51 results about "DNA ligase" patented technology

The invention includes a mutant T7 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T7 DNA ligase. The mutant T7 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary. The preferred mutant T7 DNA ligase has at least one of the following mutations: E63K (SEQ ID NO:4), K73E (SEQ ID NO:6), K137E (SEQ ID NO:7), K174E (SEQ ID NO:9), E182K (SEQ ID NO:11), K210E (SEQ ID NO: 13), E243K (SEQ ID NO:15), D245R (SEQ ID NO: 17), E268K (SEQ ID NO:19), E272K (SEQ ID NO:21), E289K (SEQ ID NO:23), K295E (SEQ ID NO:25) and D336R (SEQ ID NO:27).

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 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 belongs to the technical field of biology, and particularly relates to a T4 DNA ligase mutant with improved connection fidelity. The T4 DNA ligase mutant is obtained by mutating any one or more amino acid sites of lysine at the 365th site and lysine at the 367th site of wild type T4 DNA ligase. Compared with a wild type T4 DNA ligase, the T4 DNA ligase has higher fidelity, and the fidelity of the T4 DNA ligase is maximally improved to more than 10 times of that of the wild type T4 DNA ligase.

A number of T4 DNA ligase mutants exhibiting enhanced ligation activity in the presence of high salt concentrations compared to the wild-type ligase were engineered, characterized, and selected via gel electrophoresis of ligation products from a standard ligation assay. Ligase catalyzes the formation of phosphodiester bonds between the 5′ and 3′ ends of complementary cohesive ends or blunt ends of duplex DNA, a process that is vital to numerous molecular biology processes including cloning and sequencing.

The present invention belongs to the technical field of biological detection, and provides a fluorescence biosensor for jointly detecting glutathione and ATP, comprising: a ligation probe L with nucleotide sequences shown in SEQ ID NO: 1-4, a padlock probe M, an ATP block strand, a reporter probe S, and Mg<supgt;2+< / supgt;, phi29 DNA polymerase, T4 DNA ligase, dNTP; the 5'-end of the padlock probe M is phosphorylated; the 8th and 9th positions of the ATP block strand are modified with disulfide bonds, and the 3'-end is modified with Inverted dT; the 13th and 14th positions of the reporter probe S are ribonucleotides; a fluorescence quenching group is modified before the 13th position, and a fluorescence reporting group is modified after the 14th position. The sensor of the present invention has the advantages of fast detection speed, low detection limit, high specificity, etc., realizes the joint detection of two target substances by one method, can make up for the defects and deficiencies of the existing detection methods, and realizes fast and accurate quantitative detection.

The invention provides a method for controlling activity of deoxyribozyme through intramolecular cyclization and application of the method, and relates to application of the method in the field of biological analysis and regulation. According to the method, a ring-forming chain is subjected to intramolecular connection mainly through T4DNA ligase and a splint chain to form a ring-shaped molecule, so that active conformation cannot be formed, and catalytic activity cannot be lost. Meanwhile, by modifying functional elements (such as photosensitive groups, disulfide bonds and the like) on the deoxyribozyme, the annular deoxyribozyme can be opened and converted into a linear structure under specific conditions (such as illumination, a reducing agent and the like), so that the catalytic activity of the annular deoxyribozyme is recovered. The cyclic deoxyribozyme molecule has a good enzyme digestion resistant effect and biological stability. The method is simple and efficient, can be used for controlling the catalytic activity of deoxyribozyme in living cells, realizes space-time selective sensing analysis and metabolism regulation of target biomolecules, and is extremely high in application value.

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.

The application discloses a kind of for sweat glucose concentration monitoring method, it is related to sweat monitoring field, comprising the following steps: step 1, enzyme-fixed chain conjugate synthesis: glucose dehydrogenase is reacted with bilirubin oxidase respectively with bifunctional crosslinking agent and is purified, obtain enzyme-crosslinking agent intermediate;It is clicked with azide modified DNA fixed chain chemistry reaction again, and after purification, obtain GDH-fixed chain and BOx-fixed chain conjugate;Step 2, preparation of circular DNA template: after annealing hybridization of phosphorylated template chain and primer chain, it is connected into ring by T4 DNA ligase;Step 3, rolling circle amplification self-assembly package enzyme DNA hydrogel;The present application uses super-flexible DNA hydrogel as flexible electrode material, realizes the efficient fixation and continuous stable catalysis of GDH and BOx, and constructs flexible sweat-based EBFC self-powered sensor as flexible bioelectrode, to realize continuous non-invasive blood glucose monitoring.

The invention provides a construction method and application of bacillus amyloliquefaciens capable of enhancing yhcA expression, and the construction method comprises the following steps: taking bacillus amyloliquefaciens XH7 genome DNA as a template, carrying out PCR amplification on a transport protein coding gene yhcA, and connecting a promoter and a terminator to obtain a target gene segment; respectively carrying out double enzyme digestion on a free plasmid vector and a target gene segment by adopting the restriction enzyme to obtain a linear plasmid segment and an enzyme digestion gene segment; connecting the enzyme-digested gene fragment and the linear plasmid fragment through DNA ligase to obtain a free expression vector; and finally, transferring the free expression vector into a competent cell of bacillus amyloliquefaciens XH7, and adding a resistance selection marker to obtain a positive transformant. The research proves that the enhanced expression of the transporter gene yhcA is also a very effective method for improving the inosinic acid yield of bacillus amyloliquefaciens.

The invention provides a method for detecting Ggt in gene mutation. The invention relates to a lock type probe for detecting A mutation, which comprises a recognition sequence complementary with a target p53 R175H and a sequence recognized by CRISPR / Cas12a, and is modified with 8-oxoguanine. The invention also provides a Ggt based on the combination of the Fpg enzyme and CRISPR / Cas12a (clustered regularly interspaced short palindromic repeats / CRISPR / Cas12a). The universal type detection kit for A mutation comprises the lock-type probe, a fluorescence report probe, a primer, crRNA, a T4 DNA ligase, an Fpg enzyme, a phi29 DNA polymerase and a Cas12a protein. The invention also provides a Ggt; the invention discloses an A mutation detection method and application. According to the invention, the Ggt based on the combination of the Fpg enzyme and CRISPR / Cas12a is successfully constructed; according to the universal detection method for the A mutation, a 8-oxoG modified lock-type probe is used for recognizing a mutation site, and rolling circle amplification and CRISPR / Cas12a cascade signal amplification system are combined, so that the core bottlenecks of insufficient universality caused by restriction enzyme dependence and strict dependence of Cas protein on a target PAM site in the existing mutation detection technology are solved in a breakthrough manner, and the purpose of detecting all Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt, Ggt and Ggt mutation is achieved. And broad-spectrum and accurate detection of point A mutation is realized.

The invention relates to a method for detecting a single-stranded break (SSB) site in double-stranded (ds) DNA. A dsDNA sample is contacted with an oligonucleotide probe comprising a first sequencing linker and a tag, and then contacted with a DNA ligase. And the DNA ligase is used for covalently linking the oligonucleotide probe to the dsDNA chain at the dsDNA single-chain breakage site. The dsDNA sample is then contacted with a transposase loaded with a second sequencing linker such that the transposase cleaves the dsDNA to produce a population of DNA fragments comprising the second sequencing linker. DNA strands comprising the first and second sequencing linkers are isolated from the population and the sequence of the isolated DNA strands is determined. These methods and kits for carrying out these methods are provided.

The invention discloses an in-situ capture, amplification and sequencing method of an RNA (Ribonucleic Acid) target sequence, which is characterized in that a lock-type probe with a gap is designed aiming at the RNA target sequence, the 5'end and the 3 'end of the lock-type probe are respectively complementarily hybridized with the upstream and downstream of the target sequence, and a middle gap corresponds to the target sequence. CDNA of a target sequence is generated through reverse transcription, and the cDNA is connected with the 5'end of the probe through DNA ligase to form annular DNA. And after rolling circle amplification, target sequence information is obtained through in-situ sequencing. According to the method, the experimental process is simplified, the detection sensitivity and specificity are improved, the method is suitable for various sample types, and an efficient tool is provided for spatial transcriptomics research.

The invention relates to the technical field of biochemical analysis, in particular to a preparation method of a non-destructive exosome efficient drug loading system, an anti-tumor drug and application. The method comprises the steps of exosome separation and extraction and EVs-RCT synthesis, the exosome is used as a siRNA carrier, Primer-5 'cholesterol connects a Primer sequence to the surface of the exosome through hydrophilic and hydrophobic interaction, the Primer-5' cholesterol is hybridized with an annular template containing siRNA antisense and antisense complementary sequences, and a gap in annular DNA is chemically blocked by T4 DNA ligase; the closed-loop DNA generates RNA transcription products under the condition of T7 RNA polymerase, and a large number of hairpin RNA structures are generated. The high-density RNA structure on the efficient drug loading system not only can improve the drug loading capacity of siRNA, but also can improve the stability of the exosome in a complex environment, and has important significance in the aspects of tumor fluorescence imaging and antitumor drugs.

The invention discloses a construction method of an RNAi lentiviral vector of a targeted GINS1 gene, which comprises the following steps: designing two specific RNAi target sequences respectively shown as SEQ ID NO: 1-2 according to an mRNA (messenger ribonucleic acid) sequence of the human GINS1 gene; designing and chemically synthesizing a corresponding shRNA oligonucleotide single strand aiming at each target spot sequence; carrying out single-chain annealing treatment on the three pairs of synthesized shRNA oligonucleotides to form two kinds of double-chain shRNAs containing RNAi target spot sequences; the method comprises the following steps: inserting double-stranded shRNA into an expression vector linearized by restriction endonuclease, and connecting through DNA ligase; and respectively inserting the two double-stranded shRNAs into an expression vector, transforming competent escherichia coli DH5alpha, culturing, selecting monoclonal colonies, carrying out PCR (Polymerase Chain Reaction) identification and sequencing verification, thereby obtaining the lentiviral vector containing the shRNAs of the GINS1. The specific RNAi target sequence is carried, so that generation and development of breast cancer cells are effectively inhibited, and new guidance is provided for symptom relief and subsequent treatment of breast cancer patients.

A preparation method of a DNA drug delivery platform for ultrahigh-dose load compressed nucleic acid comprises the following steps: selecting a CpG type according to disease types, mixing a DNA template with a phosphorylated 5'end and containing a CpG sequence with a first primer solution, adding a T4 ligase buffer solution and ultrapure water, mixing to form a cyclization system, and heating and annealing in a PCR (Polymerase Chain Reaction) instrument; adding a T4DNA ligase solution, reacting overnight, and inactivating the ligase after the reaction is finished, so as to obtain a circular DNA template; the method comprises the following steps: adding phi29 DNA polymerase, a 10 * phi29 buffer solution, a mixed solution of dATP, dTTP, dCTP and dGTP and Milli-Q into a circular DNA template, mixing to form a reaction solution, and putting the reaction solution into a constant-temperature uniform mixing instrument for reaction; then, adding a second primer and a third primer, and carrying out constant-temperature reaction to form a DNA drug delivery platform; and after the reaction is finished, inactivating the polymerase, and terminating the reaction. The invention also provides application of the DNA drug delivery platform in preparation of drugs for treating cancers or inflammatory diseases. The device can be used for super-compressing and efficiently loading nucleic acid drugs.

The present disclosure relates generally to the enzymatic conversion of methylated nucleic acids in order to differentiate methylated cytosine from unmethylated cytosine in DNA, and more particularly to improved methods and compositions for enzymatic methylation sequencing. In one aspect, various compositions and methods for improving the recovery of methylation signals are provided. The methods include one or more of a nick repair step, methylation signal restoration, use of a modified methylcytosine nucleic acid adapter, and use of a helicase, an ssDNA binding protein, an engineered DNA ligase, or a combination thereof.

Provided is a reaction system for preparing DNA nanospheres (DNB), comprising: a linker; an enzyme, wherein the enzyme comprises a DNA ligase; the linkers comprise a linker A and a linker B, the linker A comprises a first chain and a second chain, the first chain comprises a sequence a, the second chain comprises a sequence a'and a sequence b, and the sequence a and the sequence a 'are complementarily paired; and the linker B comprises a third strand and a fourth strand, the third strand comprises a sequence c and a sequence e, the fourth strand comprises a sequence e ', a sequence f and a sequence b', the sequence b is complementarily paired with the sequence c, the sequence e is complementarily paired with the sequence e ', and the sequence b is complementarily paired with the sequence b'.

The invention belongs to the technical field of biological detection, and particularly discloses a background-free DNA amplification method based on CRISPR-Cas12a pretreatment and application of the background-free DNA amplification method. The background-free DNA amplification method comprises the following steps: S1, carrying out precutting on interference oligonucleotide and a pre-primer by taking double-stranded target nucleic acid in a sample as a CRISPR-Cas12 primer; adding an ASFV dsDNA target into a PCR (Polymerase Chain Reaction) tube containing a buffer solution, a magnetic bead-coated DNA / RNA pre-primer and Cas12a-crRNA for incubation; s2, magnetic separation and amplification of the pre-primer: collecting a precipitate by separating magnetic beads with the help of a magnetic frame; transferring the precipitate into another PCR tube containing a circular padlock template, T4DNA ligase, phi29 DNA polymerase and an SERS label for incubation, and then taking out the solution for enzyme inactivation treatment; and S3, signal detection of an amplification product: adding a NaCl solution with the same volume into the solution subjected to enzyme inactivation treatment obtained in the step S2, and then carrying out Raman detection. The nucleic acid pretreatment scheme provided by the invention can be used as an ideal nucleic acid treatment platform for high-efficiency and high-specificity amplification of target nucleic acid.

The application belongs to the technical field of biological analysis detection, and specifically discloses an acute myocardial infarction biological detection system based on multiplex isothermal transcription amplification, application and method thereof, the system comprises a probe group, PBCV-1 DNA ligase, T7 RNA polymerase and thioflavin T, the probe group comprises probes P1, P2, double-stranded probe P2 / A1 and double-stranded probe A2 / A3, the double-stranded probe A2 / A3 is composed of probe A2 and probe A3, and the double-stranded probe P2 / A1 is composed of probe P2 and probe A1. The application innovatively combines target recognition based on PBCV-1 DNA ligase, double-cycle transcription signal amplification mediated by T7 RNA polymerase and split G-quadruplex / ThT fluorescence signal amplification system, and constructs a triple signal cycle amplification detection system, which has the characteristics of ultra-high sensitivity, excellent specificity and simple operation.

The present invention discloses a method and application for functionalization of engineered DNA crystals after crystallization. The present invention constructs a rigid triangular tensile structural unit, including three different DNA component chains, a total of seven chains, the side chains extending in three double helix directions, and the central chain with three repeating sequences is located in the center of the structural unit. Each double helix structure has two sticky ends with two bases at both ends, which interact with each other to form a very ordered 3D crystal network. T4 DNA ligase is used to connect the sticky ends to obtain stable DNA crystals. The crystal structure of the present invention is stable, the target central chain can be added with complex modifications, and it has universality. The crystal has a wide range of uses in medical disease detection.

The present invention belongs to the field of biological detection technology, and specifically relates to a biosensor for detecting exosomes based on double rolling circle separation, comprising: template CT1 with nucleotide sequences shown in SEQ NO: 1-10, template CT2, Connector, DNA-chol, CP1, CP2, hairpin probe H1, hairpin probe H2, Reporter, crRNA, and Cas12a, T4 DNA ligase; the 3'-end of the DNA-chol is modified with cholesterol; the 5'-end and 3'-end of the Reporter are respectively connected with a fluorescent reporter group and a fluorescent quenching group. This sensor has the advantages of fast detection speed, simple operation, low price, low detection limit, high specificity, etc.

The invention includes a mutant T4 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T4 DNA ligase. The mutant T4 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary.

The invention provides a T4 DNA ligase variant which has increased T / A connection specificity, in the library construction process, the library yield and the library quality can be improved by using the T4 DNA ligase variant, and the A / T separation problem in offline data is solved to a certain extent.

The invention discloses a lentivirus sgRNA one-step method convenient vector construction kit which comprises the following steps: designing sgRNA: designing an sgRNA sequence aiming at a specific knockout or knockout site of a target gene, including an upstream primer and a downstream primer; primer annealing: carrying out annealing treatment on the designed sgRNA upstream and downstream primers to form a double-stranded sgRNA template; one-step vector construction: in a single reaction system, mixing an annealed sgRNA template, a no-load fusion plasmid, DNA ligase and a necessary reaction buffer solution, carrying out enzyme digestion and enzyme linked reaction, and directly constructing a CRISPR-Cas9 gene editing vector without a tedious gel recovery step; and plasmid transformation: transforming the constructed vector into a competent cell, and carrying out amplification to obtain a large number of CRISPR-Cas9 gene editing plasmids containing the target sgRNA sequence. According to the method, carrier construction is completed through a one-step method, a tedious gel recovery step is not needed, the experimental period is greatly shortened, the construction efficiency is improved, and the experimental cost is reduced.

The invention relates to the technical field of sequencing, in particular to a preparation method of a sequencing library and a reagent. The invention provides a preparation method of a sequencing library, which comprises the following steps of: additionally adding an additive or an additive combination capable of reducing a DNA secondary structure and / or reducing single-stranded DNA degradation in single-stranded cyclization reaction and / or rolling circle amplification reaction; an additive or an additive combination capable of reducing a DNA secondary structure, reducing single-stranded DNA degradation and / or reducing double-stranded DNA double-helix stability is additionally added in a double-stranded cyclization library rolling circle amplification reaction, so that the binding capacity of DNA ligase and / or strand displacement DNA polymerase and target nucleic acid is improved, and the situation that the rolling circle amplification efficiency of different circular DNAs is not uniform due to the secondary structure is reduced; the probability that the loose single-stranded DNA is easy to degrade is reduced, and the amplification uniformity of different GC regions and different sequence characteristic regions of the library is improved, so that the sequencing quality and the sequencing data yield are improved.

The invention relates to the technical field of gene editing, and discloses a gene editing efficiency detection vector based on double-color fluorescent protein as well as a construction method and application thereof. The construction method comprises the following steps: synthesizing a full-length DNA fragment containing a specific targeting sequence, an mCherry reporter gene and a C-terminal fused PEST degrading peptide sequence; performing double enzyme digestion and purification to obtain a target fragment; connecting the target fragment with the vector skeleton subjected to enzyme digestion linearization through T4 DNA ligase; the gene editing efficiency detection vector pLV3-CMV-RGFP-PEST-Neo is prepared by the following steps: carrying out gene editing efficiency detection on the gene editing efficiency vector pLV3-CMV-RGFP-PEST-Neo, transforming the gene editing efficiency vector pLV3-CMV-RGFP- The system containing the gene editing efficiency detection vector co-expresses red and green, during editing, an mCherry coding sequence is cut off, cells are converted from double positive (yellow) to single positive (green), cell lysis is not needed, the editing efficiency is quantified by calculating the change of the green / red fluorescence ratio, and the method is convenient and fast.

This invention discloses a highly active T4 DNA ligase mutant, its preparation method, and its applications. In this invention, through crystal structure and bioinformatics analysis combined with site-directed mutagenesis, saturation mutagenesis, and combinatorial mutagenesis, as well as rigorous experimental analysis, several T4 DNA ligase mutants with significantly improved performance were screened, including single mutants and combinatorial mutants. The improved performance includes: enzyme activity, thermal stability, and / or ligation efficiency.

B cells with an altered function. FIG. 1A Rearrangement at the Ig locus n - 250-1,000) VH1 VHP DH1-12 T FEu DNA-PKcs RAG1 / 2 VIDN Excised Ku70, Ku80 NHEJ recombination intervening DNA ARTEMIS XROC4 DNA Ligase N V DJ Cp C6 OB Cr1 0120 area Ce Cex 3' RR Eu Su Se At S regions: DNA breaks AD Class-switch DNA repair recombination IgM V D JCu Transcription Recombined CH locus V DJ Ce Ca 3' RR Cy2a Cu Eµ C8 Excised Cy2b intervening DNA IgE V DJCe Cy3 Cyl 20 26 20 47 61 19 J un 2 02 6 A B S T R A C T 1 9 J u n 2 0 2 6 2 0 2 6 2 0 4 7 6 1 F I G . 1 A R e a r r a n g e m e n t a t t h e I g l o c u s V H 1 V H n D H 1 - 1 2 7 F E u r D N A - P K c s R A G 1 / 2 V I D N N H E J V D J C p C 6 O B 0 1 2 0 O R D C e E u S u S e A D r e c o m b i n a t i o n I g M V D J C u R e c o m b i n e d V D J C e C a 3 ' R R C y 2 a C u E u r C 8 E x c i s e d C y 2 b I g E V D J C e C y 3 C y l

The invention includes a mutant T7 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T7 DNA ligase. The mutant T7 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary. The preferred mutant T7 DNA ligase has at least one of the following mutations: E63K (SEQ ID NO:4), K73E (SEQ ID NO:6), K137E (SEQ ID NO:7), K174E (SEQ ID NO:9), E182K (SEQ ID NO:11), K210E (SEQ ID NO: 13), E243K (SEQ ID NO:15), D245R (SEQ ID NO: 17), E268K (SEQ ID NO:19), E272K (SEQ ID NO:21), E289K (SEQ ID NO:23), K295E (SEQ ID NO:25) and D336R (SEQ ID NO:27).