444 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 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 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 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.

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.

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.

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 present disclosure relates to the technical field of sequencing, and in particular to a method and reagent for preparing a sequencing library. Provided in the present disclosure is a method for preparing a sequencing library. According to the method, by means of additionally adding an additive or an additive combination capable of reducing the DNA secondary structure and / or reducing the degradation of single-stranded DNA in the single-chain cyclization reaction and / or the rolling circle amplification reaction, and additionally adding an additive or an additive combination capable of reducing the DNA secondary structure and reducing the degradation of single-stranded DNA and / or reducing the stability of the double helix structure of double-stranded DNA in the rolling circle amplification reaction of the double-stranded circular library, the ability of the DNA ligase and / or the strand displacement DNA polymerase to bind to a target nucleic acid is enhanced, such that the non-uniform rolling circle amplification efficiency of different circular DNAs caused by the secondary structure is reduced, the probability that the loose single-stranded DNA is easily degraded is reduced, the amplification uniformity of regions with different GC contents and different sequence characteristics in the library is improved, and thus the sequencing quality and the yield of sequencing data are improved.

The invention relates to the technical field of biological detection, and discloses a method for double-ring detection of lung cancer L858R mutation based on CRISPR / Cas9 and DNA, and the method comprises the following steps: enriching circulating tumor cells CTC from a blood sample; extracting genomic DNA (deoxyribonucleic acid) of the enriched CTC; carrying out amplification on the DNA fragment containing the L858R mutation by adopting recombinase polymerase amplification RPA (recombinase polymerase amplification); the amplified DNA fragment is hybridized with a recognition ring in a DNA double-ring structure, a DNA double ring is composed of the recognition ring and a report ring, and a part of region of the recognition ring is hybridized with a corresponding fragment of the report ring to form a staggered double-chain region; when L858R mutation exists, the mutated DNA and a recognition ring are hybridized to activate a CRISPR / Cas9 system, and under the guidance of sgRNA, Cas9 cuts hybrid double strands and releases a report ring; carrying out rolling circle amplification RCA under the action of DNA polymerase by taking the released report ring as a template; a fluorescent probe is used for detecting an RCA amplification product, and whether L858R mutation exists or not is judged according to a fluorescence signal. The CRISPR / Cas9 system is combined with a DNA double-ring structure, so that the ultra-sensitive and specific detection on the lung cancer L858R mutation is realized.

The invention provides a method and a kit for detecting genotypes of SNP (single nucleotide polymorphism) sites in target genes by cascade amplification, and the method comprises the following steps: designing a first primer, a second primer and a medium probe aiming at the target genes containing to-be-detected SNP sites, and designing an amplification probe according to a medium sequence of the medium probe, and designing a detection probe according to the single-chain report sequence of the amplification probe, and carrying out PCR amplification and amplification product analysis in a reaction system containing the upstream first primer, the second primer, the medium probe, the amplification probe, the detection probe, a nucleic acid sample containing the target gene and DNA polymerase. The method is a non-target-dependent detection method, a melting peak with a melting point of a certain duplex body is detected in melting curve analysis, the target gene SNP typing corresponding to the duplex body can be judged, and by means of the advantages of PCR amplification and two times of PCR index amplification by combining the medium primer and the amplification probe, the SNP typing of the target gene corresponding to the duplex body can be judged. A large amount of target nucleic acid can be enriched, and the detection sensitivity is improved.

The invention discloses a single molecule detection kit for a plurality of non-coding RNAs (Ribonucleic Acid) in human breast tissues by using a programmable automatic cascade mechanism and application of the single molecule detection kit. The preparation method of the single molecule detection kit comprises the following steps: incubating piRNA-36026, lncRNA-DSCAM-AS1, a dumbbell probe-pi, a dumbbell probe-lnc, a primer-pi, a primer-lnc, a dNTP mixed solution, a ThermoPol reaction buffer solution and a reaction solution of Bst DNA polymerase (large fragment); and adding the reaction product into a reaction solution containing the signal probe 1, the signal probe 2, an NEBuffer 4 reaction buffer solution and T7 exonuclease, and incubating to obtain the single molecule detection kit. According to the present invention, the efficient automatic cascade mechanism and the high signal-to-noise ratio single molecule detection are adopted to achieve the sensitive simultaneous detection of a variety of ncRNAs, and the high sensitivity, the good selectivity and the multi-path analysis ability are provided.

The application discloses a kind of compositions, reagents and its detection method for high-throughput gene sequencing, belong to gene sequencing technical field, the composition includes Tris-HCl buffer 80-120 parts, potassium chloride 40-60 parts, ammonium sulfate 10-20 parts, magnesium chloride 15-25 parts, dNTPs mixed solution 30-50 parts, heat-stable DNA polymerase mutant 8-15 parts, PCR promoter complex 5-12 parts, modified iridium quinoline ketone complex 1-5 parts, glycerol 150-250 parts, Tween-20 1-3 parts, EDTA disodium salt 0.5-2 parts and nuclease-free ultrapure water, by introducing the modified iridium quinoline ketone complex of innovation, effectively solve the technical problems of low amplification efficiency, poor multiplex PCR reaction uniformity and high non-specific amplification background for high GC content, complex secondary structure genomic template, significantly improve the specificity of amplification, uniformity and the quality and reliability of final sequencing data.

The present invention discloses phi29 DNA polymerase mutants with improved performance, their preparation methods and applications, which relate to the field of biotechnology. Specifically, the present invention discloses phi29 DNA polymerase mutants that contain mutation sites Y163K, F181K, Y224K, W277K / W277P, Y298K, Y369K, Y439K, F526R relative to wild-type phi29 DNA polymerase; the amino acid sequence of the wild-type phi29 DNA polymerase is as shown in SEQ ID NO:1. Compared with the wild-type phi29 DNA polymerase, it has higher specific enzyme activity, stability and higher amplification efficiency, and has good application prospects in the fields of nucleic acid amplification and sequencing, etc.

The invention discloses a method for synthesizing millions of unique DNA tags through primer combination and application of the method, and relates to the field of molecular biology. According to the method, partial complementary pairing characteristics of F, X and Y primers are utilized, and under the synergistic effect of T4 DNA polymerase and ligase, millions of unique DNA tags are generated through one-time reaction through annealing-filling-ligation three-step reaction. The label structure comprises 5'end functional modification sites (such as amino and biotin) and a customizable sequencing joint, and is suitable for high-throughput labeling scenes such as single-cell multiomics, space proteomics and antibody coupling (AOC). Compared with a traditional one-by-one synthesis method, the method has the advantages that the single-tag synthesis cost is reduced by 80% or above, a reaction system is compatible with automatic operation, the tag combination complexity can reach 5.6 * 10 (based on 384 * 384 * 384 primer combination), and an efficient solution is provided for large-scale molecular marking.

The invention discloses a method for targeted enrichment of polymorphic sites by introducing thio-modified nucleic acid into a single base. According to the invention, the selector contains a segment of primer extension binding region with more than four basic groups, when the selector is extended by taking a target DNA / RNA as a template, single thio-modified dNTP and another three terminated dNTP / ddNTP, denaturation and renaturation are carried out, a hairpin structure is formed by itself, the selector without thio-modification is digested under the action of T4DNA polymerase, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and then the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA, and the target DNA / RNA is converted into the target DNA / RNA. And complementing the sequence at the incision to form an intermediate product with a hairpin structure, so as to fulfill the aim of enriching rare mutation sites. The method is suitable for DNA and RNA templates, and the obtained hairpin ring structure intermediate can be subjected to signal amplification in two ways: enzyme-mediated nucleic acid amplification or PCR amplification after linearization of a ring structure.

The present invention relates to cells comprising an orthogonal DNA replication machine, linear plasmids replicable by said machine, uses of said cells and said linear plasmids, methods of maintaining linear plasmids in cells, methods of evolving sequences of interest, and methods of preparing polypeptides or nucleic acids. The invention also relates to error-prone DNA polymerases, nucleic acids encoding said polymerases and uses thereof.