30 results about "Exonuclease I" patented technology

96 New PCT-Patent Application based on EP 24 186 635.9 Insempra GmbH Vossius Ref.: AG4141 PCT S3 Abstract The present invention relates to a linear double-stranded deoxyribonucleic acid (dsDNA) molecule comprising one or more Tus protein (Tus) binding site(s) at the 5'-terminus, one or more Lac repressor protein (LacI) binding site(s) at the 3'-terminus, and a segment comprising a DNA sequence of interest (DOI) between said Tus binding site(s) and said LacI binding site(s). The present invention further relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from the linear dsDNA molecule of the invention, and to an expression DNA cassette comprising a promoter, an RBS, a GOI encoding a POI, and one or more LacI binding site(s), wherein said expression cassette does not comprise a terminator sequence between the 3´ end of said GOI and said one or more LacI binding site(s). The present invention also relates to a non-naturally or naturally occurring RNA molecule as encoded by or transcribable / transcribed from said expression cassette. The present invention further relates to method of protecting a linear deoxyribonucleic acid (DNA) molecule from exonuclease degradation by adding one or more Tus binding site(s) at the first terminus of the DNA molecule and adding one or more LacI binding site(s) at the other terminus of the DNA molecule. The present invention further relates to a method of synthesizing a protein of interest (POI) in a cell- free protein synthesis (CFPS) reaction mixture by using the (ds)DNA and / or RNA molecules of the invention, and also to a cell-free biological system or CFPS reaction mixture comprising the (ds)DNA, expression cassette and / or RNA molecules of the invention.

The invention discloses a divalent nucleic acid aptamer fluorescence detection method based on an exonuclease I auxiliary target circulation strategy, and belongs to the field of analysis and detection.The divalent nucleic acid aptamer is a repetitive sequence composed of two monovalent nucleic acid aptamers and can form double-stranded DNA through base complementary pairing with complementary strand cDNA of the divalent nucleic acid aptamer, and the divalent nucleic acid aptamer can be used for fluorescence detection of the divalent nucleic acid aptamer. The method comprises the following steps: taking a bivalent nucleic acid aptamer as a template, forming a DNA silver nanocluster (DNA-AgNCs) under the action of silver nitrate and sodium borohydride, and when the target okadaic acid exists, combining the bivalent nucleic acid aptamer with the target, so that cDNA can be replaced from double-stranded DNA by the target. Exonuclease I is added to perform enzyme digestion on the cDNA and the divalent nucleic acid aptamer, so that the fluorescence intensity of the DNA-AgNCs can be rapidly reduced. The content of the okadaic acid can be judged by detecting the change of the fluorescence intensity. The invention provides a simple, convenient, rapid, high-sensitivity and high-specificity detection method for okadaic acid detection based on a divalent nucleic acid aptamer for the first time.

The present application relates to a kind of hAAG photoelectrochemical detection method based on sulfur vacancy engineering, belong to analytical detection technical field.The present application will prepare hairpin structure DNA to single-stranded DNA, sequentially join different concentrations of human alkyladenine DNA glycosylase (hAAG), endonuclease IV, exonuclease I and its reaction buffer;Dopamine solution is added to reaction, and the enzymatic reaction system of hAAG is obtained;CdZnS modified electrode is immersed in enzymatic reaction product solution and reacts, so that dopamine and zinc atom on the surface of CdZnS are combined by ortho-hydroxy coordination, and sulfur vacancy is induced to generate;Photoelectrochemical test is carried out to CdZnS modified electrode, the photocurrent signal is detected, the linear relationship between photocurrent signal and hAAG concentration is established, and the quantitative detection of hAAG is realized.The present application not only provides efficient strategy for hAAG detection, but also provides new ideas for performance optimization of sulfide-based photoelectrochemical device.

The invention provides a method for producing a partially closed deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease a

The present invention provides an RNA-based tumor vaccine, a preparation method, an immune adjuvant, a vector and an application. The tumor vaccine can specifically act on tumors with positive expression of GPC3, such as hepatocellular carcinoma, ovarian clear cell carcinoma, yolk sac carcinoma, etc. The tumor vaccine includes circular RNA capable of encoding an immunogenic polypeptide. The circular RNA is configured to be capable of specifically encoding the immunogenic polypeptide GPC3. The circ-GPC3 in the present invention has a covalently closed-loop structure that can protect it from exonuclease-mediated degradation, has a longer half-life and higher stability compared to mRNA, and can ensure the continuous expression of immunogenic proteins. By artificially modifying and introducing a ribosome entry site (IRES) modification, the present invention can increase the translation efficiency of tumor-specific antigen RNA.

The present invention provides methods of increasing plant productivity and tolerance to water and nutrient deficiency by overexpressing an exonuclease responsible for the d200 mutant. Also provided are plants which overexpress the exonuclease and have improved tolerance to water and nutrient deficiency.

The present invention discloses a composition, a kit and their applications for detecting lung cancer. The composition includes detection reagents for detecting the methylation status of one or more of 17 genes such as SIX2 and PANTR1. The detection reagents contain multiplex PCR pre-amplification primers, real-time fluorescence quantitative PCR primers and probes, and primers and probes for the internal reference ACTB, and are designed by nested PCR, and the primer sequences are SEQ ID No.1-90. The kit contains the above composition and also contains methylation-sensitive restriction enzymes and Exonuclease I. Among them, the real-time fluorescence quantitative PCR primers and probes for 17 genes and ACTB are分装 into 6 reaction tubes. The present invention combines methylation-sensitive restriction enzyme treatment, multiplex PCR pre-amplification and fluorescence quantitative PCR techniques to achieve high-sensitivity and high-specificity detection of lung cancer. The combined detection of 17 genes has an AUC of 0.91, is easy to operate, has low cost, is suitable for early screening and auxiliary diagnosis of lung cancer, and has good clinical application prospects. It should be noted that the word "分装" in the original text seems to be a misspelling. It should probably be "separately packaged" or something similar. The translation above keeps the original text as it is for that word.

The disclosed invention relates to a complex molecule comprising a linear double-stranded DNA product with exonuclease resistance due to the presence of internal protected nucleotides, preferably phos

The application belongs to the field of biosensors, and provides a construction method and application of an electrochemical biosensor for quantifying AFP-L3% (AFP-L3 / AFP), and the specific process is as follows: Au@UiO-66 nanocomposites are obtained by using a hydrothermal method and an immersion reduction method, TCEP can open S-S in alpha-fetoprotein to obtain sulfydryl (-SH) and connect to Au@UiO-66, in-situ synthesis of AFP aptamer-copper clusters (Apt-CuNCs) is carried out by using DNA as a template, Apt-CuNCs are used to recognize total AFP, the content of total AFP is determined by using the generated Cu electrochemical signal, then, the Apt-CuNCs are subjected to enzymatic cleavage and stripping by using exonuclease I, alpha-fetoprotein heterogeneity (AFP-L3) is labeled by using lentil agglutinin (LCA) functionalized silver nanoparticles LCA@AgNPs, the content of AFP-L3 is determined by using the generated Ag electrochemical signal, and AFP-L3% can be calculated by combining the data of the two, and the electrochemical biosensor constructed by the method can be used for determining AFP-L3% in human serum.

The invention discloses a TNT explosive detection method, and belongs to the technical field of explosive detection. The method comprises the following three parts: a first part of primer is reserved, a probe DNA 1 containing a TNT nucleic acid aptamer can be prevented from being degraded by exonuclease I after being combined with TNT, and the probe DNA 1 which is not combined with TNT is degraded by exonuclease I; the second part is circular amplification, the probe DNA 1 reserved in the previous step opens the DNA of the hairpin stem-loop structure, and the generation of circular amplification reaction with the DNA of the hairpin stem-loop structure as a template is promoted; in the third part, naked eye visual detection and / or ultraviolet spectrophotometry detection are / is carried out according to the G-quadruplex contained in the cyclic amplification product. The detection method provided by the invention is sensitive and high in visualization degree, in-situ detection of the TNT explosive can be realized, and the detection limit reaches the concentration level of pmol / L.

Multiplex PCR-based assays may be improved by using a load-balanced set of primers with different proportions of primers in the set to provide balanced amplicon read depth. The multiplex PCR amplification in such assays may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers.

Described herein are methods and compositions for improved polymerase chain reaction (PCR). In one aspect the methods and compositions include improved aptamers to reversibly inhibit polymerase and exonuclease activity of the polymerase enzyme.

Multiplex PCR-based assays may be improved by using a load-balanced set of primers with different proportions of primers in the set to provide balanced amplicon read depth. The multiplex PCR amplification in such assays may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers.

The present invention discloses a ratiometric aggregation-induced ECL / EC sensing platform, a preparation method thereof, and an application thereof for detecting circulating tumor DNA. The sensing platform uses TPA-BT nanoparticles (TPA-BT NPs) prepared by a nanoprecipitation method as an ECL luminophore, which is dripped onto a treated gold electrode surface for incubation, and then H1 and MCH are dripped for incubation, and a target circulation reaction solution assisted by exonuclease III (Exo III) is dripped, and the signal ratio of ECL to EC is measured after incubation. During detection, based on the EC signal of ferrocene (Fc) and the ECL signal of TPA-BT NPs, as the concentration of target ctDNA increases, the ECL signal gradually increases, while the EC signal gradually decreases, and the ECL and EC signals are compared, and are linearly correlated within the range of 100 aM-100 pM. It can be used to detect ctDNA and has the characteristics of high sensitivity.

The invention relates to a kit for detecting heart potassium ion channel coding gene idiopathic ventricular fibrillation risk gene SNP (Single Nucleotide Polymorphism) loci, which comprises 38 pairs of specific multiplex PCR (Polymerase Chain Reaction) primer pairs with 41 required SNP loci, 41 SNP single-base extension primers, and necessary PCR reaction liquid, SNaPshot reaction liquid, shrimp alkaline phosphatase and exonuclease I. The kit is used for detecting heart potassium ion channel coding gene idiopathic ventricular fibrillation risk gene SNP loci. According to the kit, the multiple PCR technology, the SNaPshot micro-sequencing technology and the capillary electrophoresis technology are combined to screen the risk gene SNP sites related to the idiopathic ventricular fibrillation on the heart potassium ion channel coding genes, the SNP related to the idiopathic ventricular fibrillation on 41 potassium ion channel genes can be detected only through one experiment, a clear conclusion can be obtained, and result interpretation is simple and objective.

Described are methods of producing closed linear DNA, comprising amplifying a template molecule to generate a double stranded DNA, wherein the double stranded DNA does not comprise a hairpin, loop or

The invention provides a method for producing a partially closed deoxyribonucleic acid (DNA) product, wherein the method comprises: (a) contacting a double-stranded DNA molecule with an endonuclease a

Described are methods of producing linear DNA products, comprising contacting a double stranded DNA with an endonuclease, ligase and first and second adaptors in a single contiguous aqueous volume, an

The disclosed invention relates to a linear double stranded DNA product with enhanced exonuclease resistance due to the presence of protected nucleotides, preferably phosphorothioated nucleotides. The

This invention discloses a method for preparing DNA samples for SNP chip detection, comprising the following steps: obtaining at least one library sequence, wherein the library sequence includes a capture sequence complementary to the target fragment of the DNA sample to be tested; amplifying the library sequence and then digesting it with enzymes to obtain a single-stranded library sequence; mixing the amplification product of the DNA sample to be tested with the single-stranded library sequence, denaturing and annealing; and digesting the product with Exonuclease I. The DNA sample prepared by this invention is more purified, with targeted enrichment of the target fragment and removal of a large number of non-target fragments. When applied to SNP chip detection, it improves the hybridization efficiency with the SNP chip, thereby increasing the detection signal value and accuracy.

Described is a linear double stranded DNA product comprising a first adaptor molecule ligated to a first end and a second adapter ligated to a second end. The double stranded DNA molecule is closed at

The invention belongs to the technical field of biological medicine, a gene editing system MMEJ-Exo is constructed through humanized application of exonuclease or key functional domains derived from microorganisms and eukaryotes, spCas9-mediated eukaryote cells are remarkably improved, especially long-fragment editing of primary neuronal cells is remarkably improved, and the gene editing efficiency is improved. And a new technology and a new method are provided for gene therapy and cell therapy transformation application.

The invention relates to an exonuclease III-based MC-LR rapid detection biosensor, a detection kit and a detection method. The exonuclease III-based MC-LR rapid detection biosensor comprises an Aptamer / Blocker compound, a hairpin probe H and exonuclease III, wherein the Aptamer / Blocker compound comprises a specific aptamer A and a blocking agent B of MC-LR, and two ends of the hairpin probe H are respectively modified by a fluorophore and a quencher. The detection kit comprises the MC-LR rapid detection biosensor based on exonuclease III and a buffer solution, wherein the buffer solution comprises a PBS buffer solution and an exonuclease III buffer solution. The exonuclease Exo III with the MC-LR aptamer as the recognition probe is used for assisting in catalyzing target recovery, operation is easy and convenient, sensitivity is high, and good recovery rate and accuracy are achieved in an actual water sample.

The invention provides a DNA probe system and method for aptamer-driven circulating detection of kanamycin based on G-tetramer response, belongs to the technical field of biological analysis and detection, and aims to realize target signal amplification detection by utilizing specific recognition of Aptamer and probe recycling started by exonuclease III in combination with a toehold-mediated specific strand displacement reaction. The split G tetramer is used as a chimeric receptor for specifically lightening ThT signal molecules, and signal transduction cannot be realized when no target activation cycle generates RP for downstream strand displacement reaction, so that background signals can be remarkably reduced, and the signal-to-multiple ratio can be improved. Meanwhile, a ThT label-free signal output model is constructed by adopting split type G-Quadruplex, so that the specific and high-sensitivity detection of the target Kana is realized.

The invention belongs to the technical field of medicines, and particularly relates to application of an NSP14 small-molecule inhibitor in preparation of a medicine for preventing and treating novel coronavirus infection. The invention provides application of an NSP14 small-molecule inhibitor in preparation of a medicine for preventing and treating novel coronavirus infection. The NSP14 small-molecule inhibitor comprises at least one of DEL1 as shown in a structural formula I, DEL2 as shown in a structural formula II and DEL3 as shown in a structural formula III. The NSP14 small-molecule inhibitor can effectively inhibit the exonuclease activity of novel coronavirus NSP14 protein, blocks a virus RNA processing mechanism on the molecular level, shows a remarkable antiviral effect, and is beneficial to being widely applied to preparation of drugs for preventing and treating novel coronavirus infection.

The disclosed invention relates to a complex molecule comprising a linear double-stranded DNA product with exonuclease resistance due to the presence of internal protected nucleotides, preferably phos

The invention relates to nucleic acid constructs which comprises a DNA molecule encoding a recombinant L or S segment of a Mopeia virus (MOPV), as well as an expression cassette or a vector comprising them, an eukaryotic cell comprising them, and a method of expressing one or more polypeptide(s) or protein(s) that is(are) heterologous to Mopeia virus (MOPV) or fragments thereof and / or producing recombinant live attenuated Mopeia virus (MOPV) expressing one or more polypeptide(s) or protein(s) that is(are) heterologous to MOPV or fragments thereof, in a eukaryotic host cell, using in particular the nucleic acid constructs of the invention. The invention also relates to a composition comprising viral particles obtained from such a method, a viral particle obtained from such a method and a set of nucleic acid constructs or expression cassettes or vectors comprising them, wherein the set comprises at least one nucleic acid construct or expression cassette or vector comprising it, which encodes a recombinant L segment of a Mopeia virus (MOPV), and one nucleic acid construct or expression cassette or vector comprising it, which encodes a recombinant S segment of a Mopeia virus (MOPV) that encodes a MOPV nucleoprotein (NP) having attenuated exonuclease activity, wherein one or both nucleic acid constructs or expression cassettes or vectors comprise(s) a polynucleotide encoding a polypeptide or a protein that is heterologous to MOPV and the latter is different from a non-MOPV GPC when on the S segment, and wherein the positions where the polynucleotide encoding at least one polypeptide or protein that is heterologous to MOPV is inserted in the said nucleic acid constructs or expression cassettes or vectors are peculiar.

The invention belongs to the field of biosensors, and provides a construction method and application of an electrochemical biosensor for quantifying AFP-L3% (AFP-L3 / AFP), and the specific process is as follows: an Au-coated UiO-66 nano composite material is obtained by using a hydrothermal method and an impregnation reduction method, TCEP can open S-S in alpha fetoprotein to obtain sulfydryl (-SH), the sulfydryl (-SH) is connected to the Au-coated UiO-66, and then the Au-coated UiO-66 nano composite material is obtained. The method comprises the following steps: by taking DNA as a template, carrying out in-situ synthesis of an AFP aptamer-copper cluster (Apt-CuNCs), identifying total type AFP by using the Apt-CuNCs, determining the content of the total type AFP by using a generated Cu electrochemical signal, then carrying out enzyme digestion stripping on the Apt-CuNCs by using exonuclease I, marking an alpha fetoprotein variant (AFP-L3) by using lentil lectin (LCA) functionalized silver nanoparticles LCA-AgNPs, and determining the content of subtype AFP-L3 by using a generated Ag electrochemical signal. The AFP-L3% can be calculated through the combination of the two data, and the electrochemical biosensor constructed by the method can be used for determining the AFP-L3% in human serum.

Disclosed is a method for removing extracellular antibiotic resistance genes in water, which belongs to the field of environmental management. The method comprises adding a cell lysate, especially a cell lysate of E. coli, to water, wherein the cell lysate comprises various enzymes such as ribonucleotide exonuclease I, ribonucleotide exonuclease III, and DNA topoisomerase I, and can degrade extracellular antibiotic resistance genes. In addition, in view of the problems of poor stability of the cell lysate in an aqueous solution and the enzymes in the cell lysate being easily deactived due to complicated water ingredients, the present invention provides a cell lysate immobilized with a polyacrylamide (PAM) hydrogel, such that extracellular antibiotic resistance genes in sewage can be effectively reduced and the cell lysate can be reused by means of protecting the cell lysate.