1204 results about "Polymerase" patented technology

The invention discloses a hemoglobin-resistant Taq DNA polymerase mutant and a construction method thereof, and relates to the field of biology, and the hemoglobin-resistant Taq DNA polymerase mutant is characterized in that a nucleotide sequence for coding the Taq DNA polymerase is shown as SEQ ID NO.1, and the mutant comprises at least one mutation or all mutations selected from S623D and E721A sites. On the basis of natural Taq DNA polymerase, the molecular structure of the Taq DNA polymerase is modified through rational design and combination with a site-specific mutagenesis biotechnology, so that the Taq DNA polymerase mutant is more suitable for PCR amplification of samples containing hemoglobin and the like than a wild type, and the result judgment accuracy of a sample containing a hemoglobin inhibitor is more facilitated.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology, the enzyme specific activity of the variant is obviously improved compared with that of a wild type, and the addition amount of polymerase is reduced, so that the cost is saved. Meanwhile, the mutant also effectively reduces the generation of incomplete fragment impurities and improves the integrity. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

Disclosed herein are certain thiadiazolyl derivatives Formula (I):that inhibit DNA Polymerase Theta (Polθ) activity, in particular inhibit Polθ activity by inhibiting ATP dependent helicase domain activity of Polθ. Also, disclosed are pharmaceutical compositions comprising such compounds and methods of treating and / or preventing diseases treatable by inhibition of Polθ such as cancer, including homologous recombination (HR) deficient cancers.

The invention discloses application of iron ruthenium nano-enzyme in preparation of nucleic acid detection products, and belongs to the technical field of biological detection and nano-materials. The RIN has stable peroxidase-like (POD) activity, and can catalyze a chromogenic reaction between 3, 3 ', 5, 5'-tetramethyl benzidine (TMB) and hydrogen peroxide (H2O2). The catalytic activity of the nano-enzyme can be selectively inhibited by pyrophosphate (PPi) generated in a nucleic acid amplification reaction, so that visual colorimetric analysis on whether target nucleic acid exists or not is realized. In combination with a polymerase chain reaction (PCR) system, the invention constructs a new nucleic acid detection strategy which does not need fluorescence labeling, is simple and convenient to operate and has high sensitivity. Through verification, the system can be widely applied to rapid on-site screening and visual interpretation of related nucleic acid of drug-resistant pathogens (such as ESKAPE flora) and the like, and has a good application prospect.

The invention discloses a Taq DNA polymerase mutant as well as a preparation method and application thereof, and relates to the technical field of biology. The Taq DNA polymerase mutant is mutation on the basis of a wild type Taq DNA polymerase amino acid sequence as shown in SEQ ID NO.1, and comprises at least one of amino acid mutation L345E, L352D and S438M. The Taq DNA polymerase mutant has excellent tolerance to urea, and solves the dual technical problems that complex biological samples containing endogenous inhibitors such as urea can be efficiently amplified, and the Taq DNA polymerase mutant is compatible with difficult PCR reactions which take urea as an additive to optimize a high GC template and the like. Therefore, the mutant is a molecular biology tool enzyme which is wider in applicability and more stable and reliable in performance.

The invention belongs to the technical field of genetic engineering and enzyme engineering, and particularly relates to a genetically engineered bacterium for stably and efficiently producing L-isoleucine as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes E. coliTHRD as a host and contains an RNA (Ribonucleic Acid) polymerase beta-subunit mutant coding gene rpoBM; compared with a parent RNA polymerase beta-subunit, the RNA polymerase beta-subunit mutant has the advantage that the amino acid residue at the 618th site of the amino acid sequence is sequentially replaced with leucine from the N terminal to the C terminal. The genetically engineered bacterium disclosed by the invention can tolerate L-isoleucine and accumulate less alpha-ketobutyric acid, and has a very good industrial application prospect.

The invention relates to the technical field of phytopathogen detection, and particularly discloses a primer, a kit and a method for detecting crop root rot fusarium solani and application, the primer comprises a specific RPA primer RPA-F and a specific RPA primer RPA-R; the specific RPA primer RPA-F is as shown in SEQ ID NO. 1, and the specific RPA primer RPA-R is as shown in SEQ ID NO. 2; the kit further comprises a guide RNA primer crRNA, and the guide RNA primer crRNA is as shown in SEQ ID NO. 3. The kit comprises a specific RPA primer RPA-F, a specific RPA primer RPA-R, a Cas12 protein, a T7 transcriptase, a crRNA DNA template, a DNA polymerase and a Cas12 / 13 nucleic acid detection test strip. The kit has the characteristics of good sensitivity, relatively good specificity and enough reliability.

The invention discloses an oil palm U6 promoter gene and application thereof, and belongs to the technical field of biology. The nucleotide sequence of the promoter gene is shown as SEQ ID NO.1. The oil palm RNA polymerase III type promoter gene, namely the oil palm endogenous U6 promoter gene EgU6, is obtained by cloning in an oil palm genome for the first time, and the promoter gene has high transcriptional activity and can drive downstream fluorescent protein mNeonGreen expression. The candidate oil palm endogenous U6 promoter gene can be provided for subsequently establishing a high-efficiency oil palm gene editing technology system based on a CRISPR / cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / Cas9) system.

The present disclosure provides compositions, apparatus and methods for conducting separate sequencing batches on a support having nucleic acid template molecules immobilized thereon, where the separate sequencing batches can be conducted using any massively parallel sequencing technology. In some embodiments, a plurality of sub-populations of nucleic acid template molecules are immobilized to the support including at least a first and second sub-population. In some embodiments, the first sub-population of template molecules undergo first batch sequencing reactions and a region of the support is imaged to detect the first sequencing reactions, wherein the second sub-population of template molecules do not undergo sequencing reactions. In some embodiments, the second sub-population of template molecules undergo second batch sequencing and the same region of the support is imaged to detect the second sequencing reactions, wherein the first sub-population of template molecules do not undergo sequencing reactions.

The invention discloses a CRISPR / Cas12a-RPA (clustered regularly interspaced short palindromic repeats / CRISPR associated protein 12a-recombinase polymerase amplification)-based sequence combination for rapidly detecting mouse hepatitis virus and a kit thereof, and belongs to the technical field of biological detection The sequence combination comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence; the kit comprises an RPA primer pair sequence, a crRNA sequence and an ssDNA probe sequence, the kit further comprises a mouse hepatitis virus negative control standard substance, RNase-free water, an RPA reaction buffer solution, an NEB buffer solution, Lba Cas12a nuclease, dry powder RPA reaction microspheres containing recombinase polymerase and magnesium acetate with the concentration of 280 mM. The kit can be used for rapidly detecting the mouse hepatitis virus. The primer sequence combination provided by the invention is good in conservative property and specificity and relatively high in sensitivity, and rapid detection of the mouse hepatitis virus can be realized.

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 invention provides an RNA polymerase variant, the enzyme specific activity of the variant is obviously improved compared with that of a wild type, and the addition amount of polymerase is reduced, so that the cost is saved. In addition, the invention further provides a method for generating RNA through in-vitro transcription.

The present disclosure relates to Taq DNA Polymerase mutants comprising at least three mutations which are capable of high levels of DNA amplification in the presence of several PCR inhibitors and compositions comprising the same. Also disclosed are methods of performing mutagenesis on wild-type Taq DNA polymerase to obtain the disclosed Taq DNA polymerase mutants, DNA amplification using the same, and cDNA amplification using the same.

The invention discloses a method for quickly rescuing bovine coronavirus epidemic strains and application of the method. The bovine coronavirus SHZ isolate is subjected to efficient segmented cloning, the obtained segment and a Linker sequence are subjected to a cyclic polymerase extension reaction, and the obtained reaction product can be directly transfected to 293T cells for virus packaging without purification. Different from a traditional method, the method does not need to amplify full-length virus cDNA clone by means of bacteria and yeast, but directly obtains a sufficient amount of preliminary products through PCR, and avoids the problems of instability and low efficiency when partial sequences of a virus genome proliferate in a bacteria or yeast host. By adopting the reverse genetic system, the recombinant bovine coronavirus expressing the foreign protein is successfully rescued, a brand new technical platform is provided for dynamic visualization research of in-vivo and in-vitro replication of the virus, and an efficient and flexible tool is also provided for virology research, development of virus vector vaccines and screening of antiviral drugs.

The present disclosure provides compositions and methods for prime editing with improved editing efficiency and / or reduced indel formation by inhibiting the DNA mismatch repair path way while conducting prime editing of a target site. Accordingly, the present disclosure provides a method for editing a nucleic acid molecule by prime editing that involves contacting a nucleic acid molecule with a prime editor, a pegRNA, and an inhibitor of the DNA mismatch repair pathway, thereby installing one or more modifications to the nucleic acid molecule at a target site with increased editing efficiency and / or lower indel formation. The present disclosure further provides polynucleotides for editing a DNA target site by prime editing comprising a nucleic acid sequence encoding a napDNAbp, a polymerase, and an inhibitor of the DNA mismatch repair pathway, wherein the napDNAbp and polymerase is capable in the presence of a pegRNA of installing one or more modifications in the DNA target site with increased editing efficiency and / or lower indel formation. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure. The present disclosure also provides compositions and methods for prime editing with improved editing efficiency and / or reduced indel formation with modified prime editor fusion proteins. The disclosure further provides, vectors, cells, and kits comprising the compositions and polynucleotides of the disclosure.

The invention provides an RNA polymerase variant and application thereof, and relates to the technical field of biology. The RNA polymerase variant provided by the invention has the performance of remarkably improving the integrity of a super-long fragment mRNA transcription product, can be used for preparing mRNA through in-vitro transcription, particularly can improve the integrity of the product when preparing saRNA exceeding 10000 nt, and has important significance for industrial production of saRNA.

The present document describes methods and systems for amplifying and quantifying amplification of a nucleic acid molecule, with a polymerase chain reaction (PCR) or a loop-mediated isothermal amplification (LAMP), by irradiating, with a heating activation light beam from a continuous wave laser a biological enzymatic reaction mixture in solution comprising a nucleic acid template, a polymerase enzyme, and chemically modified nanoparticles. Quantification of the amplification is achieved by irradiating the biological enzymatic reaction mixture during an annealing and / or elongation steps with an ultraviolet (UV) light source and measuring with a photodetector a transmission change in UV light transmission.

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 discloses a variable region amino acid sequence of a monoclonal antibody of a West Nile virus non-structural protein NS1 and application of the variable region amino acid sequence, and belongs to the technical field of medicines. According to the invention, West Nile virus non-structural protein NS1 expressed by human embryo kidney 293 cells is used as an antigen to immunize a rabbit, B cells capable of being specifically combined with the West Nile virus non-structural protein NS1 are screened from rabbit spleen cells through flow sorting, and a signal peptide and a variable region gene fragment of an antibody are cloned through reverse transcription-polymerase chain reaction; according to the present invention, the non-structural protein NS1 of flaviviridae flaviviridae virus is taken as a template, and is connected with a constant region gene to an expression vector, and after mammalian cell expression and purification, the monoclonal antibody which has high affinity and is not combined with the non-structural protein NS1 of other eight viruses of flaviviridae flaviviridae virus is obtained through enzyme-linked immunosorbent assay; the monoclonal antibody has application value in diagnosis and prevention and treatment of West Nile virus infection.

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.

The invention belongs to the field of nucleic acid detection and molecular biology, and particularly relates to a method and a kit for eliminating false positive results in nucleic acid amplification reaction. According to the method, after a nucleic acid amplification reaction (taking recombinase polymerase amplification, namely RPA, for example) is completed, a target amplification product is subjected to selective enzyme digestion by utilizing restriction endonuclease, non-target products (such as primer dimers and non-specific amplification products) are not cut, and meanwhile, the non-target products which are not subjected to enzyme digestion are removed by combining solid-phase separation, so that the target amplification product is obtained. Therefore, the false positive result is eliminated. The invention also discloses a kit containing the restriction enzyme. According to the method, the specificity and the signal-to-noise ratio of a nucleic acid amplification reaction, especially RPA, are remarkably improved, the cost is low, operation is easy, the method is compatible with an existing technical platform, the method is suitable for detection scenes such as clinical molecular diagnosis, environmental monitoring and food safety, and the problem of misjudgment caused by false positive signals is effectively avoided.

The invention discloses an anti-Taq DNA polymerase antibody or an antigen binding fragment thereof and application thereof, and relates to the technical field of biology. The antibody or the antigen binding fragment of the antibody can be specifically bound with Taq DNA polymerase, the polymerization activity and / or excision activity of the Taq DNA polymerase are / is blocked, and the stability of the Taq DNA polymerase is improved.

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 present invention provides a process for the production of a closed linear DNA comprising the steps of (a) providing a DNA template comprising a DNA sequence of interest; (b) amplifying DNA from the DNA template of step (a) wherein the amplification is primed with a primase / polymerase enzyme; (c) generating a closed linear DNA with the amplified DNA produced in step (b); and (d) purifying the closed linear DNA produced in step (c). The invention also provides a closed linear DNA obtainable according to the process of the invention, a pharmaceutical composition comprising a therapeutically effective amount of the closed linear DNA of the invention, and a concatameric DNA comprising repeats of a DNA sequence of interest.

The invention discloses an anti-inhibition Taq DNA polymerase and application thereof, and relates to the technical field of biology. According to the anti-inhibition Taq DNA polymerase, amino acid mutations of K206R, F667Y and the like are carried out on an amino acid sequence of a wild type Taq DNA polymerase with an amino acid sequence as shown in SEQ ID No.1, so that a certain degree of anti-inhibition property is obtained. In addition, the invention also discloses application of the anti-inhibition Taq DNA polymerase in preparation of reagents or kits, and the like. Compared with a wild type Taq DNA polymerase, the anti-inhibition Taq DNA polymerase can maintain the PCR amplification capability in the presence of an inhibitor such as polysaccharide.

The invention provides an RNA (Ribonucleic Acid) polymerase variant and a preparation method thereof, and an RNA product with low dsRNA pollution can be obtained by applying the RNA polymerase variant disclosed by the invention to in-vitro transcription. In addition, the invention further provides a method for preparing RNA through in-vitro transcription.

Provided herein are modified Archaeal family B polymerases derived from the Archaeal microorganism Pyrococcus abyssi that exhibit improved incorporation of nucleotide analogues utilized in DNA sequencing.

The invention belongs to the technical field of DNA amplification, and relates to a mixed enzyme system capable of amplifying circular DNA as well as application and a product of the mixed enzyme system. The invention provides a mixed enzyme system capable of amplifying circular DNA (deoxyribonucleic acid). The mixed enzyme system comprises a polymerase III tau clip loader and a catalytic core compound. The mixed enzyme system provided by the invention is remarkably superior to a traditional method in the aspects of efficiency, cost, safety, applicability, sensitivity and the like, provides a brand new solution for efficient preparation of the circular DNA, and has a wide application prospect in the field of biotechnology.

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.

The invention relates to a plant having an increased proportion of edible biomass resulting from genetic modification of gene that encodes a poly(adenosine 5′-diphosphate (ADP)-Ribose) Polymerase (PARP) enzyme.