According to the detection method, the optimal PMA concentration is 50 [mu] mol L <-1 >, the optimal exposure time is 15 min, dead and surviving fusarium pseudograminearum spores are distinguished, and the lowest detection limit is 628 spore g <-1 >. The invention provides a rapid, sensitive and reliable method which is used for quantitatively detecting the surviving fusarium pseudograminearum in the soil, and the method has the advantages of high sensitivity, high sensitivity and high sensitivity. Accurate and reliable data are provided for monitoring and early warning, early diagnosis and prevention and control measures of the wheat basal stem rot.

The present disclosure relates to Taq DNA Polymerase mutants comprising at least three mutations which are capable of high levels of DNA amplification in the presence of several PCR inhibitors and compositions comprising the same. Also disclosed are methods of performing mutagenesis on wild-type Taq DNA polymerase to obtain the disclosed Taq DNA polymerase mutants, DNA amplification using the same, and cDNA amplification using the same.

The invention relates to the technical field of plant breeding and biology, in particular to a loquat transgenic method mediated by a magnetic nano material. According to the method, a red fluorescent plant expression vector containing a loquat EjIPUT gene is constructed, and the full length of the gene sequence is as shown in SEQ ID No.1. EjIPUT genes and magnetic nano Fe3O4 particles form a compound through electrostatic force, the compound and loquat pollen are mixed in a pollen culture solution by standing on a magnetic frame, pollen precipitates containing the compound are obtained after standing, the precipitates are dried and pollinated to castrated stigmas, and reproductive isolation is performed. Finally, DNA amplification identification, fluorescence identification, expression quantity identification and the like show that a loquat transgenic plant over-expressing the EjIPUT gene is obtained.

Disclosed herein are DNA amplification methods for quantifying DNA fragments of a target DNA in a sample by size. This can be used, for example, to detect tumor-derived viral DNA in blood sample and distinguish it from larger viral DNA from non-tumor sources. In particular, disclosed herein are methods of detecting, monitoring or treating a human papilloma virus (HPV)-associated malignancy in a subject that involves detecting a presence or absence of at least one circulating tumor-derived HPV DNA in a sample from the subject. Kits for accomplishing the same are also provided.

The application provides a method and application for improving DNA yield in vitro amplification, and belongs to the technical field of biology. The application provides a method for improving DNA yield in vitro amplification, which comprises the following steps: (1) reacting a reaction system at 27-38 DEG C for 40-60 min; (2) adding the reaction liquid into a dialysis bag, adding dialysis liquid into an outer tube, and dialyzing at 30-38 DEG C; (3) supplementing tRNA into the reaction liquid for sealing after dialyzing for 1-3 h; the reaction system in step (1) comprises 5x synthesis enzyme mix, 5x synthesis reaction buffer, NTP, dNTP, a template and water; and the concentration of the dNTP is 0.1-0.5 mM. The method can significantly improve the DNA yield in vitro amplification, the concentration of the amplification product is increased by more than about 134 %, and the core problem of insufficient yield in the in vitro DNA amplification is effectively solved.

The invention provides an enhanced colorimetric method for in-situ generation and catalysis of multiple horseradish peroxidase clusters based on allosteric probe triggering and a kit, and the method comprises the following steps: S1, designing a nucleic acid aptamer sequence of a to-be-detected target object, and designing an allosteric probe AP with a hairpin structure according to the nucleic acid aptamer sequence; s2, coupling the allosteric probe on the granular carrier to obtain a carrier-AP; s3, purifying a sample containing a to-be-detected target object, adding the carrier-AP, the primer for amplifying the allosteric probe, DNA polymerase, one of the deoxyribonucleotides with biotin-labeled dNTP and a reaction solution required for DNA amplification to form a mixed solution, and reacting for a certain time to obtain a carrier-dsDNA; s4, sequentially adding streptavidin and horse radish peroxidase labeled by biotin into the carrier-dsDNA (double-stranded deoxyribonucleic acid); and S5, adding a substrate for color development, and calculating the concentration of a target object according to a colorimetric method. According to the method disclosed by the invention, the detection sensitivity is remarkably improved through cascade amplification of signals.

Provided are a DNA analysis device and a DNA analysis method which have reduced cost. A DNA analysis device (100) according to the present invention amplifies DNA in a sample to be measured, and analyzes the DNA by electrophoresis, said device comprising a first light source which emits light to a sample that has been introduced into a DNA amplification chamber, a second light source which emits light to the sample that has been sent from the DNA amplification chamber to an electrophoresis flow path, and a detector (111) to which the DNA amplification chamber and the electrophoresis flow path are connected in common, wherein: the detector (111) detects a first fluorescence intensity from the DNA amplification chamber; the same detector (111) detects a second fluorescence intensity from the electrophoresis flow path; and a control device (121) estimates, on the basis of the first fluorescence intensity, the amplification product amount resulting from the amplification, and analyzes the DNA on the basis of the second fluorescence intensity.

The invention belongs to the technical field of biological detection, and discloses a primer probe composition for detecting EBV (Epstein-Barr Virus) and application of the primer probe composition. The primer probe composition comprises a forward primer EBV-F and a reverse primer EBV-F, wherein the nucleotide sequence of the forward primer EBV-F is shown as SEQ ID NO.1; a reverse primer EBV-R with a nucleotide sequence as shown in SEQ ID NO.2; the nucleotide sequence of the fluorescent labeled probe EBV-Probe is as shown in SEQ ID NO.3. The invention also discloses a detection method of the fluorescent labeled probe. The invention also discloses a product for detecting the EBV virus. The product comprises the primer probe composition, an EBV negative control, an EBV positive control (plasmid containing a target sequence) and a DNA amplification premix solution. The primer probe composition for detecting the EBV has relatively good detection sensitivity and accuracy, is simple and convenient to operate, and can be widely applied to EBV detection in each production stage of cell therapy products.

This invention discloses a kit for amplifying the whole genome of bovine embryonic cells and its applications. The invention provides a primer set for amplifying the whole genome of bovine embryonic cells, consisting of 20 primers, each 6 nt in length. This invention can utilize 4-8 bovine embryonic cells or genomic DNA down to the pg level to amplify and generate whole-genome DNA amplification products with high coverage and low mismatch rate. These products can be directly adapted to downstream applications such as next-generation sequencing and targeted capture sequencing, while reducing detection costs. Furthermore, the three-step operation process of this invention (cell lysis-lysis termination-isothermal amplification) can complete efficient amplification within 3.5 hours.

A rapid detection and visualization method for triple RPA-LFA targeting necrophilic flies includes the following steps: Step 1: DNA amplification reaction; DNA is extracted from *Gynostemma pentaphyllum*, *Gynostemma spp.*, *Variegata spp.*, *Variegata spp.*, *Bulbus rubrothes*, *Gynostemma pentaphyllum*, *Flyflies fasciatus*, and *Heterodon spp.* using the SteadyPure Universal Genomic DNA Extraction Kit; the concentration and purity of the genomic DNA of the above species are assessed using a Nano Drop 2000 spectrophotometer; the reaction is performed according to the instructions of the DNA isothermal rapid amplification kit; the reaction system uses Buffer A, a primer mixture consisting of 3 pairs of primers; the mixture is incubated in a metal bath to perform the RPA reaction and obtain the amplified product; Step 2: Visual detection; Mixed amplification product: the amplified product is mixed with dd water to obtain a mixed amplification product; the mixed amplification product is dropped onto the LFD test strip sample pad, and then the result is displayed. This invention features no dependence on a sequencer, simple operation, rapid detection, and visualization.

The invention relates to a DNA polymerase and application thereof. Wherein the DNA polymerase is 1) a protein with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2; or 2) a protein which has an amino acid sequence having more than 80%, more preferably more than 90%, and more preferably more than 95% homology with the amino acid sequence as shown in SEQ ID NO: 1 or 2 and has DNA polymerase activity. The DNA polymerase has the advantages of high amplification rate, good thermal stability, 5 '-3' polymerization activity and 3 '-5' excision activity, DNA amplification can be carried out at a fast PCR extension rate during DNA amplification, market requirements can be well met, and the DNA polymerase has great application potential.

Provided is a method to remove double-stranded DNA with sequence errors, which can occur in various stages of DNA production such as chemical synthesis, hybridization, and amplification, from double-stranded DNA without sequence errors, thereby providing double-stranded DNA with a low proportion of sequence errors. Specifically, this invention is a method for producing double-stranded DNA, which includes: (1) providing a double-stranded DNA mixture containing double-stranded DNA with sequence errors and double-stranded DNA without sequence errors; (2) adding a mismatch repair-related enzyme group to the double-stranded DNA mixture, where the mismatch repair-related enzyme group includes MutS and MutL, or MutS and single-strand specific exonuclease; and (3) subjecting the double-stranded DNA mixture to a double-stranded DNA amplification reaction.

Methods and kits for probing chromatin accessibility, including Deaminase Assisted single-molecule chromatin Fiber sequencing (DAF-seq) methods and kits. The disclosed approaches leverage DNA deaminase toxin A (DddA) to efficiently stencil protein occupancy along DNA molecules via selective deamination of sterically accessible cytosine bases. These deamination events result in C-to-T transitions during DNA amplification, such that the resultant sequences preserve the chromatin architectures of the underlying individual chromatin fibers as per-molecule DNA alterations. The disclosed approaches can be used for identifying where proteins are bound to genomic DNA, mappable at a single-cell, single-molecule, and single-nucleotide level, and as diagnostic tools in clinical settings.

The invention relates to a specific probe for a branched DNA amplification system and application of the specific probe, and belongs to the technical field of pathological diagnosis. On the basis of an original target gene probe, a pre-amplification probe, an amplification probe and a marking probe of a branched chain DNA amplification system probe system, a closing probe for closing the pre-amplification probe is added, and the closing probe can be complementarily combined with the pre-amplification probe. Probes used in the branched DNA signal amplification (bDNA) technology are all single-stranded DNA (ssDNA), and the combination of a closed probe and a pre-amplification probe can reduce the combination of non-target fragments in a sample and the ssDNA, so that the non-specific hybridization is reduced, and the specificity of the bDNA is improved.

A method for amplifying a DNA template, comprising: a) providing a composition comprising a DNA template, an RNA polymerase, a DNA polymerase, and a deoxyribonucleotide, wherein said composition does not comprise a ribonucleotide; and b) incubating said composition in a suitable condition to allow amplification of said DNA template. Also provided is a method for priming DNA amplification from a DNA template, comprising: incubating a DNA template, an RNA polymerase and a deoxyribonucleotide in a suitable condition to allow the initiation of DNA amplification; wherein said method does not comprise adding a ribonucleotide.