651 results about "DNA polymerase" patented technology

The invention belongs to the technical field of biology, and particularly provides UCRT X heat-resistant reverse transcriptase mutants with improved thermal stability, the UCRT heat-resistant reverse transcriptase mutants provided by the invention comprise four single-point mutants, six double mutants and four three mutants, and compared with wild-type UCRT X heat-resistant reverse transcriptase, the mutant has longer half-life period at 65 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about four times of that of wild type DNA polymerase. The invention also provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for synthesizing DNA (Deoxyribonucleic Acid) by reverse transcription at higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and particularly provides UCRT VI heat-resistant reverse transcriptase mutants with improved thermal stability, the mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild-type UCRT VI heat-resistant reverse transcriptase, the mutants have longer half-life period at 65 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about three times of that of wild type DNA polymerase. The invention also provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for synthesizing DNA (Deoxyribonucleic Acid) by reverse transcription at higher temperature, and has higher application potential.

The invention discloses a preparation method of polydeoxyribonucleotide, and relates to the technical field of synthetic biology, and the preparation method comprises the following steps: S1, randomly selecting n ssDNAs from a salmon genome sequence; s2, carrying out cyclization on the ssDNA, so as to obtain cssDNA; s3, carrying out amplification on the cssDNA to obtain a repeated long single-stranded DNA (Deoxyribose Nucleic Acid); and S4, breaking the repeated long single-stranded DNA to obtain the PDRN. According to the method, a large amount of repeated long single-stranded DNA can be synthesized only by introducing a small amount of foreign proteins (T4DNA ligase and Phi29 DNA polymerase), the repeated long single-stranded DNA is broken by a physical breaking method, and a large amount of PDRN products with ideal molecular weight can be obtained in a short time.

Recombinant DPO4-type DNA polymerase variants with amino acid substitutions that confer modified properties upon the polymerase for improved single molecule sequencing applications are provided. Such properties may include enhanced binding and accurate incorporation of bulky nucleotide analog substrates into daughter strands and the like. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids which encode the polymerases with the aforementioned phenotypes.

The invention discloses a fusion protein of Bst DNA polymerase and ferritin as well as a mutant and application of the fusion protein. The invention firstly provides a Bst DNA polymerase fusion protein obtained by connecting a C end of a monomer ferritin subunit with an N end of a wild type Bst DNA polymerase through a connecting peptide, and large fragments of the wild type Bst DNA polymerase are repeatedly and orderly displayed on the surface of intense pyrococcus ferritin with strong heat resistance, so that the enzyme activity and the heat stability of the fusion protein are improved; the fusion protein is further subjected to single-site and double-site mutation, compared with a wild type Bst DNA polymerase and the fusion protein, the enzymatic activity, specific activity and thermal stability of the obtained mutant are remarkably improved or improved, LAMP specific amplification reaction can still be carried out at the highest temperature of 85 DEG C, and the application prospect is wide. The reaction preciseness is improved, the use cost of the enzyme is reduced, and the method can be applied to a loop-mediated isothermal amplification technology in a more complex scene.

This invention relates to recombinant nucleic constructs comprising a sequence-specific DNA binding protein, DNA-dependent DNA polymerase and a DNA encoded repair template, optionally a DNA endonuclease or wherein the sequence-specific DNA binding protein comprises DNA endonuclease activity, and methods of use thereof for modifying nucleic acids in cells and organisms.

The invention discloses a kit for detecting miRNAs of multiple extracellular vesicles based on a TtAgo-driven rolling circle amplification technology and application of the kit, a platform combines TtAgo protein and RCA reaction, cooperates with endonuclease activity and DNA polymerase activity of TtAgo, accurately controls reaction kinetic balance between temperature and enzyme activity, not only does not damage the cutting activity of TtAgo, but also can detect miRNAs of multiple extracellular vesicles. The method provided by the invention can be used for detecting multiple extracellular vesicles miRNAs, ensures strong polymerization amplification performance, creates a strong one-step single-tube experiment, greatly simplifies the experimental process, shortens the detection time, realizes ultra-sensitive and specific detection of nucleic acid markers, and can be used for detecting multiple extracellular vesicles miRNAs.

The present disclosure relates to compositions, methods, and systems for genomic editing. In certain aspects, the compositions, methods, and systems provided herein can incorporate a SpyCas9 nickase, a DNA-dependent DNA polymerase, and / or a template guide RNA for template-based genome editing applications.

The invention belongs to the technical field of biology, and particularly provides UCRT heat-resistant reverse transcriptase mutants with improved thermal stability, the mutants comprise four single-point mutants, six double mutants and four three mutants, and compared with wild-type UCRT III heat-resistant reverse transcriptase, the mutants have longer half-life period at 65 DEG C; the double mutant has a better effect, and the half-life period of the double mutant is about four times of that of wild type DNA polymerase. The invention also provides a genetically engineered bacterium containing the mutant. The UCRT heat-resistant reverse transcriptase mutant obtained by the construction method provided by the invention has better thermal stability, shows higher thermal stability when being used for synthesizing DNA (Deoxyribonucleic Acid) by reverse transcription at higher temperature, and has higher application potential.

The invention belongs to the technical field of biology, and provides a primer group, a kit and a method for detecting aeromonas hydrophila of micropterus salmoides. The primer group comprises outer primers F3 and B3, inner primers FIP and BIP, and loop primers LF and LB. The kit comprises a reaction solution, and the reaction solution comprises the primer group. The detection method comprises the step of carrying out LAMP detection by using the kit. The designed LAMP special specific primer group is used for recognizing a plurality of specific areas of the aeromonas hydrophila target gene, and the target gene can be successfully detected by carrying out amplification reaction at a constant temperature through strand displacement DNA polymerase. The detection method disclosed by the invention is strong in specificity, high in sensitivity, convenient and rapid in detection and strong in applicability, and lays a foundation for on-site rapid and accurate detection of micropterus salmoides infected with aeromonas hydrophila.

The invention relates to the technical field of molecular biology, and provides a DNA origami structure with enhanced stability in a DNA polymerase environment as well as preparation and application of the DNA origami structure. An optional number of conventional staple chains; one or more anti-enzymolysis staple chains, wherein the anti-enzymolysis staple chain comprises a staple section and a suspension section for preventing DNA polymerase degradation; wherein staple sections of the skeleton chain, the conventional staple chain and the anti-enzymolysis staple chain form a DNA origami body, and the DNA origami body comprises a plane or curved surface structure; in addition, the hanging section of the anti-enzymolysis staple chain is exposed out of the DNA origami body, and the hanging section is provided with a polyT block. The stability of the DNA origami structure in a DNA polymerase environment is effectively improved, characterization and analysis are facilitated, and the application range of the DNA origami structure in different enzyme systems is widened.

The purpose of the present invention is to provide a Bst DNA polymerase that can reduce the effects of interfering substances and reactivity at temperatures lower than the optimum reaction temperature. In addition, the invention aims to provide a reagent composition containing the Bst DNA polymerase, a kit containing the Bst DNA polymerase, and a nucleic acid amplification method using the Bst DNA polymerase. The problem is solved by a Bst DNA polymerase which contains an amino acid sequence represented by SEQ ID NO: 1, or a consensus sequence Asn-X-(Ser / Thr) (X is any amino acid residue other than a proline residue), contains an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 1, has 5 '-3' DNA polymerase activity and chain substitution activity, and has a consensus sequence containing an N-binding sugar chain, and the N-binding sugar chain contains more than three mannose residues.

The invention relates to the technical field of biology, in particular to a cis-trans typing method for drug-resistant gene mutation EGFR T790M / C797S. The invention provides an EGFR (Epidermal Growth Factor Receptor) T790M / C797S cis-trans typing method based on a locked nucleic acid LNA (Low Nucleic Acid) probe, ultrahigh-specificity DNA (Deoxyribose Nucleic Acid) polymerase and a primer probe combination. The typing method can be used for accurately distinguishing EGFR T790M / C797S double-mutation cis-configuration and trans-configuration; meanwhile, the kit has the advantages of high sensitivity, high specificity, simple steps and low cost, is suitable for detecting low-abundance samples such as circulating tumor DNA (ctDNA) and can meet the clinical rapid detection requirement.

To provide a Bst DNA polymerase capable of reducing the effects of interfering substances and reactivity at temperatures lower than an optimal reaction temperature, to provide a reagent composition and a reagent kit containing the Bst DNA polymerase, and to provide a method for amplifying nucleic acid using the Bst DNA polymerase.SOLUTION: The above problems are solved by a Bst DNA polymerase, which contains a specific amino acid sequence or a consensus sequence Asn-X-(Ser / Thr) (X is any amino acid residue other than a proline residue), contains an amino acid sequence that has 80% or more identity with the specific amino acid sequence, has 5'→3' DNA polymerase activity and strand displacement activity, and contains an N-linked glycan in the consensus sequence, the N-linked glycan containing three or more mannose residues.SELECTED DRAWING: Figure 4

Provided is a recombinant KOD polymerase, which is the following A) or B): the polymerase shown in A) is a protein having DNA polymerase activity that is obtained by modifying amino acid residues in at least one of the following 18 positions in a wild-type KOD DNA polymerase amino acid sequence: 675th, 385th, 710th, 674th, 735th, 736th, 606th, 709th, 347th, 349th, 590th, 676th, 389th, 589th, 680th, 384th, 496th and 383rd; the polymerase described by B) is a protein having DNA polymerase activity that is derived from A) by adding a tag sequence to an end of the amino acid sequence of the protein shown in A).

The invention discloses a method for determining the absolute activity of Bst DNA polymerase, which comprises the following steps: by taking annular single-stranded DNA as a template, in a reaction system containing an amplification primer and dATP, initiating an extension replication reaction by using Bst DNA polymerase to be detected, and consuming dNTPs in the reaction system; the consumption of dATP is calculated according to a pre-constructed standard curve f1 representing the relationship between the concentration of dATP and the fluorescent value, the concentration of Bst DNA polymerase is calculated through a pre-constructed standard curve f2 representing the relationship between the concentration of Bst DNA polymerase and the consumption of dATP, and finally the absolute activity of the Bst DNA polymerase to be detected is calculated according to the absolute activity definition of Bst DNA polymerase. The method is free of radioactive contamination, and has the advantages of determination of the polymerization activity of the Bst DNA polymerase, absolute constant activity, simplicity and convenience in operation, rapidness, sensitivity, low cost and the like.

Disclosed herein are composition and methods of modifying a target double-stranded DNA (dsDNA) molecule in a cell. Systems, compositions, and methods may comprise a CRISPR-associated (Cas) protein, an RNA-dependent DNA polymerase, and / or one or more guide nucleic acids or uses thereof.

The invention relates to the technical field of food safety detection, in particular to a food crop pathogenic mycotoxin detection method based on double signal amplification. The core of the method is a section of linear lock-type probe containing a mycotoxin specific nucleic acid aptamer, under the condition that target mycotoxin exists in a sample, the aptamer is combined with the mycotoxin, the probe is induced to generate conformational change, and the probe is closed into annular DNA under the catalysis of DNA ligase; then, the circular DNA is used as a template, isothermal rolling circle amplification is carried out through Phi29 DNA polymerase, and a long-chain DNA product containing a large number of repetitive sequences is generated; subsequently, the long-chain product is used as a molecular scaffold, and two kinds of gold nanoparticles of which the surfaces are modified with different complementary probes are cross-linked at the same time, so that the nanoparticles are quickly gathered, the color of the solution is changed from wine red to blue or purple, and convenient visual detection is realized. The method has the outstanding advantages of ultrahigh sensitivity, rapid detection and the like, and is suitable for on-site rapid screening of mycotoxin pollution.

The invention discloses a Tth DNA polymerase mutant. On the basis of Tth wild type polymerase, the mutant has a group of mutations as follows: S515K, I640F, S741G and L789F; a D734G unit and an M763T unit; or E617D, D734G, and M763T, and the like. The reverse transcription activity of the Tth DNA polymerase mutant disclosed by the invention is close to the activity of an M-MLV RT wild type under the condition that the Tth DNA polymerase mutant does not depend on manganese ions. The Tth DNA polymerase mutant provided by the invention is suitable for being prepared into a kit, and has a wide application prospect.

The invention relates to an on-site synchronous quantitative determination method for three pathogenic bacteria in marine sediments, and belongs to the technical field of pathogenic bacteria detection.The three pathogenic bacteria are vibrio parahaemolyticus, vibrio alginolyticus and escherichia coli O157, and the method comprises the steps that marine sediment samples are collected and put into a centrifugal tube filled with sterile seawater, and are evenly mixed and centrifuged; taking the supernate as a template for LAMP amplification, respectively adding the supernate into detection tubes of an LAMP reaction system preloaded with the three pathogenic bacteria, and sealing. Placing the detection tube into a detection channel of a portable electronic isothermal gene amplification instrument, and determining a delta C4-t curve, a dv / dt-t curve and Tp; and after the determination is finished, substituting the Tp value into a working curve formula to calculate the content / concentration of the three pathogenic bacteria of the corresponding marine sediment sample in the detection tube. According to the method disclosed by the invention, Bst DNA polymerase and betaine are not needed in the reaction, so that the cost of LAMP analysis is reduced by at least 50%, the operation is simple and convenient, and simultaneous quantitative determination of the three pathogenic bacteria can be completed on site within 1h.

The invention relates to a multiple miRNA (micro Ribonucleic Acid) detection method based on deoxyribozyme or CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) / The detection method provided by the invention comprises the following steps: designing a probe 1 and a probe 2 matched with different targets according to the different targets; when a target exists, the probe 1 and the probe 2 are connected to miRNA through T4DNA liguase, then DNA polymerase is introduced for amplification of a DNA chain, the released miRNA is further hybridized with a new probe, the next round of reaction is activated, and signals are circularly amplified. The S1 duplex body generated after amplification can be subjected to multiple characterization through two design modes: the first mode is based on deoxyribozyme detection; and the second mode is to carry out detection based on CRISPR / Cas12a. The method provided by the invention can realize detection of multiple tumor miRNAs, is short in detection time, and provides an important theoretical basis for treatment of tumor diseases.

Disclosed herein, inter alia, are mutant enzymes, kits, and methods of use thereof.

The invention relates to the technical field of biology, in particular to a phi29 DNA polymerase mutant and application thereof. The protein provided by the invention is selected from at least one of the following items: (A), the protein is an amino acid sequence compared with a wild type phi29 DNA polymerase and comprises at least one of the following mutation sites: M8R, D12A, N62D, M97K, G197D, L253A, T368M, Y369E, E375D, Q380L, A382L, A382F, A484R, A484E, E508F, D510M, V514Q, V514D, V514S and / or F526L; (B) the protein is an amino acid sequence compared with phi29 DNA polymerase and has at least one palm structural domain; according to the mutation form of the phi29 DNA polymerase, the amplification efficiency and capability of non-natural substrates under the catalysis of Mg < 2 + > can be remarkably improved.

The present invention relates to engineered DNA polymerase polypeptides and compositions thereof, and polynucleotides encoding the engineered DNA polymerase polypeptides. The invention also provides methods of using the engineered DNA polymerase polypeptides, or compositions thereof, for diagnostic and other purposes.

The invention relates to novel DNA polymerases of the polX family, and in particular Terminal deoxynucleotidyl Transferase (TdT) variants comprising specific mutations or substitutions, and to their uses.

The invention provides a kit for detecting human BRAF gene V600E and TERT gene promoter mutation, and belongs to the technical field of biology. The kit comprises a primer, a probe, a 5 * PCR (Polymerase Chain Reaction)-Enhancer, a 10 * PCR Buffer, dNTP (Deoxyribonucleoside Triphosphate) Mix, MalTaq DNAPlyase, magnesium ions, DEPC (Diethylpyrocarbonate) water, a positive control and a negative control. According to the present invention, the ARMS-PCR detection technology is adopted to detect the specific gene mutation, and the target mutation site is identified by designing the specific primer and the probe, such that the detection specificity and the detection sensitivity are improved; by combining with a Taqman probe technology, the fluorescent signal change in the PCR amplification process can be monitored in real time, so that whether mutation of a BRAF V600E site, a TERT C228T site and a TERT C250T site exists or not is accurately judged; according to the method, pre-amplification of 15 cycles is adopted, then quantitative amplification of 30 cycles is adopted, and when the initial amount of the target DNA is very low, the copy number of the target DNA can be increased, so that the detection accuracy and sensitivity are further improved.

The invention discloses a method for detecting accessibility of space chromatin and application of the method. The method comprises the following steps: (1) slicing a tissue sample to be detected on a chip with spatial information; (2) fixing, permeabilizing and dyeing a tissue sample to be detected; (3) preparing fragmented genome DNA (deoxyribonucleic acid); (4) performing DNA hybridization based on a transfer mode; (5) carrying out connection and terminal repair reaction on the fragmented genome DNA by using DNA polymerase and DNA ligase; (6) denaturation and melting; and (7) sequencing. According to the method, the transcriptional activity state of chromatin can be reflected through chromatin accessibility sequencing analysis, staining imaging and chromatin accessibility information can be obtained on the same tissue slice at the same time without complex micro-fluidic equipment, experimental operation is easy, repeatability is high, and the analysis result is more remarkable.

Methods are provided for nucleic acid amplification using primers that contain DNA oligonucleotide segments and at least one ribonucleotide. In PCR reactions, this increases the amount of amplified product with each amplification cycle, resulting in greater sensitivity and speed than conventional PCR. In isothermal reactions, this reduces the time required to detect the target. Also provided are methods for monitoring amplification reactions using probes that contain ribonucleotides, without relying on the 5'-nuclease activity of DNA polymerase.

The present disclosure provides an aptamer capable of inhibiting the activity of a strand-displacing DNA polymerase, the aptamer including: an oligonucleotide region 1 comprising a sequence in which a sequence X1a is linked to the 3'-end of a first sequence or the sequence X1a is linked to the 3'-end of a mutant sequence of the first sequence; and an oligonucleotide region 2 comprising a sequence in which a sequence X1b is linked to the 5'-end of a second sequence or the sequence X1b is linked to the 5'-end of a mutant sequence of the second sequence. The present disclosure also provides: a composition and a kit each containing the aptamer; and a method for amplifying a nucleic acid using the composition or the kit.

Provided are a DNA polymerase θ inhibitor and a use thereof. Specifically disclosed are a nitrogen-containing heterocyclic compound represented by formula (I), or a stereoisomer, deuterated compound, or pharmaceutically acceptable salt or co-crystal thereof, and a pharmaceutical composition comprising same. In addition, provided is a use in the preparation of a drug for treating DNA polymerase θ-mediated diseases. Groups in formula (I) are as defined in the description.