1967 results about "Polymerase" patented technology

The present invention provides a method and a kit for detecting a target nucleic acid to be detected by using a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a nicking enzyme and a DNA polymerase, generating a reporter primer that is complementary to the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by the easy mutation of the target nucleic acid sequence to be detected.

The present invention provides a method and a kit for detecting a target nucleic acid to be detected through a melting curve. The method includes designing a first primer, a second primer and a detection probe for the target nucleic acid sequence to be detected, performing PCR amplification in a PCR amplification system containing the first primer, the second primer, the detection probe, the sample to be detected, a restriction endonuclease and a DNA polymerase, generating a reporter primer that is complementary to and pairs with the detection probe to form a double-stranded product, obtaining the melting curve of the double-stranded product, and the melting curve of the double-stranded product is the melting curve corresponding to the target nucleic acid to be detected. The method of the present invention is a non-target-dependent melting curve method, and the melting point (T m value) of each double-stranded body can be calculated in advance, solving the problems of melting curve peak shift and easy misjudgment caused by easy mutation of the target nucleic acid sequence to be detected.

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 discloses a kit for detecting tomato brown crinkled fruit virus based on enzyme-mediated dual-amplification nucleic acid amplification and a detection method. The detection system comprises a primer pair DNA (deoxyribonucleic acid) upstream primer (F5) and a DNA downstream primer (R6) for amplifying the tomato brown crinkled fruit virus movement protein gene, a probe (RNA primer (RNA1)) for detecting the MP gene, a nucleic acid amplification group enzyme system (recombinase, single-chain binding protein, polymerase and ATP (adenosine triphosphate) energy regeneration enzyme), a signal amplification group enzyme system (positioning enzyme and cutting enzyme), activating enzyme NTP, a buffer solution and RNase-free Water. The system composed of the freeze-drying protective agent is prepared into the pre-subpackaged freeze-dried balls. The kit disclosed by the invention can realize DNA detection of aM concentration, and besides rapid qualitative detection of the tomato brown crinkled fruit virus, early screening of the tomato brown crinkled fruit virus can also be realized due to high detection sensitivity.

The invention discloses a tea tree CsU3 gene promoter and application thereof, and belongs to the technical field of plant biotechnology and gene engineering. Tea tree RNA polymerase III type promoters-tea tree endogenous U3 gene promoters CsU3a, CsU3b, CsU3c and CsU3d are successfully cloned for the first time, a tea tree U3 gene promoter activity detection carrier is successfully constructed, the transcriptional activities of the four CsU3 promoters are compared through instantaneous tobacco leaf conversion, GUS dyeing and GUS enzyme activity determination, and the CsU3 gene promoter activity detection carrier can be used for detecting the activity of the tea tree U3 gene promoter. CsU3a and CsU6b promoters with strong transcriptional activity are selected to be used for constructing a tea tree gene editing carrier, the feasibility of the promoter applied to a tea tree CRISPR / Cas9 gene editing system is verified in a tea tree protoplast, and CRISPR / Cas9 mediated tea tree genome targeted editing is realized.

The invention relates to the field of nucleic acid detection, in particular to a multifunctional dumbbell type nucleic acid probe, a kit, a target detection method and application thereof. The multifunctional dumbbell type nucleic acid probe provided by the invention comprises two or more of the following functional areas: a promoter area used for being combined with RNA polymerase; the target recognition area is used for recognizing and combining a target sequence and opening the dumbbell type nucleic acid probe; the dumbbell type nucleic acid probe can be transcribed and combined with a rolling ring, a specific sequence is subjected to mass transcription, amplification of the specific sequence is achieved, finally, signal amplification is achieved during detection, and the dumbbell type nucleic acid probe has the advantages of being simple in reaction system, low in background signal and high in detection specificity; and the dumbbell type nucleic acid probe can be designed according to different target sequences, so that the controllable detection of different targets can be realized.

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 relates to the technical field of biology. The RNA polymerase variant provided by the invention can be used for remarkably reducing the generation of dsRNA impurities in an in-vitro transcription process and remarkably improving the integrity of an RNA transcription product, especially in transcription of long-fragment RNA. In addition, the invention provides an application of the RNA polymerase variant in in-vitro transcription, and also provides a method for preparing ultra-long saRNA (greater than 10000 nt).

The invention includes a mutant Taq polymerase, which can significantly extend and amplify a target sequence where the extension conditions are time limited to as little as one second. The mutant Taq polymerase, or a biologically active fragment thereof, has one or more substitutions differing from the wild type as shown in Table I.

Systems and methods for sequencing polynucleotides using nanopores are disclosed. In some embodiments, a polynucleotide including a single-stranded region and a double-stranded region, in which the single-stranded region is disposed through a nanopore. The polynucleotide can be moved relative to the nanopore by electric forces while one or more structural locks keep the polynucleotide close to the nanopore. A characteristic signal based on nanopore ionic current blockade and associated with the regions of the polynucleotide at or near the nanopore recognition zone is measured and used to infer the nucleobase sequence of the polynucleotide. In some examples, the double-stranded region is extended by a polymerase, and the polymerase is removed from the polynucleotide. In some examples, signals measured under different applied voltages provide nonredundant information regarding the polynucleotide sequence.

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 present disclosure provides methods for reducing sequencing errors comprising one or any combination of: (i) removing deaminated bases in any nucleic acid molecule throughout a library preparation workflow which includes immobilised splints which bind to the library, the use of a compaction oligonucleotide, optionally with an intervening sequence, formation of closed circular nucleic acids, creating gaps using glycosylase and lyase activities at positions with deaminated bases. The library may be sequenced using pairwise sequencing, e.g. with dark sequencing and / or sequencing using a multivalent labelled probe for the formation of an avidity molecule and soluble primer and polymerase. Method for sequencing concatemers in which the concatermers are contacted with polymerases, soluble primers and a multivalent labelled molecule which forms a complex with the polymerase. Detecting polymerase position and nucleobase bound to the polymerase in the complex. These methods generate higher quality base calls during downstream sequencing workflows.

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 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.

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and / or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

The invention belongs to the technical field of gene engineering, and particularly relates to an RNA polymerase beta subunit mutant and application thereof. The RpoB mutant is obtained through a gene editing technology, after the 560th amino acid of the RpoB is changed into leucine from proline, the number of living cells during strain fermentation is greatly increased, the tolerance of the RpoB mutant in the later environment of a fermentation tank is improved, the RpoB mutant is applied to escherichia coli for producing human milk oligosaccharide, and finally the yield of the human milk oligosaccharide is greatly increased.

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas-based kit for isothermal rapid visual detection of H1N1 influenza virus and mycoplasma pneumoniae and a detection method thereof. The kit and the detection method provided by the invention can be used for timely, sensitively and specifically detecting a nucleic acid target in a sample. The invention provides a lateral flow analysis (LFA) detection platform of a three-stage amplification system integrating recombinase polymerase amplification (RPA), CRISPR / Cas12a and catalytic hairpin assembly (CHA) circulation, the detection platform is used for sensitively and accurately detecting mycoplasma pneumoniae DNA (MP DNA) and influenza A virus subtype H1N1RNA (H1N1RNA), and compared with other researches based on an LFA-CRISPR / Cas12a system, the detection platform has the advantages that the detection efficiency is greatly improved, and the detection cost is reduced. According to the kit and the detection method provided by the invention, nucleic acids of two pathogens can be visually detected on a single LFA at the same time, and the sensitivity is high.

The present disclosure provides compositions and methods for the targeted modification of RNA molecules by RNA prime editing. The compositions and methods may be conducted invitro or in vivo within cells (e.g., human cells) for the therapeutic correction of disease-causing mutations and / or installation of motifs or mutations in RNA molecules of interest as a tool for scientific research. The disclosure provides compositions and methods for conducting RNA prime editing of a target RNA molecule (e.g., an RNA transcript) that enables the incorporation of one or more nucleotide changes and / or targeted mutagenesis of a target RNA molecule. The nucleotide change can include a single-nucleotide change, an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides a variety of configurations of the RNA prime editors each comprising a nucleic acid programmable RNA binding proteins (napRNAbp), such as Cas13, and an RNA-dependent RNA polymerase (RDRP), which are provided as fusion proteins or which can be separately provided in trans. The RNA prime editors are guided to a target RNA site by a guide RNA, which can be a rpegRNA that includes a template region for the synthesis of an RNA sequence to be installed on the RNA molecule attached to an available 3′ terminus. In others embodiments, the RNA template can be provided in trans.

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 present disclosure relates in some aspects to methods, systems, and kits for sequencing a template nucleic acid molecule, where the methods comprise: (i) contacting a priming strand bound to the template nucleic acid molecule with a first plurality of nucleotide molecules and a polymerase coupled to a heterologous polynucleotide-binding moiety to form a complex comprising a 3′ terminus of the priming strand, the template nucleic acid molecule, the polymerase, and a nucleotide molecule of the first plurality of nucleotide molecules, wherein the polynucleotide-binding moiety enhances stability of the complex, and wherein the priming strand comprises a reversibly-terminated nucleotide at its 3′ end such that the nucleotide molecule of the transient complex is not incorporated; and (ii) detecting a presence of the nucleotide molecule in the complex to identify a complementary nucleotide in the template nucleic acid molecule.

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 present disclosure provides compositions and methods that employ the compositions for conducting pairwise sequencing and for generating concatemer template molecules for pairwise sequencing. The concatemers can be generated using a rolling circle amplification reaction which is conducted either on-support, or conducted in-solution and then distributed onto a support. The rolling circle amplification reaction generates concatemers containing tandem copies of a sequence of interest and at least one universal adaptor sequence. An increase in the number of tandem copies in a given concatemer increases the number of sites along the concatemer for hybridizing to multiple sequencing primers which serve as multiple initiation sites for polymerase-catalyzed sequencing reactions. When the sequencing reaction employs detectably labeled nucleotides and / or detectably labeled multivalent molecules (e.g., having nucleotide units), the signals emitted by the nucleotides or nucleotide units that participate in the parallel sequencing reactions along the concatemer yields an increased signal intensity for each concatemer.

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.

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

The present invention provides methods of reducing oncogene transcription, or of reducing phosphorylated RNA polymerase 2, in a subject in need thereof by administering to the subject of Compound (I), or a pharmaceutically acceptable salt thereof.

Provided herein are engineered variants of archaeal, prokaryotic, and eukaryotic polymerases that exhibit enhanced thermostability, enhanced incorporation of 3′ modified nucleotides, and improved uracil-tolerance, in polymerase-catalyzed nucleotide extension reactions relative to wild type polymerase enzymes. Also provided are uses of the engineered polymerases for forming complexed polymerases, forming binding complexes and forming ternary complexes, and uses for conducting nucleic acid sequencing reactions.

The invention relates to the technical field of rapid detection methods, in particular to a one-step detection method for urinary tract infection escherichia coli with a non-disease diagnosis or non-treatment purpose. According to recombinase polymerase amplification provided by the invention, target DNA is specifically amplified through primers, CRISPR / Cas12a specifically recognizes a target sequence through crRNA, and through double screening of RPA and CRISPR / Cas12a, the target sequence is specifically recognized through crRNA; the rapid, sensitive and specific detection on the Escherichia coli infected with the urinary tract can be realized. Meanwhile, an RPA primer, crRNA and reaction conditions are optimized, so that an RPA reaction system and a CRISPR / Cas12a reaction system can be premixed in a tube, and the reaction can be completed only by a one-step method. The method can specifically detect urinary tract infection escherichia coli and has no cross reaction with common pathogenic bacteria, optimized conditions can avoid the competitive effect of CRISPR / Cas12a and RPA, the reaction rate and sensitivity are guaranteed, and meanwhile, the risk of nucleic acid aerosol pollution is also avoided due to the fact that a cover does not need to be opened.

The invention provides a composition, a kit and a detection method for detecting staphylococcus aureus or a drug-resistant gene thereof based on RPA-CRISPR / Cas12a and application of the composition, the kit and the detection method, and belongs to the technical field of gene detection. A CRISPR / Cas12a system is combined with recombinase polymerase amplification (RPA), an RPA-CRISPR / Cas12a detection method for rapidly detecting the staphylococcus aureus in the wastewater and the drug-resistant gene of the staphylococcus aureus is established, a specific primer and crRNA are designed, the high-specificity nucleic acid cutting capability of a gene editing enzyme CRISPR / CAS system and the isothermal rapid amplification advantage of a nucleic acid isothermal amplification technology are fully fused, and the detection method has the advantages of high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity, high specificity The fluorescent probe has high-specificity sequence recognition capability, realizes efficient detection of target nucleic acid, is strong in detection specificity, high in sensitivity and simple and convenient to operate, does not need complex equipment and professionals, and is suitable for rapid detection in an on-site environment with limited resources.

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.