102 results about "Exonuclease" patented technology

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 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.

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.

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 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 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 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.

Multiplex PCR amplification may be improved by using primers with exonuclease-resistant bonds or nucleotides to reduce the formation of primer dimers. 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.

This invention discloses a magnetic sensor device based on DNA binding force and its application in bacterial detection. This invention relates to the field of magnetic sensing technology, specifically to a magnetic sensor device based on DNA binding force and its application in bacterial detection. The device for biological sample analysis of this invention includes a magnetic sensor, a DNA walking strand (WS), a nucleic acid aptamer specific to the biological sample to be detected, and an exonuclease III. The magnetic sensor is an array containing N types of double-stranded DNA molecules named TP-AP-FP immobilized on a substrate and a magnetic solid modified with streptavidin. When used for bacterial detection, this invention eliminates the need for bacterial labeling and bacterial nucleic acid extraction, allowing direct interaction with the bacterial surface. It is simple to operate, saves time, has high accuracy, can construct magnetic sensors for detecting different targets, and is versatile with a wide detection range.

Disclosed is an OTA analysis method based on a high-efficiency homogeneous electrochemiluminescence aptamer sensor, which comprises the following steps: preparing a mixed solution of L / S*-Ru, IA / B, hairpin probe H1, hairpin probe H2 and exonuclease Exo-III; putting OTA into the mixed solution for reaction to obtain a reaction solution; pouring the reaction solution into a solution containing 0.02M TPrA to obtain a test solution; and immersing an electrode in the test solution for ECL detection and reading the detection result. The analysis method has the advantages of high stability, high sensitivity and good selectivity, and can be used for detecting other toxins, proteins and other biomolecules by changing the aptamer sequence and blocking chain.

The invention discloses a single cell whole genome methylation and transcriptome biomics library construction method, and relates to the technical field of sequencing methods.The method comprises the steps that a single cell is added into a sorting tube with a preset lysis buffer for lysis, a nucleophilic coating is arranged on the inner wall of the sorting tube to enable cell nuclei to be adsorbed, and supernate containing mRNA is obtained; carrying out reverse transcription on supernate to obtain cDNA (complementary deoxyribonucleic acid) and constructing a transcriptome library; splitting the cell nucleus to release DNA (Deoxyribonucleic Acid) and carrying out hydrosulfite conversion; the transformed DNA is connected with a molecular tag-containing joint to obtain a methylated intermediate product, after exonuclease treatment, the intermediate products with different molecular tags are combined, and the methylated library is obtained through magnetic bead purification, single chain, connection with a sequencing joint at the other end, amplification and purification. According to the technical scheme, RNA and DNA are separated in the same tube and a library is established in parallel, separation and transfer steps are reduced, RNA degradation and nucleic acid loss are reduced, and methylation library establishment flux and biomics data consistency are improved.

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

The application discloses a method for replacing high-temperature denaturation to produce single strands in a multiplex PCR detection reaction and a method for improving the sensitivity and accuracy of liquid-phase hybridization detection of multiplex PCR products and a matching kit. The method mainly comprises the following steps: a 5' end of a primer is designed and modified to have a phosphate group, so that a PCR product with a phosphorylated end of one strand is amplified; and the PCR amplification product is subjected to digestion treatment by using Lambda exonuclease, so that the DNA strand with the phosphorylated end is completely removed, and a single strand is obtained. The method for replacing high-temperature denaturation to produce single strands in the ordinary multiplex PCR detection reaction eliminates the negative influence of DNA recombination on hybridization of a probe and a target sequence, improves the hybridization efficiency, and improves the accuracy and sensitivity of a liquid chip technology platform in application of molecular detection.

The invention provides a method for improving the recombination efficiency of denitrifying hyphomicrobe and application, and belongs to the technical field of gene engineering. On the basis of a pK18mobsacB-Tc vector, a vector capable of simultaneously expressing exonuclease-recombinase and upstream and downstream homologous arms of a target gene is constructed, the vector is transformed into denitrifying hyphomicrobe for homologous recombination, mediation of other plasmids is not needed, traceless knockout of the target gene can be completed only through one round of transformation, and the method is simple and convenient to operate. Compared with existing gene knockout plasmids and transformation methods of denitrifying hyphomicrobe, the method is more efficient. Experiments prove that when the vector is used for constructing a denitrifying hyphomicrobium engineering strain, compared with plasmids without recombinase, the number of recombinants is remarkably increased, and the vector has the characteristics of simplicity, convenience and high efficiency.

The present invention provides a polymerase mutant applicable to single-molecule sequencing, which has weakened 3'→5' exonuclease activity relative to a wild-type polymerase, while substantially maintaining or even improving the extension processivity, sequencing accuracy, and read length. The present invention also provides use of the polymerase mutant in single-molecule sequencing.

Linear DNA with improved resistance against exonucleases and methods for its production. The present invention relates to methods for producing a linear deoxyribonucleic acid (DNA) product (e.g., a closed linear DNA product) with improved resistance to nuclease digestion. The present invention relates to methods comprising the steps of: (a) contacting a double-stranded DNA molecule with an endonuclease, a ligase, and first and second terminal adaptor molecules and intermediate adaptor molecules to form a single contiguous aqueous volume; and (b) incubating the single contiguous aqueous volume to generate a linear DNA product (e.g., a closed linear DNA product). The present invention also relates to linear deoxyribonucleic acid (DNA) products (e.g., a closed linear DNA product) with improved resistance to nuclease digestion and uses thereof.

Compositions and methods for enriching DNA from any loci of interest are provided. Endonuclease(s) such as RNA-guided nucleases Cpf1 / Cas9 can cut the flanking regions of the gene of interest in, for example genomic DNA, followed by ligation of loop adapters. The loop adapters protect the gene of interest for subsequent digestion with one or more exonucleases. Multiple genes of interest can be digested and ligated with loop adapters at the same time. The undigested target sequence can be further purified by, for example, gel extraction using, for example, a commercial DNA purification kit. The enriched DNA can be used for sequencing.

The present invention relates to enzymes having DNA polymerase and 3′-5′ exonuclease activities. In particular, the present invention relates to a heat labile enzyme possessing a DNA polymerase II activity and a 3′-5′ exonuclease activity of marine origin. Furthermore, the present invention relates to a DNA polymerase primarily exerting a 3′-5′ activity, i.e. where the polymerase activity is absent. The present invention furthermore relates to the use of the exonuclease activity to degrade the 3′-5′ strand of double stranded DNA to perform single stranded overhang, e.g. in recombinant cloning processes, or in processes for removal of contaminating nucleic acid molecules.

The present invention relates to methods and compositions for preparing a sample enriched for single stranded nucleic acids. The single stranded nucleic acids find use as templates for synthesizing Xpandomer copies of the nucleic acids. The Xpandomer copies may be passed through a nanopore sensor to determine the nucleic acid sequence of the nucleic acids. The methods include asymmetric PCR, amplicon immobilization melt, exonuclease-mediated partial or complete digestion of one or both strand of an amplicon and linear amplification.

The invention relates to the technical field of biological medicine, and particularly discloses synthesis and application of oligonucleotide with a spacer group embedded in C4-oxygen and thiophosphoric acid at the tail end. The chemical modification strategy comprises the following steps: connecting C4-oxygen of nucleotide with thiophosphoric acid through a spacer group Y, and combining O3-hydroxyl with a nucleotide monomer of phosphoramidite; carrying out solid-phase synthesis on the nucleotide phosphoramidite to construct an oligonucleotide molecule; c4-oxygen at the tail end of the oligonucleotide is connected with thiophosphoric acid through a spacer group Y, the formed O4-Y-PSO22 < 2-> structure is phosphonic acid in a non-natural form and does not belong to a substrate of phosphatase, and the modified oligonucleotide can resist exonuclease degradation and improve the biological activity of the oligonucleotide.

The application discloses an Aeromonas phage recombinant system, an expression vector, a recombinant strain and application thereof, and the recombinant system comprises a recombinase operon 109TES derived from a salmonella Aeromonas phage vB_AsaM-56; the operon comprises at least one gene with exonuclease function and one single-stranded DNA annealing protein gene. Through function verification in Escherichia coli, 109TES can efficiently mediate linear circle recombination and linear linear recombination by using a short homologous arm (50 bp), and has stable and reliable recombination function in a heterologous host, indicating that the recombinase operon from a non-Escherichia coli phage can also exert efficient homologous recombination function in Escherichia coli, and can be used as a powerful candidate for constructing a new type of recombination tool, and provides a new choice for developing a multi-host available gene editing system.

The present invention relates to recombinant DNA technology, in particular to methods for assembling two or more double stranded (ds) nucleic acid molecules with overlapping terminal sequences. In particular, the present invention relates to the use of a thermolabile DNA polymerase II derived 3′-5′ exonuclease isolated from Moritella viscoa and a thermolabile DNA polymerase I of marine origin in multi DNA assembly processes.