97 results about "Pyrophosphatase" patented technology

Convenient techniques for discriminating the base type in a base sequence of a nucleic acid are provided. The technique includes the step (a) of preparing a sample solution containing a nucleic acid, a primer having a base sequence that includes a complementary binding region which complementarily binds to the nucleic acid, and a nucleotide; the step (b) of allowing the sample solution to stand under a condition to cause an extension reaction of the primer, and producing pyrophosphate when the extension reaction is caused; the step (c) of bringing the sample solution into contact with the front face of a H⁺ hardly permeable membrane having H⁺-pyrophosphatase, which penetrates from front to back of the membrane, of which active site that hydrolyzes pyrophosphate being exposed to the front face; the step (d) of measuring the H⁺ concentration of at least either one of the solution at the front face side of the H⁺ hardly permeable membrane or the solution at the back face side of the H⁺ hardly permeable membrane, in a state where the H⁺-pyrophosphatase is immersed in the solution; the step (e) of detecting the extension reaction on the basis of the result of measurement in the step (d) ; and the step (f) of discriminating the base type in the base sequence of the nucleic acid on the basis of the result of detection in the step (e).

Method for sequencing a nucleic acid molecule in a thermocycling reaction which initially comprises a nucleic acid molecule, a first primer, a second primer, a reaction buffer, a first thermostable DNA polymerase, (optionally) a thermostable pyrophosphatase, deoxynucleotides or derivatives thereof and a dideoxynucleotide or a derivative thereof and which is characterized in that the thermocycling reaction additionally contains a second thermostable DNA polymerase which, in comparison to the said first thermostable DNA polymerase, has a reduced ability to incorporate dideoxynucleotides as well as the use of the said method.

The invention discloses a method for heightening the salt tolerance and the drought tolerance of cotton by polymeric stress-resistant gene, comprising the following steps of: recombining phosphatidyl inositol synthetase gene PIS from a signal transduction pathway of a plant, tonoplast pyrophosphatase gene Ppase isolated from an ion region, and choline dehydrogenase gene betA which is from colon bacillus and gives glycine betaine synthesis capability into a plant expression carrier; transplanting the recombinant into cotton cells to be effectively expressed; regenerating transgene plantlet; selecting transgene homozygote with the obviously heightened salt tolerance and drought tolerance from the transgene plantlet and filial generation thereof; and breeding out new cotton germplasm with good salt tolerance and salt tolerance, so as to create new material for breeding the cotton with good salt tolerance and salt tolerance for breeding the conventional variety or hybrid variety of the cotton.

The present invention is directed to a method and a kit for amplifying and detecting a target nucleic acid, wherein the composition containing reagents to perform and monitor nucleic acid amplification in real time comprises at least a first hybridization probe labeled with a first fluorescent entity and a pyrophosphatase.

The present invention relates to a transgenic plant which is tolerant to a salt, comprising one or more plant cells transformed with exogenous nucleic acid which alters expression of vacuolar pyrophosphatase in the plant. Also encompassed by the present invention are transgenic progeny and seeds of the transgenic plants described herein. Progeny transgenic plant grown from seed are also described. The present invention also relates to a construct comprising an AVP1 gene operably linked to a chimeric promoter designed to overexpress AVP1 or designed to down regulate endogenous pyrophosphatase. Plant cells (e.g., root cells, stem cell, leaf cells) comprising exogenous nucleic acid which alters expression of vacuolar pyrophosphatase in the plant cell are also the subject of the present invention. Also encompassed by the present invention are methods of making a transgenic plant described herein. Transgenic plants produced by the methods of making a transgenic plant as described herein are also a subject of the present invention. The present invention also relates to a method of bioremediating soil, a method of increasing the yield of a plant, a method of making a plant which is larger than its corresponding wild type plant, and a method of producing a transgenic plant which grows in salt water comprising introducing into one or more cells of a plant nucleic acid which alters expression of vacuolar pyrophosphatase in the plant. The transgenic plants of the present invention can also be used to produce double transgenic plants which are tolerant to a salt.

The invention relates to a fast and efficient construction method of a normalized full-length cDNA library. The method consists of: using specific 5-OH oligonucleotide to close non-full-length mRNA, then employing tobacco acid pyrophosphatase to remove a cap structure from the mRNA5' end to make a phosphate group at the cap structure of the mRNA5' end exposed, adopting an efficient RNA connection system to connect a section of optimized oligomeric RNA to the mRNA 5' end to serve as a primer binding site for triggering synthesis of second strand cDNA, finally conducting reverse transcription, two-strand synthesis as well as graded purification, and cloning the double-stranded DNA into a vector. The method can concisely and efficiently screen full-length mRNA, and realizes full-length mRNA capture of eukaryotes. The mRNA5' tail end contains a biotin label, which can be used for full-length mRNA separation. The used oligonucleotide primer contains optimized modification method and a nucleotide sequence, thus being able to realize full-length mRNA capture of eukaryotes, reverse transcription and DNA synthesis.

The invention discloses a thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and the application of a deletion mutant thereof. A clone carrying a TsVPI promoter is screened from a genomiclibrary of halophyte thellungiella, a 2200 nucleotide sequence positioned on the upstream of a TsVPI coding frame 5' is truncated as an overall-length promoter, promoter snippets with different lengths are obtained through PCR amplification, and then the promoter snippets are respectively blended with gus genes to be recombined into a plant expression vector to be converted into Arabidopsis; the TsVPI promoter and a part of the deletion mutant thereof are determined as salt-stress inducible type promoters by detecting the GUS enzymatic activity of transgenic plants, wherein a T5 promoter not only has a short sequence (667bp) but also has root specificity, and the TsVPI promoter and the T5 promoter are respectively connected with betA genes from colibacillus to be transmitted into tobaccosand corns so as to determine that the TsVPI promoter and the T5 promoter can normally exert functions in the transgenic tobaccos and the transgenic corns, are salt-stress inducible type strong promoters and have important application value in the industrialization development of plant gene engineering.

The invention relates to an enzymatic preparation method of an oxidized coenzyme II. Nicotinamide nucleotide (NR) and adenosine triphosphate disodium salt (ATP-Na2) are used as the raw material and subjected to one-pot reaction with nicotinamide nucleoside kinase (NRK), inorganic pyrophosphatase, NAD (Nicotinamide Adenine Dinucleotide) kinase and poly-pyrophosphokinase in a buffer solution with pH being 4.0-8.5 and at the temperature being 10-40 DEG C to obtain the oxidized coenzyme II. With the adoption of the enzymatic preparation method, the technical problems that in the existing enzymatic preparation method, the nicotinamide nucleotide is expensive and not easily obtained, the reaction time is long, the technical cost is relatively high and the technical conditions are not suitable for the industrialized scale-up production are solved; and the oxidized coenzyme II can be obtained with high efficiency and low lost in an industrialized scale-up production way.

This invention provides a recombinant yeast that can produce branched alcohol appropriate for automotive fuel and the like and a branched alcohol production method whereby branched alcohol can be produced at low cost with the use of the recombinant yeast.

A recombinant yeast in which a hydroxymethyl glutaryl-CoA reductase gene has been expressed to a high degree and the ADP-ribose pyrophosphatase gene and/or the yhfR gene are introduced so as to be expressed therein is provided.

Aortic valve stenosis (AS) is a chronic process related to a progressive mineralization of the aortic root and valve cusps. We found in human AS valves a high level of expression and enzymatic activity of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP-1), which correlated to the degree of mineralization. In vitro, inhibition of ENPP activity with ARL 67156 significantly reduced calcification of isolated valve interstitial cells. In a rat model of cardiovascular calcification, ARL 67156 significantly reduced calcification of the aortic root and valve cusps. This is the first study to demonstrate that increased expression and activity of ENPP-1 promotes the mineralization process in AS valves. Hence, inhibition of ectonucleotidase may represent a novel target of therapy for this frequent and serious cardiovascular disease.

The invention discloses application of a pyrophosphatase gene and application of pyrophosphatase gene folQ in improvement of lactobacillus plantarum folic acid synthesis. The nucleotide sequence of the pyrophosphatase gene folQ is as shown in SEQ ID NO: 1. The application comprises the following steps of carrying out enzyme digestion connection on the gene folQ and a constitutive vector pMG36e to obtain a recombinant expression vector, transferring the recombinant expression vector into lactobacillus plantarum YM-4-3, and realizing overexpression of the gene folQ in a YM-4-3 strain body to obtain an overexpression strain. An LC-MS method is adopted to measure the folic acid producing capacity of the strain, it is found that compared with a wild type strain, the content of folic acid monoglutamic acid produced by the BDQ strain is increased, the folQ gene plays a key role in folic acid synthesis, and therefore the pyrophosphatase gene has huge potential in the fields of folic acid biosynthesis research and application.

The present invention relates to a transgenic plant which is tolerant to a salt, comprising one or more plant cells transformed with exogenous nucleic acid which alters expression of vacuolar pyrophosphatase in the plant. The present invention also relates to a transgenic plant with increased Pi uptake, comprising one or more plant cells transformed with exogenous nucleic acid which alters expression of vacuolar pyrophosphatase in the plant. Also encompassed by the present invention are transgenic progeny and seeds of the transgenic plants described herein. Progeny transgenic plant grown from seed are also described. Plant cells (e.g., root cells, stem cell, leaf cells, flower cells, fruit cells and seed cells) comprising exogenous nucleic acid which alters expression of vacuolar pyrophosphatase in the plant cell are also the subject of the present invention. Also encompassed by the present invention are methods of making a transgenic plant described herein. Transgenic plants produced by the methods of making a transgenic plant as described herein are also a subject of the present invention. The present invention also relates to a method of bioremediating soil, a method of increasing the yield of a plant, a method of making a plant which is larger than its corresponding wild type plant, a method of producing a transgenic plant which grows in salt water, and a method of producing a transgenic plant with increased Pi uptake. The transgenic plants of the present invention can also be used to produce double transgenic plants which are tolerant to a salt, or have increased Pi uptake.

The invention relates to novel temperature-response inorganic pyrophosphatase conjugate synthesis and application of the conjugate to enhanced polymerase chain reaction, belongs to the field of polymer-protein coupling techniques and application of the polymer-protein coupling techniques to biology, and provides a strategy of enhancing PCR (polymerase chain reaction) efficiency. By the aid of the polymer-protein coupling techniques, temperature-response PNIPAM (polymer N-isopropylacrylamide) is modified in a site-directed manner near the active center of inorganic PPase (pyrophosphatase), heat stability of protein is enhanced to adapt to the condition of PCR high-temperature long-time circulation, and PCR is enhanced by the aid of the capacity of catalytic decomposition of pyrophosphoric acid.

The invention, which belongs to the technical field of alkaline phosphatase detection, discloses a room-temperature phosphorescence detection method of alkaline phosphatase and application thereof, thereby solving problems of complex detection process, high cost and large interference of the existing alkaline phosphatase. At a room temperature, a prepared beta-cyclodextrin-modified Mn: ZnS room-temperature phosphorescent quantum dot is used as a phosphorescent probe and pyrophosphate is used as a molecular recognition unit; and analysis and detection of pyrophosphatase are realized by detecting room-temperature phosphorescence changes of a system after alkaline phosphatase is added. The response range of the phosphorescence detection system to pyrophosphatase is 0.22-10.4 U/L, and the detection limit is 0.045 U/L. The method can be used for detecting alkaline phosphatase in serum; and a complex sample pretreatment process is not needed during measurement. A deoxidant and an inducer donot need to be added for generation of room-temperature phosphorescent, and interference of background fluorescence and scattered light of an actual sample can be avoided.

The invention puts forward a method for preparing [beta]-nicotinamide mononucleotide. In the method, [beta]-nicotinamide adenine dinucleotide is taken as a substrate, and reacts under a catalytic function of [beta]-nicotinamide adenine dinucleotide pyrophosphatase and/ or a recombinant cell containing the [beta]-nicotinamide adenine dinucleotide pyrophosphatase to generate the [beta]-nicotinamidemononucleotide. Compared with an existing preparation method for the [beta]-nicotinamide mononucleotide, the method disclosed by the invention is simple to operate, is efficient, has a short production period, is environmentally-friendly, is low in cost and has an important application value in fields, including health care products, biological medicines and the like, and a product can be easily purified.

The invention provides an LC-MS/MS method for detecting IPP, GPP, FPP and GGPP in a fresh tobacco leaf. The LC-MS/MS method comprises the following steps: 1) preparing an extraction solvent; 2) conducting liquid nitrogen flash freezing on the fresh tobacco leaf, and conducting freeze drying and crushing; 3) adding the extraction solvent to the crushed sample, and conducting ultrasonic extraction at low temperature; 4) carrying out refrigerated centrifugation, taking supernatant, and filtering; 5) conducting LC-MS/MS method to detect IPP, GPP, FPP and GGPP in the detected sample. The LC-MS/MS method has the advantages that the pretreatment method of the sample is simple and only comprises ultrasonic extraction and filtration; compared with the existing method, the method is high in sensitivity, good in precision, and high in recovery of standard addition; the sample can be directly fed and analyzed through filtration, but according to the methods in the existing documents, the pretreatment method is complicated and time-consuming, and requires pyrophosphatase and alkaline phosphatase for carrying out biochemical reaction on the sample. Therefore, the LC-MS/MS method disclosed by the invention can effectively increase the analysis efficiency, and is suitable for analysis of the ultralow content target in the complex substrate.

The invention belongs to the technical field of biotechnology and particularly relates to a preparation method of biotin labeled inorganic pyrophosphatase. The method includes the steps of dissolving inorganic pyrophosphatase, activating biotin, combining the biotin with the inorganic pyrophosphatase, performing dialyzing, measuring a protein content, and storing. The inorganic pyrophosphatase prepared by the method is very suitable for application in pyrosequencing reactions; high activity of the inorganic pyrophosphatase ensures accurateness of the pyrosequencing reactions.