54 results about "DNA polymerase" patented technology

This invention discloses direct amplification qPCR reagents, kits, and applications for whole blood samples. The amplification reagents include: a composite hot-start DNA polymerase system, a buffer system, a surfactant, an anti-contamination system, and an anti-inhibition enhancer system. The composite hot-start DNA polymerase system is obtained by mixing antibody-modified hot-start Taq enzyme and antibody-modified Taq1C2 mutant Taq enzyme in a predetermined ratio. The buffer system includes Tris-HCl, MgCl2, (NH4)2SO4, and KCl. The surfactants include Tween-20 and NP-40. The anti-contamination system includes UDG enzyme and dNTPs, wherein the dNTPs are composed of dATP, dGTP, dCTP, dTTP, and dUTP. The anti-inhibition enhancer system includes D-trehalose, L-carnitine, DMSO, BSA, and SSB single-chain binding protein.

The present disclosure relates, in general terms, to directed evolution, and more specifically to compositions and methods for phage-assisted directed evolution in microbes. In one embodiment, there is provided a method for directed molecular evolution, the method comprising: a) introducing a propagation-defective phage vector into a first host cell (mutagenic host) competent to propagate the phage vector, wherein the first host cell comprises a gene construct of interest (GOI) to be evolved; and wherein the phage vector allows for (i) expression of an error-prone DNA polymerase in the first host cell for mutagenesis, and (ii) replication and packaging of the GOI into infectious phage particles; b) incubating the first host cell under conditions for replication and mutagenesis of the GOI and release of phage particles comprising mutated GOIs; c) infecting a second host cell (selection host) with phage particles from step b); and d) selecting for a desirable function of the GOI in the second host cell.

This invention discloses a reaction tube for recombinase polymerase amplification (RPA), belonging to the field of nucleic acid detection. The reaction tube of this invention includes a tube body and a cap separate from the tube body. The tube body contains lyophilized bulbs or lyophilized powder containing premixed nucleic acid detection reagents, and the cap contains lyophilized powder containing an activator. The tube body and cap are sealed with a thin film. The lyophilized bulbs or lyophilized powder containing the premixed nucleic acid detection reagents include: recombinase, single-stranded DNA-binding protein, strand displacement DNA polymerase, buffer components, ATP, ATP regeneration components, and dNTPs. The lyophilized powder containing the activator includes: potassium ions, magnesium ions, and polyethylene glycol. This invention enables magnesium acetate to stably adhere to the cap, solving the problem of easy spillage of the lyophilized activator in the cap. In use, only the nucleic acid of the sample to be tested needs to be added; the operation is simple, easy to promote and apply, and suitable for batch detection.

The present disclosure relates to compounds of Formula (I):and to their prodrugs, pharmaceutically acceptable salts, pharmaceutical compositions, methods of use, and methods for their preparation. The compounds disclosed herein are useful for modulating DNA polymerase Θ activity and may be used in the treatment of disorders in which DNA polymerase Θ activity is implicated, such as cancer.

This invention provides a DNA polymerase and its applications. The DNA polymerase comprises: (a) a protein having the sequence shown in SEQ ID NO: 1; (b) a protein having at least one or more amino acids substituted, deleted, and / or added at at least one of the following sites in the sequence shown in SEQ ID NO: 1: D147, E149, L424, Y425, P426, and A503; or (c) a protein having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to the sequence in (a) or (b) and possessing DNA polymerase activity. This invention addresses the problem of the limited variety of DNA polymerases in the prior art and is applicable to the field of DNA polymerases.

This invention discloses a dual-linker head, its introduction method, and its applications, relating to the field of biotechnology. The dual-linker head of this invention comprises a long nucleic acid chain and a short nucleic acid chain; the long nucleic acid chain includes a first guide sequence and a support sequence; the long nucleic acid chain is complementary to the short nucleic acid chain through the support sequence; the first guide sequence is 2-30 nt long; the first guide sequence is used to complementarily pair with a second guide sequence at the end of the target nucleic acid. This dual-linker head can freely carry various nucleic acid or nucleic acid aptamer functional modules and is compatible with different sample types, different sequencing platforms, and one-tube or step-by-step workflows. After the end of the target nucleic acid forms or exposes a second guide sequence complementary to the first guide sequence, the dual-linker head can undergo directional annealing with the target nucleic acid to construct a controllable anchoring complex, and further complete the adapter incorporation under the action of DNA ligase and / or DNA polymerase.

Provided is use of a glyconanomaterial for inhibiting a DNA polymerase. The glyconanomaterial is generated by means of dry heating of a saccharide, and the glyconanomaterial comprises a graphene-like nanosheet and the saccharide, wherein the graphene-like nanosheet comprises a carbonization product of at least a part of the saccharide, and the graphene-like nanosheet and the saccharide are combined to form a cross-linked supramolecular structure. The glyconanomaterial can be applied to a hot-start polymerase chain reaction, thereby improving the sensitivity and specificity of the reaction.

Provided are methods of treating a Primary Mitochondrial Disorder (PMD) or Mitochondrial DNA Depletion Syndrome (MDDS) in a subject in need thereof comprising: administering a therapeutically effective amount of a compound listed in Table 1, Table 2, Table 3, Table 4, or Table 5. The subject may have one or more mutations or deletions in the DNA polymerase γ gene (POLG).

This invention discloses a method and kit for quantitative detection of circularized rolling circle amplified miRNAs. The method utilizes T4 RNA ligase to ligate the 3'-OH and 5'-phosphate ends of the target miRNA to form a circular miRNA. Using the circularized miRNA as a template, isothermal rolling circle amplification is performed using phi29 DNA polymerase to generate a large number of single-stranded DNA sequences with repetitive sequences. A universal molecular beacon is used to monitor the fluorescence signal of the amplification products in real time, achieving highly sensitive quantitative detection of miRNAs. This method has advantages such as high sensitivity, high specificity, and a wide linear detection range. It eliminates the need to design specific reverse transcription primers for each miRNA, can be extended to multiplex detection, and is suitable for clinical applications such as early tumor diagnosis, drug efficacy evaluation, and single-cell miRNA expression profiling.

The application relates to the technical field of biological detection, in particular to an N-MSSRCA-CRISPR cascade amplification system for specific recognition of HPV16 E7 mRNA, a construction method and application. Specific recognition is realized through target-dependent connection of Nick probes and Bind probes, a large number of RCA primers are generated through the 'extension-cutting-displacement' cycle of Bst DNA polymerase and nicking endonuclease, long-chain ssDNA products are generated through Phi29 polymerase-mediated MSSRCA rolling circle amplification, and finally secondary amplification of a fluorescence signal is realized through competitive crRNA-mediated CRISPR / LbCas12a cascade activation. The system exhibits a three-level cascade amplification effect, a double-specificity guarantee mechanism and a modular general design in HPV16 E7 mRNA detection, and has clinical transformation value for early screening of cervical cancer.

The application discloses a light-responsive nucleic acid aptamer, a light-activated DNA polymerase, a kit, a method for preparing the light-activated DNA polymerase, a method for activating the light-activated DNA polymerase, and application of the light-activated DNA polymerase. The light-responsive nucleic acid aptamer comprises an aptamer nucleotide sequence and a photo-cleavable group modified in the aptamer nucleotide sequence, the photo-cleavable group is used for being broken after ultraviolet light irradiation to break the aptamer nucleotide sequence into two nucleotide sequences; the aptamer nucleotide sequence comprises a G-quadruplex nucleotide sequence capable of forming a G-quadruplex and a side nucleotide sequence capable of forming a hairpin structure, the G-quadruplex nucleotide sequence is used for being combined with a G-quadruplex binding peptide fused on a DNA polymerase, and the side nucleotide sequence is used for being combined with an active site of the DNA polymerase. By using the light-activated DNA polymerase provided in the application, the difference in amplification time between samples is reduced, non-specific amplification is avoided, and the accuracy of nucleic acid detection is improved.

The present application relates to the technical field of biological detection, in particular to a one-tube pathogen detection method based on PAM-Disruptor regulated CRISPR-Cas12a system, the detection method comprising: constructing a PAM regulated CRISPR-Cas system; mixing and incubating PAM-Disruptor with LbCas12a effector protein, guide RNA, fluorescent reporter, F-primer, R-primer, recombinase, single-stranded DNA binding protein (SSB), DNA polymerase and reaction buffer solution to prepare a premixed reaction solution, and then performing reaction and judgment. The present application realizes light control and non-light control dual-mode one-tube detection, effectively avoids the problems of complicated operation, cross contamination, aerosol pollution and the like caused by step-by-step reaction, and provides a new strategy for one-tube detection of pathogens.

Provided are methods of treating a subject having a Primary Mitochondrial Disorder (PMD) or Mitochondrial DNA Depletion Syndrome (MDDS) comprising administering a therapeutically effective amount of a compound listed in Table 1, Table 2, Table 3, Table 4, or Table 5, wherein the subject does not have a mutation in the DNA polymerase γ gene (POLG).

The application discloses a kind of quick separation and detection methods of pathogenic bacteria in aquaculture water body, belong to the field of microorganism detection.The method reduces sample amount through enrichment of microorganism, does not need complex equipment, is lysed at room temperature and isothermal amplification LAMP detection, significantly shortens detection period, meets the demand of early warning and drug instruction of aquaculture;LAMP detection is carried out using mutant Bst-WK30 DNA polymerase containing specific 7 amino acid mutation sites, with high amplification efficiency and strong specificity, can accurately identify pathogenic bacteria, and can detect a variety of common harmful pathogenic microorganisms.Kit components are synergistic, efficient lysis and specific amplification are realized;The separation device is synergistic through physical filtration and electrochemical enrichment, rapidly separates target microorganisms, reduces impurity interference, enriches microorganisms to filter membrane within 20min, has simple structure and is convenient to carry, and is suitable for on-site rapid sample processing.

The application relates to a rapamycin-G4H nanocapsule, a preparation method and application thereof. The preparation method of the rapamycin-G4H nanocapsule comprises the following steps: S1. preparing a rapamycin micelle by adopting a film hydration method; S2. modifying a dibenzocyclooctyne amplification primer on the surface of the rapamycin micelle through a click chemistry reaction to obtain a rapamycin micelle modified with the amplification primer; S3. taking a linear DNA molecule as a template, and synthesizing a circular amplification template through an intramolecular cyclization reaction; and S4. incubating the circular amplification template, the rapamycin micelle modified with the amplification primer, a thermophilic DNA polymerase, a pyrophosphatase, dNTP and Co-G4-haem DNAzyme under the condition of a thermophilic DNA polymerase reaction buffer. The rapamycin-G4H nanocapsule can play an efficient antioxidant stress and immune inflammatory regulation function, can improve the intraocular microenvironment of a glaucoma patient, and can inhibit the continuous apoptosis of retinal ganglion cells.

The application belongs to the technical field of biology and relates to a phi29 DNA polymerase mutant with reduced polymerization rate and a preparation method and application thereof. The application designs and provides a mutant of phi29 DNA polymerase. The amino acid residues at at least one of positions 190, 324, 411, 49, 83, 375, 526, 85, 149, 284, 287, 35, 115, 178, 102, 246, 336, 506, 257, 502, 55, 113, 187, 289, 306, 517, 527, 140, 203, 368, 372, 89, 54, 277, 327, 163, 224 in the amino acid sequence of the phi29 DNA polymerase are modified, a new protein with reduced polymerization rate of the polymerase and good polymerase activity is generated, problems caused by the too fast polymerization rate of the phi29 DNA polymerase in practical application are successfully solved, the accuracy, stability and reliability of the phi29 DNA polymerase in DNA amplification, sequencing and other molecular biology experiments are improved, and the sequencing quality is greatly improved.

The present application relates to the construction and application of the EcORep-based continuous directed evolution reporter system of aminoacyl-tRNA synthetase, and specifically, the reporter system comprises: a green fluorescent protein mutant sfGFP containing amber stop codon TAG N150TAG ; a chloramphenicol acetyltransferase mutant CAT containing amber stop codon TAG D112TAG ; a linear replicon containing aminoacyl-tRNA synthetase; a reporter plasmid containing a plus-strand mismatch DNA polymerase to continuously mutate the linear replicon, thereby generating an in-vivo library of aminoacyl-tRNA synthetase mutants; and a tRNA, which is orthogonal to the aminoacyl-tRNA synthetase and enables the incorporation of unnatural amino acids and the complete expression of proteins. The present application proves that the evolution reporter system can realize the efficient directed evolution of aminoacyl-tRNA synthetase, and further expands the application range of genetic code expansion technology.

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

The present application relates to a kind of Bst DNA polymerase mutant and its preparation method and application.The Bst DNA polymerase mutant is the mutant obtained on the basis of enzyme shown in amino acid sequence such as SEQ ID No.1;The mutation site includes at least one of K2, T54, M74, S77, C92, D112, D113, K121, E124, A140, Q228, N231, L251, T256, N277, S289, K304, L334, S359, D373, N404, R407, D422, T481, N484, A540, C549 or A579.The mutant library is constructed in the present application, by enzyme activity detection, heat resistance detection and inhibitor screening, and the Bst DNA polymerase mutant with excellent performance is obtained.

The present application relates to chimeric DNA polymerases and methods of making the same. The chimeric DNA polymerases comprise 2-8, e.g., 3, domains or segments derived from different polymerases. The chimeric DNA polymerases of the present application have improved properties, e.g., superior extension properties, superior DNA binding properties, superior proofreading activity, superior fidelity, faster amplification speed, superior tolerance to inhibitors, superior long fragment amplification ability, etc.

A method of amplifying a target nucleic acid in a DNA sample comprising (a) contacting the DNA sample containing the target nucleic acid with a DNA polymerase, at least two oligonucleotide primers designed to flank the target nucleic acid, a mixture of dATP, dGTP, dCTP, and dTTP, and a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer, (b) heating the output of step (a) to a temperature at which the conjugate precipitates and thus the DNA polymerase is no longer inhibited and (c) amplifying the target nucleic acid, e.g. by performing PCR steps of denaturing the target nucleic acid, annealing the primers to the target nucleic acid, and extending the primers, wherein step (c) is repeated at least two times. Also provided is a conjugate comprising a DNA polymerase inhibitor covalently attached to a negative temperature sensitive polymer and kits and aqueous compositions comprising the same.

The application discloses a non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metal beta-lactamase and a screening method thereof. The screening method uses dNaMTP and dTPT3TP, or dCNMOTP and dTPT3TP as non-natural base nucleoside triphosphate substrates, uses OneTaq DNA polymerase to amplify a DNA library containing non-natural bases, and uses lamda exonuclease to degrade 5' end phosphorylated DNA chains in PCR products, so as to obtain two ssDNA libraries. The non-natural base DNA aptamer targeting Bacillus cereus 5 / B / 6 metal beta-lactamase is screened based on the libraries, and three non-natural base DNA aptamers with a length of 70 nt are obtained. The results of a biological membrane layer interference analysis show that the three non-natural base DNA aptamers have good binding affinity to Bacillus cereus 5 / B / 6 metal beta-lactamase.

The technical field relates to methods for promoting gut microbiome health using low G+C Directed (LDN) analogs. More particularly, the technical field relates to methods of using LDN analogs as inhibitors of DNA Polymerase IIIC (DNA pol IIIC) enzyme to selectively reduce physiologically harmful Gram-positive bacteria with a genome having low amounts of Guanine and Cytosine (rather the genomes comprise a greater amount of adenine and thymine / uracil nucleotides) in the gut microbiome while simultaneously maintaining or increasing the proportions of beneficial microflora.

This invention discloses an Ago-DNA polymerase simultaneous dual-cycle amplification system and its applications. The Ago-DNA polymerase simultaneous dual-cycle amplification system comprises: Ago enzyme, DNA polymerase, amplification template, dNTPs, MnCl2, and gDNA; the sequences of the amplification template and gDNA are obtained using the design method of this invention. This system can be applied to amplify and / or detect microRNA. This system enables integrated simultaneous dual-cycle amplification, simplifying the operation steps, reducing reaction complexity, avoiding aerosol contamination, reducing costs, and eliminating the requirement for specific sequences by restriction enzymes such as Cas and Nt.BstNBI. The amplification and / or detection methods using this system are highly versatile, accurate, and efficient, breaking through the bottlenecks of current molecular diagnostic technologies and holding promise for widespread application.

Disclosed herein are mutants of bacteriophage Phi29 DNA polymerase with improved primer recognition, compared to the wild-type enzyme. Certain mutants comprise one or both of the mutations K64R or M97K. The provided mutants are capable of using more efficiently shorter and longer random synthetic DNA primers than wild-type Phi29 DNA polymerase, generating more amplification product in Multiple Displacement Amplification (MDA) reactions. The inventive mutants amplify human genomic DNA with less bias and better coverage in comparison to reactions carried out with wild-type Phi29 DNA polymerase.

This invention discloses the use of nano-magnesium oxide particles as a PCR amplification modifier in hot-start PCR, and also discloses a polymerase hot-start PCR reagent based on nano-magnesium oxide. The magnesium ion release of the nano-magnesium oxide is temperature-dependent, with a release rate of less than 5% below 25°C and a release rate of more than 90% above 95°C. It activates DNA polymerase, initiates PCR amplification, and improves the specificity and amplification efficiency of the PCR reaction. It is suitable for conventional PCR, long-fragment PCR, and amplification of templates with high GC content, and has broad application prospects.

A kind of recombinant KOD polymerase.A KOD polymerase mutant, a protein, as follows: for the amino acid residue in the 267, 326, 347, 349, 353, 375, 378, 379, 380, 385, 451, 452, 453, 454, 457, 461, 465, 470, 474, 477, 478, 479, 480, 482, 484, 485, 486, 493, 496, 497, 514, 574, 584, 605, 610, 630, 665, 666, 667, 674, 676, 680, 682, 698, 707, 718, 723 and 729 of KOD DNA polymerase GH78 amino acid sequence at least one of the 48 positions is modified, other amino acid sequences are unchanged, obtain the protein with DNA polymerase activity;Compared with KOD DNA polymerase GH78, the recombinant DNA polymerase shows stronger reaction rate, better catalytic efficiency, better affinity and other advantages in catalysis, improves the reaction rate of DNA polymerase in sequencing, increases reaction read length.

The application discloses a recombinant Bst DNA polymerase with high DNA polymerase activity and reverse transcriptase activity, and preparation and application thereof. Sulfolobus tokodaii The application carries out sequence rearrangement on a Headpiece domain truncated body Hp47 derived from chicken villin and a Sto7d domain derived from a thermophilic archaea, and fuses the amino terminal end of the Bst DNA polymerase large fragment to construct a novel Bst DNA polymerase rearrangement mutant HpSt-G23. The recombinant polymerase HpSt-G23 obtained by the application has high polymerase activity and reverse transcriptase activity, and can resist inhibitors such as urea and sodium chloride. Tth The HpSt-G23 can be used in a RecA detection system, and only two primers are needed to complete DNA or RNA amplification in the same system, and the detection sensitivity of the RNA template is greatly improved, so that the HpSt-G23 has a good development prospect in the field of nucleic acid detection.

The application discloses a Bst DNA polymerase activity detection method and a kit thereof, and comprises the following steps: isothermal amplification is carried out by using Bst DNA polymerase, a standard curve of maximum fluorescence increment per unit time in a linear growth period of an amplification curve corresponding to double-stranded DNA of an amplification product of a Bst DNA polymerase standard is drawn, the maximum fluorescence increment per unit time in the linear growth period of an amplification product of a Bst DNA polymerase sample to be detected is detected, and the enzyme activity of the sample to be detected is calculated according to the standard curve. 2 The detection range of the enzyme activity is wider than 0.99, the detection accuracy is high, the repeatability is good, and the operation is simple when the enzyme activity is detected in the range of 10-80 U, and the Bst DNA polymerase activity can be quickly and accurately determined.

Recombinant DNA polymerase variants of the DPO4 type are provided having amino acid substitutions that confer improved properties to the polymerase for improved single molecule sequencing applications. Such properties may include enhanced binding to bulky nucleotide analog substrates and precise incorporation thereof into sub-chains. Also provided are compositions comprising such DPO4 variants and nucleotide analogs, as well as nucleic acids encoding the polymerases having the above phenotypes.