265 results about "Exonuclease" patented technology

The invention discloses an optimized NlovFz2-omega RNA (Ribonucleic Acid) editing system, the optimal NlovFz2 system enNlovFz2 is obtained by modifying NlovFz2 protein and an omega RNA skeleton, and on the basis of remarkably improving the editing efficiency compared with a parent NlovFz2 system, the NlovFz2 mutant protein and T5 exonuclease are fused to obtain an enNlovFz2-T5 system with higher editing efficiency. The method has important significance and value for promoting wide application of a gene editing technology in the aspects of functional mechanism research and clinical treatment.

Disclosed herein is a composition for nuclease treatment of hybridized probes in experiments to profile and analyze biological samples. Also disclosed herein, is a kit for nuclease treatment of hybridized probes in experiments to profile and analyze biological samples. Also disclosed herein, is a method for nuclease treatment of hybridized probes in experiments to profile and analyze biological samples.

The invention relates to a T7RNA polymerase mutant for synthesizing single-stranded DNA as well as a screening method and application of the T7RNA polymerase mutant, and belongs to the technical field of enzyme engineering. The invention provides a method for directed evolution and screening of a T7RNA polymerase mutant, which comprises the following steps: knocking out exonuclease and mismatch repair protein from a chassis strain genome, knocking in single-stranded annealing protein, resistance gene containing deletion mutation and a single-stranded DNA synthesis template, and constructing to obtain a genetically engineered bacterium for screening; a T7RNA polymerase mutant library is constructed, recombinant plasmids with the mutant library are transferred into engineering bacteria, T7RNA polymerase mutants synthesize single-stranded DNA to repair deletion mutation of resistance genes, the transformed engineering bacteria can grow on a resistance plate, the T7RNA polymerase mutants obtained through multiple rounds of screening can efficiently synthesize the single-stranded DNA through in-vitro verification, and the T7RNA polymerase mutant can be used for preparing the single-stranded DNA. A brand new tool is provided for synthesis of single-stranded DNA, and the method has the potential of being applied to the fields of gene editing, evolutionary engineering and the like.

The invention discloses a circulating tumor DNA biosensor and a construction method and application thereof, and belongs to the technical field of biosensors. The dual-mode ratio type circulating tumor DNA biosensor comprises a sensing substrate assembled with a triangular DNA probe, a probe solution and exonuclease. The triangular DNA probe is used as an anchoring substrate to support target cycle amplification, and the sensitivity of the biosensor is effectively improved by using an ECL-RET effect and an enzyme-assisted target cycle signal amplification strategy; and the FL / ECL ratio is used as a dual-mode signal output strategy, so that the accuracy and feasibility of the detection technology are improved. The biosensor is high in accuracy, high in sensitivity and good in selectivity, quantitative detection of PIK3CA E545K ctDNA in blood of a breast cancer patient can be achieved, and the biosensor has important application value in the fields of clinical diagnosis and drug research of breast cancer.

The invention relates to the technical field of gene editing, in particular to a Cas9 protein mutant and application thereof. According to the present invention, the mutation is introduced at the position far away from the catalytic site to induce the Cas9 protein conformation change so as to obtain the new Cas9 protein mutant, only the Cas9-sgRNA target chain is cut, and the Cas9-sgRNA non-target chain is not cut, such that the new Cas9 protein mutant can be used for constructing the novel base editor, and the high-efficiency base editing can be performed on the mammalian cell genome. The Cas9 protein mutant disclosed by the invention is fused with a mutant TX with DNA binding deletion of 3 '-> 5' exonuclease TREX2, so that the gene editing efficiency of a 3'terminal generated by paired single-nick enzyme cutting can be greatly improved, and the safety of the Cas9 protein mutant is maintained.

The invention discloses a Taq DNA polymerase mutant with improved excision activity and application thereof, and belongs to the technical field of enzyme engineering. The invention aims to solve the technical problems of low probe cutting efficiency, weak fluorescence signal, limited detection sensitivity (especially for low-abundance targets), low reaction speed (high Ct value) and reduced specificity caused by insufficient 5 '-> 3' excision enzyme activity of wild Taq DNA polymerase in TaqMan probe method qPCR (quantitative polymerase chain reaction). According to the technical scheme, the Taq DNA polymerase mutant with the high 5 '-3' exonuclease activity is provided, the amino acid sequence of the Taq DNA polymerase mutant is shown as SEQ ID NO.2, the exonuclease activity of the mutant is improved by 77%, the cutting speed of a TaqMan probe is greatly increased, accumulation of fluorescence signals is accelerated, the Ct value is reduced, and the detection time is remarkably shortened.

The invention relates to an efficient homologous recombination engineering bacterium and a method for improving gene editing efficiency. The genome editing work is limited by the homologous recombination efficiency of the host bacteria, so that the engineering bacteria with higher homologous recombination efficiency are of great significance for improving the efficiency of the genome editing work. In order to achieve the purpose, exonuclease VII of host bacteria is modified, an xseA subunit in double subunits is knocked out, meanwhile, an xseB subunit is overexpressed, the dsDNA recombination mediated single region editing efficiency can be improved by 220%, the bacteria homologous recombination efficiency is remarkably improved, and universality and convenience are achieved.

The invention provides a method for constructing a sequencing library, the sequencing library, a sequencing method and a kit for constructing the sequencing library. The method for constructing the sequencing library comprises the following steps: performing digestion treatment on a to-be-detected sample connected with a joint under the action of exonuclease, so as to obtain a target sequencing library.

Provided herein are products and processes for primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested. Also provided herein are products and processes for the detection of the presence, absence or amount of one or more variants of a target nucleic acid species or a plurality of target nucleic acid species comprising primer extension reactions using chain terminating reagents and enzymes having 3′ to 5′ exonuclease activity, whereby unextended oligonucleotides are digested and extended oligonucleotides are not digested.

The invention provides a method for removing a non-target nucleic acid template. According to the method provided by the invention, on the basis of UDG / AAG + AP site endonuclease, incision site-dependent endonuclease or 5 '-phosphoric acid-dependent exonuclease is further added, and a non-target nucleic acid template with dU / dI is subjected to digestion treatment, so that fragments cut by glycosylase can be prevented from being repaired into a complete template in a positive and negative chain mutual overlapping manner; amplification of a non-target nucleic acid template is caused. Compared with a traditional anti-pollution system, the method provided by the invention has the advantages that polluted non-target amplified nucleic acid fragments can be thoroughly removed, the detection rate of the polluted fragments is effectively reduced, and false positive is reduced.

The invention belongs to the field of gene engineering, and particularly relates to a novel recombination system in aeromonas and application of the novel recombination system. A novel recombination system in the aeromonas disclosed by the invention contains a homologous recombination system expression vector, the expression vector expresses an operon under the control of an inducible promoter, and the operon contains three genes of a coding host specific exonuclease RedAahy, a single-stranded DNA annealing protein RedBahy and a single-stranded DNA binding protein RedSahy; the operon is derived from an aeromonas hydrophila (Aeromonas hydrophila) plasmid pR148, and is characterized in that the operon is derived from the aeromonas hydrophila (Aeromonas hydrophila) plasmid pR148. By utilizing the recombination system, efficient, simple and convenient gene insertion and deletion of aeromonas are successfully realized, and the application prospect is wide.

The present invention relates to methods for producing a protected deoxyribonucleic acid (DNA) product with enhanced resistance to nuclease digestion. The methods comprise digesting a double-stranded DNA molecule with an endonuclease to generate a digested double-stranded DNA molecule comprising a first strand and a second strand, wherein the first strand has a 5' overhang; and extending the second strand 5' to 3' using a polymerase in the presence of one or more nuclease-resistant deoxynucleotide triphosphates to generate the protected DNA product, wherein the protected DNA product comprises an extended second strand comprising at least one nuclease-resistant nucleotide incorporated into the extended second strand by the polymerase using the 5' overhang of the first strand as template. Also provided are protected DNA products and uses thereof.

The invention relates to a cell-free method for synthesizing bacteriophages, comprising: (a) in vitro assembling overlapping DNA fragments into a phage genome, in a ligase-free assembling reaction, comprising: (a1) digesting the overlapping DNA fragments by an exonuclease, and (a2) annealing the overlapping DNA fragments to assemble a phage genome, and (b) cell-free transcribing and translating the phage genome. The invention also relates to phages synthesized by the method and to their applications.

The invention discloses a single molecule detection kit for a plurality of non-coding RNAs (Ribonucleic Acid) in human breast tissues by using a programmable automatic cascade mechanism and application of the single molecule detection kit. The preparation method of the single molecule detection kit comprises the following steps: incubating piRNA-36026, lncRNA-DSCAM-AS1, a dumbbell probe-pi, a dumbbell probe-lnc, a primer-pi, a primer-lnc, a dNTP mixed solution, a ThermoPol reaction buffer solution and a reaction solution of Bst DNA polymerase (large fragment); and adding the reaction product into a reaction solution containing the signal probe 1, the signal probe 2, an NEBuffer 4 reaction buffer solution and T7 exonuclease, and incubating to obtain the single molecule detection kit. According to the present invention, the efficient automatic cascade mechanism and the high signal-to-noise ratio single molecule detection are adopted to achieve the sensitive simultaneous detection of a variety of ncRNAs, and the high sensitivity, the good selectivity and the multi-path analysis ability are provided.

The invention is entitled "Formation of Synthons". The present disclosure provides, among other things, a composition comprising: a 5'-3' exonuclease; a strand displacement polymerase; and optionally, a single-stranded DNA binding protein and / or a ligase. Also described are polynucleotide assembly methods for forming synthons and kits for performing the methods.

Disclosed is a DNA assembly mix, comprising a 3′-5′ exonuclease enzyme which is XthA; and a buffer. Also disclosed is a DNA assembly mix, comprising a polymerase and ligase free composition comprising a 3′-5′ exonuclease enzyme; and a buffer. Also disclosed is a method of assembling a plurality of DNA fragments, comprising: (a) mixing the plurality of DNA fragments with the DNA assembly mix as disclosed herein; and (b) incubating the mixture from step (a) at a temperature for a period of time suitable for assembling the plurality of DNA fragments. Further disclosed is use of the DNA assembly mix as disclosed herein in high-throughput DNA assembly, wherein the DNA assembly mix is used in a microfluidic platform to assemble DNA.

Environmental and water pollution caused by highly toxic heavy metals has always been a serious problem facing mankind. 3+ , Pb 2+ and Hg 2+ As are highly toxic and widely distributed environmental pollutants that pose a serious threat to the environment and humans even at low concentrations. The World Health Organization has set strict limits on drinking water standards. Therefore, it is necessary to establish an efficient and sensitive method for simultaneously detecting As 3+ , Pb 2+ and Hg 2+ The analysis method is crucial for environmental monitoring, food safety and other fields. The present invention proposes a nanocapsule-nucleic acid molecule complex based on nuclease cyclic amplification technology and molecular recognition technology and a preparation method thereof. The use of the complex can not only achieve As 3+ , Pb 2+ and Hg 2+ Simultaneous detection is achieved, and the detection sensitivity is significantly improved while ensuring high specificity. At the same time, a wider linear detection range is obtained.

The present invention discloses a circular gRNA and its related biomaterials and applications. The coding gene of the circular gRNA includes a 3' intron, a 3' splice site, a gRNA coding gene, a 5' splice site and a 5' intron in sequence, wherein the 3' splice site sequence is as shown in positions 2810-2814 of SEQ ID No.1, and the 5' splice site sequence is as shown in positions 2917-2930 of SEQ ID No.1. The circular gRNA is not attacked by nucleases, thereby improving the stability of the CRISPR / cas9 system.

The invention relates to the technical field of biological medicines, and particularly discloses synthesis and application of oligonucleotide with a spacer group embedded between 5-terminal oxygen and thiophosphoric acid. The chemical modification strategy comprises the following steps: preparing a modified nucleotide phosphoramidite monomer in which a spacer group is embedded between O5'and thiophosphoric acid P (V); performing solid-phase synthesis on the modified nucleotide phosphoramidite monomer to construct a target oligonucleotide molecule; a spacer group is embedded between oxygen at the 5 '-terminal of the oligonucleotide and thiophosphoric acid P (V) to form a 5'-terminal thiophosphoric acid structure; as the terminal modified thiophosphoric acid belongs to a non-phosphatase substrate, the terminal modified thiophosphoric acid can resist exonuclease degradation and improve the biological activity of siRNA after being modified.

Provided are a method for constructing a library based on an RNA sample and uses thereof. The method includes: step 1 of subjecting the RNA sample to a reverse transcription reaction to obtain DNA-RNA hybrid strands; step 2 of performing reaction of the DNA-RNA hybrid strands with an endoribonuclease, a first DNA polymerase, a second DNA polymerase, and dATPs to obtain a double-stranded DNA added with dA-tail, where the first DNA polymerase has a 5′-3′ exonuclease activity and a 3′-5′ exonuclease activity, and the second DNA polymerase has no 3′-5′ exonuclease activity; step 3 of ligating the double-stranded DNA added with dA-tail and a sequencing adaptor to obtain a ligated product; and step 4 of subjecting the ligated product to PCR amplification to obtain a sequencing library.

A method of sequencing a nucleic acid characterised by the steps of (1) generating a stream of single nucleoside triphosphates by progressive enzymatic digestion of the nucleic acid; (2) producing at least one oligonucleotide used probe by reacting, in the presence of a polymerase, at least one of the single nucleoside triphosphates with a corresponding biological probe comprising (a) a first single-stranded oligonucleotide including an exonuclease blocking-site, a restriction endonuclease recognition-site located on the 5' side of the blocking-site and including a single nucleotide capture-site for capturing the single nucleoside triphosphate, and at least one fluorophore region located on the 5' side of the recognition-site arranged so as to render the fluorophore(s) quenched and (b) a second and optionally a third single-stranded oligonucleotide each separate from the first oligonucleotide and capable of hybridising to complementary regions on the first oligonucleotide flanking the 3' and 5' sides of the capture-site; (3) cleaving the first oligonucleotide strand of the used probe at the recognition-site with a restriction endonuclease to create a first oligonucleotide component bearing the fluorophores; (4) digesting the first oligonucleotide component with an enzyme having 3'-5' exonucleolytic activity to yield fluorophores in a detectable state and (5) detecting the fluorophores released in step (4). A corresponding method for use with enzymes having 5'-3' exonucleolytic activity as well as new biological probes and a kit derived therefrom is also provided.

The present invention provides a method of stabilizing a phi29 DNA polymerase by contacting the phi29 DNA polymerase with a stabilized oligonucleotide that is free from degradation by a 3'exonuclease. The phi29 polymerase exhibits improved temperature stability in the presence thereof compared to the absence of the stabilized oligonucleotide. The method involves preparing a composition comprising a phi29 DNA polymerase and a stabilized oligonucleotide comprising one or more modified nucleotides. The compositions can be used in methods for performing polymerase reactions, nucleic acid replication, and detection of a target nucleic acid or target analyte in a sample. The compositions are particularly useful in rolling circle amplification reactions in which the targets are produced by proximity ligation assay, in particular cyclized lock probes.

Provided are compositions comprising recombinant DNA polymerases that include amino acid substitutions, insertions, deletions, and / or exogenous features that confer modified properties upon the polymerase for enhanced single molecule sequencing. Such properties can include enhanced metal ion coordination, reduced exonuclease activity, reduced reaction rates at one or more steps of the polymerase kinetic cycle, decreased branching fraction, altered cofactor selectivity, increased yield, increased thermostability, increased accuracy, increased speed, increased readlength, and the like. Also provided are nucleic acids which encode the polymerases with the aforementioned phenotypes, as well as methods of using such polymerases to make a DNA or to sequence a DNA template.

The invention discloses a preparation method of an RCA-Cas12a-DNA (deoxyribonucleic acid) tetrahedron sensor for detecting vancomycin in blood serum. The sensor comprises a target object vancomycin, a DNA chain TSP1, a DNA chain TSP2, a DNA chain TSP3 and a vancomycin aptamer Aptamer which are used for preparing a DNA tetrahedron, a DNA chain T, a DNA chain S, a T4 DNA ligase, exonuclease I and exonuclease III which are used for preparing circular DNA, a DNA chain P and phi29 DNA polymerase which are used for carrying out rolling circle amplification reaction, and LbCas12a nuclease, crRNA and ssDNA-FQ probes which are used for preparing a reporter. The aptamer is specifically combined with a target object, a non-nucleic acid target is converted into nucleic acid detection, and the application range of RCA and Cas12a is expanded; rCA is combined with Cas12a, so that a signal is amplified twice, the detection limit is lower, and the sensitivity is higher; compared with a traditional detection method, expensive instruments and equipment, complex and time-consuming sample pretreatment and skilled operators are not needed, and the method is more convenient and economical.

The application discloses a TdT signal amplification PAM-independent Cas12a lateral chromatography nucleic acid detection method and application. The method comprises the following steps: performing a first reaction on a first reaction system comprising a to-be-detected nucleic acid, a recombinase polymerase amplification reaction component, a recombinase polymerase amplification primer pair and an exonuclease, so as to obtain a single-stranded target nucleic acid; performing a second reaction on a second reaction system comprising the single-stranded target nucleic acid, a CRISPR / Cas12a reaction component, a reporter probe, a terminal deoxynucleotidyl transferase and a deoxynucleotide triphosphate substrate, so as to obtain an extension product; and contacting and hybridizing the extension product with a gold nanoparticle-labeled detection probe in a lateral chromatography process, and when a visual detection signal is obtained, it is judged that the to-be-detected nucleic acid is a target nucleic acid. The method of the application can significantly improve the detection sensitivity and signal contrast while getting rid of the PAM restriction, the detection process does not need complex instruments, the result can be judged by naked eyes, and the method is suitable for on-site rapid visual detection of pathogenic microorganism nucleic acid.

The invention relates to the technical field of gene engineering, and particularly discloses a chitinase truncated body, a recombinant expression vector thereof, and a construction method and application of engineering bacteria. The chitinase truncated body is Cq181NC, the amino acid sequence of the Cq181NC is as shown in SEQ ID NO: 1, and the nucleotide sequence for coding the chitinase is as shown in SEQ ID NO: 2. According to determination, the chitinase truncation provided by the invention has the advantages that the specific enzyme activity is obviously improved by 1.8 times compared with that of a wild type, the chitinase truncation is high in environmental change tolerance, and the thermal stability of the chitinase truncation is still 1.43 times that of the wild type (incubation is performed for 8 hours at 35 DEG C). More importantly, the spectrum of the enzymolysis product is changed: the wild type is mainly disaccharide and shows a typical excision enzyme mode; and the truncated Cq181NC generates a plurality of oligosaccharides including monosaccharide, disaccharide, trisaccharide and hexasaccharide, shows double catalytic characteristics of endonuclease and exonuclease, and has a better application prospect.

The invention discloses algin lyase Alg0392, DNA, recombinant plasmid, recombinant bacteria, application and an algin cracking preparation, and relates to the technical field of microorganisms, the amino acid sequence of the algin lyase Alg0392 is shown as SEQ ID No.1, the algin lyase Alg0392 has endonuclease activity and exonuclease activity, can degrade sodium alginate into monosaccharide, disaccharide and trisaccharide, and the algin lyase Alg0392 can be used for preparing the algin lyase Alg0392. Meanwhile, the method has substrate specificity on polymannuronic acid, and sodium alginate can be well and thoroughly degraded. In addition, dithiothreitol and beta-mercaptoethanol have a promoting effect on the enzyme activity of alginate lyase Alg0392, and can be prepared into an alginate lysing preparation to improve the degradation efficiency of an alginate substrate.

This invention discloses a method for producing single-stranded DNA using Cas nickase. This method solves the problem of limitations on the enzyme cleavage sites when using nicking endonucleases, and improves the nicking efficiency of plasmids by performing multiple nicking using multiple gRNAs in a single enzyme cleavage system, while also effectively improving the efficiency of subsequent digestion by exonucleases.

The invention provides a plasmid and application thereof. The plasmid comprises a recognition site of a first endonuclease, a target gene and a recognition site of a second endonuclease from a 5 '-3' direction. According to the invention, the plasmid is amplified by using a rolling circle amplification method, the rolling circle amplification product is cleaved by using the first endonuclease and the second endonuclease, the enzyme cleaved product is digested by using exonuclease, and the product is further purified by using oligonucleotide. The obtained product can be used for in vitro transcription or directly applied to eukaryotic expression. According to the scheme provided by the invention, a simple, high-yield, economic, rapid and amplifiable preparation process of the full chemical synthesis DNA template for escherichia coli fermentation is established.

The invention relates to a CRISPR-Cas12-Cas3 gene editing system. The CRISPR-Cas12-Cas3 gene editing system comprises Cas12 nuclease, a coding gene of the Cas12 nuclease or an expression vector of the Cas12 nuclease; crRNA, a coding gene thereof or an expression vector thereof; and Cas3 nuclease, and a coding gene or an expression vector thereof. The system is characterized in that Cas12 only cuts a non-target chain to generate a specific single-chain gap, and accurate starting of Cas3 process excision activity is realized through direct compounding with Cas3, so that directional large fragment deletion is induced under the condition that double-chain breakage is not generated. Compared with a traditional I-type CRISPR system depending on a Cascade complex, the system has the advantages that the components are obviously simplified, and the operation is more convenient. Compared with an editor composed of single Cas12, the method can be used for deleting long-fragment genes and other modifications.