65 results about "Dna amplification" patented technology

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 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 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 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.

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.

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.

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.

A method for sample preparation and / or DNA amplification from urine, saliva and / or mouthwash samples is disclosed. The method involves providing a sample preparation system comprising a filter and providing a urine, saliva and / or mouthwash sample, then filtering at least a portion of the sample through the filter, thereby retaining bacteria, fungi, viruses protists and / or cancerous or precancerous cells in the filter. Subsequently a lysis reagent is applied to the filter. Finally, the DNA found in the lysate is amplified.

The application belongs to the technical field of biology and relates to a phi29 DNA polymerase mutant with reduced polymerization rate and a preparation method and application thereof. The application designs and provides a mutant of phi29 DNA polymerase. The amino acid residues at at least one of positions 190, 324, 411, 49, 83, 375, 526, 85, 149, 284, 287, 35, 115, 178, 102, 246, 336, 506, 257, 502, 55, 113, 187, 289, 306, 517, 527, 140, 203, 368, 372, 89, 54, 277, 327, 163, 224 in the amino acid sequence of the phi29 DNA polymerase are modified, a new protein with reduced polymerization rate of the polymerase and good polymerase activity is generated, problems caused by the too fast polymerization rate of the phi29 DNA polymerase in practical application are successfully solved, the accuracy, stability and reliability of the phi29 DNA polymerase in DNA amplification, sequencing and other molecular biology experiments are improved, and the sequencing quality is greatly improved.

The invention belongs to the technical field of biology, and particularly relates to application of enzyme in isothermal nucleic acid amplification, a mixture, a kit and a detection method, in particular to application of human AP endonuclease 1 (hereinafter referred to as hAPE1) in the isothermal nucleic acid amplification technology. HAPE1 is applied to a multi-enzyme synergistic isothermal real-time amplification system such as RAA, RPA, ERA and MIRA; the fluorescent probe containing the AP site is efficiently cut by utilizing the high AP incision enzyme activity of hAPE1, so that real-time detection is realized; meanwhile, by virtue of the extremely low excision enzyme activity, the completeness of a DNA amplification product is maintained to the maximum extent, so that subsequent analysis and application are facilitated. While efficient real-time detection is ensured, it is ensured that an amplification product can be used for follow-up analysis, and therefore the application range of the technology is widened.

The invention belongs to the technical field of biology, and relates to a phi29 DNA polymerase mutant losing excision enzyme activity as well as a preparation method and application of the phi29 DNA polymerase mutant. According to the present invention, at least one amino acid residue in the 54th site, the 55th site, the 90th site, the 96th site, the 102th site, the 140th site, the 163th site, the 203th site, the 212th site, the 277th site, the 284th site, the 327 site, the 439th site, the 502th site, the 505th site, the 534th site and the 552th site in the phi29DNA polymerase amino acid sequence are modified to generate the novel protein having low excision enzyme activity but maintaining the polymerase activity, such that the problem possibly caused by the too high excision enzyme activity in the practical application of the phi29DNA polymerase is successfully solved; and the stability and the reliability in DNA amplification, sequencing and other molecular biology experiments are improved.

The invention belongs to the technical field of biology, and particularly relates to a single-point mutation Phi29 DNA polymerase and a recombinant expression system thereof. The Phi29 DNA polymerase mutant provided by the invention comprises five single-point mutants at any one single-point mutation site in T204E, K208E, R236E, R415G and K478D on an amino acid sequence shown as SEQ ID NO.2. Compared with a wild type Phi29 DNA polymerase, the Phi29 DNA polymerase mutant has the advantage that the thermal stability of the Phi29 DNA polymerase mutant at 40-70 DEG C is better. The Phi29 DNA polymerase mutant obtained by the construction method provided by the invention has better thermal stability, can show higher thermal stability when being used for DNA amplification at a higher temperature, and has higher application potential in the fields of molecular biology research, clinical diagnosis and the like.

To provide compounds, compositions, kits, and methods for DNA amplification reactions.SOLUTION: To provide compounds, compositions, and methods for nucleic acid amplification reactions, particularly for viability PCR (vPCR) applications of compounds that selectively bind to nucleic acids from non-living cells.SELECTED DRAWING: None

The invention discloses a Phi29 DNA polymerase mutant suitable for high-temperature amplification as well as a coding gene, a genetically engineered bacterium construction method and application of the Phi29 DNA polymerase mutant. The Phi29 DNA polymerase mutant is a single-point mutant, and compared with a wild type Phi29 DNA polymerase, the Phi29 DNA polymerase mutant has the advantage that the thermal stability of the Phi29 DNA polymerase mutant in a temperature interval of 40-70 DEG C is obviously improved. The Phi29 DNA polymerase mutant obtained by the construction method disclosed by the invention has excellent thermal stability, so that the enzyme activity can be stably maintained when a DNA amplification reaction is carried out under a relatively high temperature condition. The mutant can effectively meet the application requirements on high-stability DNA polymerase in the fields of molecular biology research, clinical diagnosis and the like, and has important practical application value and wide industrial development potential.

The invention belongs to the technical field of biology, and discloses a DNA polymerase single-site mutant with improved thermal stability, a construction method of the DNA polymerase single-site mutant and a genetically engineered bacterium, and the DNA polymerase single-site mutant comprises a single-site mutant of any single-site mutation site in K311D, K317E, K407D, R415W, S527T and D570E on an amino acid sequence shown in SEQ ID NO.2. Compared with a wild type Phi29 DNA polymerase, the thermal stability of the six single-point mutants provided by the invention is better at the temperature of 40 DEG C to 70 DEG C. The DNA polymerase single-site mutant obtained by the construction method provided by the invention is better in thermal stability, can show higher thermal stability when being used for DNA amplification at a higher temperature, and has higher application potential in the fields of molecular biology research, clinical diagnosis and the like.

The invention belongs to the technical field of bioengineering, and particularly relates to a recombinant plasmid for promoting synthesis of tylosin, an engineering bacterium and a preparation method thereof, the genetically engineered bacterium (Streptomyces fradiae G3-F2) is preserved in China Center for Type Culture Collection on February 24, 2025, and the preservation number is CCTCC NO: M 2025278. According to the streptomyces fradiae G3-F2 disclosed by the invention, through site-specific recombination, a recombinant plasmid is constructed by utilizing an optimized ZouA gene DNA amplification system, so that multi-copy increase of a tylF gene is realized, and further, streptomyces fradiae G3-F2 is prepared; the enzymatic reaction rate of the methyltransferase is greatly improved; the efficient conversion of the component C is realized; and low-temperature culture fermentation is combined, so that the tylosin synthesis rate and efficiency are greatly improved. The titer of the strain in a 50L tank can reach 18500U / mL under the low-temperature culture condition of 28 DEG C, so that the production cost can be greatly reduced.

The present invention relates to a method for amplifying a DNA template, the method comprising incubating a DNA template with a DNA-dependent RNA polymerase in the presence of ribonucleotides, and amplifying the DNA template with a strand-displacing DNA polymerase. The invention further relates to the use of an RNA polymerase to generate ribonucleotide primers on a DNA template and subsequently amplifying the DNA template with the strand-displacing DNA polymerase, a kit of parts comprising an RNA polymerase and a strand-displacing DNA-dependent DNA polymerase, and the use of the kit of parts for amplifying a DNA template.