196 results about "Exonuclease" patented technology

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 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 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.

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.

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 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.

The present application relates to nucleic acid molecules for CRISPR cascade nucleic acid detection system and application, and relates to the field of biological detection. The nucleic acid molecules comprise a polymerase binding leading region with a DNA polymerase binding site, a trigger nuclease cleavage region with a trans-cleavage endonuclease binding cleavage site, and an exonuclease polymerization blocking region with a modified 3' end; after the trigger nuclease cleavage region is cleaved, the polymerase binding leading region and the exonuclease polymerization blocking region are connected, the DNA polymerase bound to the polymerase binding leading region can extend the exonuclease polymerization blocking region from the 5' end to the 3' end, and a target sequence that can be recognized by the reporter sgRNA of the CRISPR reporter system is synthesized. The CRISPR cascade nucleic acid detection system constructed by using the nucleic acid molecules can realize two-stage cyclic amplification reaction, and improve the reporting speed, reporting intensity and reporting sensitivity of the CRISPR reporter system.

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 application discloses a hairpin probe for detecting DNA polymerase exonuclease activity and a preparation method and application thereof. The hairpin probe comprises a region a, a region b, a region c, a region d, a region e, a region f and a region g from the 5' end in sequence, wherein the region a and the region c are reversely complementary to each other, the 5' end of the region a is marked with a first fluorescent group, and the 3' end of the region a is marked with a first quenching group; the region e and the region g are reversely complementary to each other, the 5' end of the region g is marked with a second quenching group, and the 3' end of the region g is marked with a second fluorescent group; the first fluorescent group and the second fluorescent group are different from each other. The hairpin probe can be used for detecting the 3'-5' and 5'-3' exonuclease activity of the DNA polymerase separately or simultaneously in a single system, and has the advantages of simple operation steps, high throughput, good specificity, avoidance of radioactive pollution and excellent application prospect.

The application provides a composition, a kit and a method for human cytomegalovirus detection, and relates to the technical field of molecular diagnosis.The composition comprises at least one set of human cytomegalovirus detection primer probe groups and multiplex DNA polymerase, wherein the primer probe groups comprise oligonucleotide upstream external primers, downstream external primers, upstream internal primers, downstream internal primers, upstream loop primers and downstream loop primers; the probe is a neck ring structure probe, and the multiplex DNA polymerase comprises strand displacement DNA polymerase and DNA polymerase with 5' end to 3' end exonuclease activity.The composition for human cytomegalovirus detection alleviates the technical problems that the LAMP amplification scheme in the prior art is poor in specificity and cannot realize single-tube multiplex detection.

The present disclosure relates to novel methods and compositions for a measuring exonuclease enzyme activity. In particular, the methods entail contacting a sample with a fluorescently-labeled substrate to create a test mixture, incubating the test mixture for a time sufficient for cleavage of the substrate, and measuring the fluorescence signal from the test reaction mixture. In certain aspects, the methods disclosed herein are useful for identifying and / or assessing a modulator of an exonuclease that may be used either alone or in combination with other compounds. The present disclosure also relates to fluorescently-labeled double-stranded nucleic acid compositions useful in the practice of such methods and, still further, to kits for performing the methods of the disclosure. The present disclosure further relates to compounds and methods useful for treating a viral infection.

The invention relates to the technical field of biological medicine, and particularly discloses synthesis and application of oligonucleotide with threose nucleic acid 3-oxygen at the tail end indirectly connected with thiophosphoric acid. The chemical modification strategy comprises the following steps: O < 3->-oxygen of threose nucleic acid is connected with thiophosphoric acid through a spacer group, and O < 2->-hydroxyl is combined with a nucleotide monomer of phosphoramidite; carrying out solid-phase synthesis on the threose nucleic acid O2 '-phosphoramidite monomer to construct an oligonucleotide molecule; the oxygen of the threose nucleic acid at the terminal of the oligonucleotide is linked to the thiophosphoric acid through a spacer group, and since the type of phosphonic acid does not belong to a substrate of phosphatase, the modified oligonucleotide can resist exonuclease degradation and improve its in vivo biological activity.

The present invention provides, among other things, linear mRNA compositions comprising a poly A tail and a stem loop structure downstream to the poly A tail. Also provided herein are methods of producing mRNA by ligating a stem loop exonuclease blocker using a double-stranded RNA ligase.

The invention provides an exonuclease gene editor and a gene editing system and application thereof, and particularly provides an exonuclease editor formed by fusing exonuclease and Cas9 or nicking enzyme, the exonuclease editor has very good gene editing activity, and compared with Cas9 nuclease, the exonuclease editor can remarkably improve accurate gene editing efficiency. The exonuclease editor provided by the invention can also be used for accurately deleting long-fragment genes.