20 results about "DNA ligase" patented technology

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 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 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.

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 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 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).

The invention provides a multifunctional nano-composite, which is formed by electronegative DNA nano-flower and cationic liposome through electrostatic self-assembly, the DNA nano-flower takes single-stranded DNA containing CpG motif as a template, and is synthesized through Phi29 DNA polymerase rolling circle amplification reaction after T4 DNA ligase cyclization; the cationic liposome is composed of a single component cationic lipid NGPE, and an NR1D1 agonist SR9011 is entrapped in the cationic liposome. The invention also provides a preparation method of the nanocomposite. The invention also provides an application of the nano-composite in preparation of drugs for radioimmunotherapy of triple negative breast cancer. According to the invention, the technical problems of insufficient immune activation, high tumor radiation resistance, low hydrophilic / hydrophobic drug co-delivery efficiency, tumor microenvironment immunosuppression and the like in the existing radiotherapy are solved, and the radioimmunotherapy curative effect of the triple negative breast cancer is improved.

The present invention relates to a method for detecting single-strand breaks (SSBs) in double-stranded (ds) DNA. A sample of dsDNA is brought into contact with an oligonucleotide probe containing a first sequencing adapter and tag, and then into contact with a DNA ligase. The DNA ligase covalently binds the oligonucleotide probe to the dsDNA strand at the single-strand break sites in the dsDNA. The dsDNA sample is then brought into contact with a transposase loaded with a second sequencing adapter, resulting in the cleavage of the dsDNA by the transposase and the generation of a population of DNA fragments containing the second sequencing adapter. DNA strands containing the first and second sequencing adapters are isolated from the population, and the sequences of the isolated DNA strands are determined. A method and a kit for carrying out the method are provided.

The present application relates to a product based on dumbbell-shaped probe and RCA for KRAS gene detection, the product comprises a dumbbell-shaped probe, a DNA ligase and RCA amplification reagents, the dumbbell-shaped probe comprises a stem and two loop parts, the ends of the two loop parts are connected through the stem, one of the loops of the dumbbell-shaped probe contains a G-quadruplex aptamer PW17 complementary sequence, and the other loop contains a G-quadruplex aptamer T30695 complementary sequence, the RCA amplification reagents comprise Phi29 DNA polymerase, dNTPs and Phi29 DNA polymerase reaction buffer, under the mediation of the DNA ligase and the detected gene, the stem of the dumbbell-shaped probe is opened, the nick ligation forms a closed loop structure, and serves as a template for RCA. By adopting the technical scheme, the present application provides a product based on dumbbell-shaped probe and RCA for KRAS gene detection, compared with the traditional method, when testing single nucleotide mutation of the gene, it is more convenient and more efficient.

This invention discloses a PSA detection method based on a black phosphorus / bismuth vanadate composite material, comprising: S1, coating a microplate with a PSA monoclonal antibody, blocking it, adding the PSA sample to be tested and incubating it, then incubating it with a PSA polyclonal antibody, a thiolized oligonucleotide sequence 1, and a gold nanoparticle conjugate to form a sandwich immune complex; S2, adding a circular DNA template, DNA ligase, and other reagents, and incubating to complete RCA amplification; S3, adding a thiolized oligonucleotide sequence 2 and a conjugate with alkaline phosphatase, and adding a substrate for hydrolysis to generate ascorbic acid; S4, using a biosensor to perform photoelectrochemical testing under simulated sunlight and voltage, recording the oxidation current signal to quantify the PSA concentration. The PSA detection method disclosed in this invention, using a biosensor modified with this composite material and employing a signal amplification strategy, achieves highly sensitive and stable detection of PSA, which has significant clinical implications for the early diagnosis of prostate cancer.

The invention discloses a plant insect-resistant related protein as well as a coding gene and application thereof. According to the invention, the full length of the SlJAIB14 gene is amplified from tomato, and two restriction enzyme cutting sites of BamHI and Sac I are introduced to the 5'and 3 'ends of the gene through primer sequences. The newly synthesized gene is connected with a pYPX158 plant expression vector containing double 35S promoters through a T4 DNA (Deoxyribose Nucleic Acid) ligase, so that a recombinant plasmid pYPX-S1JAIB14 containing a target gene S1JAIB14 is obtained. According to the invention, plasmids are introduced into agrobacterium tumefaciens (GV3101) by using an electric shock method. The SlJAIB14 gene is transformed into tomato by using an agrobacterium-mediated method, and the resistance of the transgenic plant to cotton bollworm is verified through a feeding experiment.

A preparation method of a super high dose loaded compressed nucleic acid DNA drug delivery platform, according to the disease type, select CpG type, mix the 5' end phosphorylated DNA template containing CpG sequence with the first primer solution, add T4 ligase buffer, mix with ultrapure water to form a circularization system, heat annealing in a PCR instrument; add T4 DNA ligase solution, overnight reaction, after the reaction is completed, inactivate the ligase, obtain the circular DNA template; add phi29 DNA polymerase, 10x phi29 buffer, mixed solution of dATP, dTTP, dCTP, dGTP, Milli-Q to the circular DNA template to form a reaction solution, and the reaction solution is placed in a constant temperature mixing instrument for reaction; then, the second primer and the third primer are added, and constant temperature reaction is carried out to form a DNA drug delivery platform; after the reaction is completed, the polymerase is inactivated, and the reaction is terminated. The application also provides application of the above-mentioned DNA drug delivery platform in preparation of treatment of cancer or inflammatory diseases. The application can super-compress and efficiently load nucleic acid drugs.

The invention designs a preparation method of an electrochemical sensor based on a multi-branched chain annular DNA signal probe. C0 and S1 form an annular template under the action of T4 DNA ligase, and S2 is added to form a multi-branched chain annular DNA structure. Thi / Au (at) PBA and S3 are incubated to synthesize a signal probe, and the signal probe and the multi-branched-chain annular DNA structure are incubated to form the multi-branched-chain annular DNA signal probe. In a magnetic separation system, a histamine aptamer chain Apt is connected to a magnetic bead, a multi-branched chain annular DNA signal probe is added to be connected with an aptamer, when histamine exists, histamine and the aptamer chain Apt are combined to enable the multi-branched chain annular DNA signal probe to fall off, and the multi-branched chain annular DNA signal probe is detected. Dropwise adding a multi-branched chain annular DNA signal probe in the supernate to the surface of an electrode modified by the gold-palladium core-shell nano material, connecting through a palladium-sulfur bond, and determining the signal change of Thi to realize quantitative detection of histamine. The multi-branched chain annular DNA signal probe and the nano material are introduced, so that the accuracy and the sensitivity of the sensor are improved.

The application relates to a preparation method and application of a pH-driven aggregation death receptor 5 combined enzyme cascade DNA nanospring, which can effectively solve the problem that the prior art cannot respond to pH aggregation DR5 while promoting DR5 expression, and the technical solution is that the DNA nanospring comprises DR5TP, glucose oxidase and horseradish peroxidase; different DNA chains are annealed, hybridized and connected by T4 DNA ligase to synthesize the DNA nanospring coding i-motif, the DR5TP, GOX and HRP are modified through biotin-streptavidin mutual bonding to obtain the pH response aggregation death receptor 5 combined enzyme cascade DNA nanospring; the application is based on a pH response nucleic acid self-delivery strategy, tumor cell apoptosis signal activation is induced by regulating ligand spacing, contraction is realized by responding to a tumor micro-acid environment, effective activation of the DR5 receptor is realized, and the enzyme cascade reaction is ingeniously combined to promote cell apoptosis, which is an innovation on DNA nanomaterials.