24 results about "DNA Ligases" patented technology

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 invention relates to the technical field of biochemistry, and discloses a DNA chimera preparation and application method, which comprises the following steps: synthesizing phosphorylated linear annular L-rTDNA and template DNA, mixing in a 1 * T4 DNA ligase buffer solution according to an equal molar concentration of 10 [mu] M, heating at 95 DEG C for 5 minutes, and then carrying out gradient cooling to room temperature for renaturation for 120 minutes; adding T4DNA ligase to close a tail end notch, and carrying out enzyme digestion treatment to obtain annular rTDNA; the method comprises the following steps: synthesizing hairpin DNAHP (deoxyribonucleic acid), mixing rTDNA and HP according to an equal molar concentration of 2 mu mol / L, denaturating at 95 DEG C for 5 minutes, slowly cooling to 25 DEG C within 2 hours, and self-assembling to obtain the DNA topology limited chimera. According to the invention, a DNA chimera with dynamic response capability is constructed, an integrated detection process of enzyme-free assembly-target triggering-signal cascade amplification is realized, and meanwhile, the problems of anti-interference and ultra-sensitive detection in a complex matrix are solved.

The invention discloses a cell-free cloning reaction system, a kit and application of the cell-free cloning reaction system. The system comprises a cell lysis solution, an inserted DNA fragment and a linear vector, the cell lysis solution is obtained from Escherichia coli which is subjected to induced expression and then subjected to lysis, and a lambda Red system and a ligase system are expressed in the Escherichia coli; the lambda Red system comprises Exo protein, Beta protein and Gam protein; and the ligase system comprises DNA ligase. The optimized lambda Red system and the efficiently expressed DNA ligase system can complete efficient splicing of multi-fragment DNA within 15 minutes at 37 DEG C, still maintain a high positive rate in a multi-fragment assembly system, show an effect superior to Gibson cloning, and expand the applicability of the Gibson clone in complex pathway synthesis and gene line reconstruction.

This invention relates to a hairpin primer for detecting miRNA and its applications. The invention develops a reassembly ligation detection scheme for miRNA detection, designing hairpin primers and developing a two-step detection process. The first hairpin primer loop structure binds to the target miRNA, opening the stem structure. The opened stem structure attracts the remaining miRNA sequence, reassembling with the second hairpin primer to form a T-shaped double-stranded structure. The two hairpin primers, using the DNA strand as a clamp, can be ligated by DNA ligase to form a long fragment containing both the miRNA's reverse-specific and universal sequences for qPCR detection. The stem-loop design improves ligation specificity, eliminates Splint ligase dependence, reduces cost, and increases efficiency. The ligated long fragment contains both specific and universal sequences, facilitating multiplex qPCR detection.

The invention discloses a DNA coding compound library screening method based on an adjacent ligation strategy and application, and belongs to the technical field of biological medicine. According to the method, a DNA coding protein strategy is introduced on the basis of a traditional DNA coding compound library screening technology; when the compound is combined with the protein or induces the two proteins to form a compound, the nucleic acid tags carried by the protein and the compound are close to each other and can be connected by DNA ligase; amplifying the connected nucleic acid tag by using primers which are respectively combined with the protein and the compound nucleic acid tag; and determining the compound and corresponding protein information through a high-throughput sequencing technology. The method not only can be used for simultaneously screening binding agents of multiple proteins, but also can be used for simultaneously screening compounds for inducing protein-protein proximity, and has application prospects in drug research and development, disease treatment mechanism research and biological signal channel regulation and control.

A method of ligating a ligation product for detecting abasic sites on DNA target sequence is described. The ligation method uses a pyrene probe that ligates with a second probe in the presence of a ligase. A pyrene may comprise 5′-phosphate 1′-pyrene deoxyribonucleotide at the 5′-terminus. The second probe may comprise a terminal 3′-hydroxy, wherein the 3′-hydroxy is ligated with the pyrene probe. The ligase may be T4 DNA Ligase or PBCV-1 as the latter has been found to improve selectivity. The abasic on the DNA target sequence may be naturally occurring or may be a generated abasic that is formed by the addition of an enzyme. Furthermore, selectivity may be increase by the addition of adenosine triphosphate (ATP) or ligating at a temperature above the probe:target sequence thermal disassociation temperature.

The present disclosure relates to engineered DNA ligase polypeptides and compositions thereof, and polynucleotides encoding the engineered DNA ligase polypeptides. The disclosure also provides methods of using the engineered DNA ligase polypeptides, or compositions thereof, in diagnostics and as molecular biology tools.

We described herein are click edits, which are a series of genome editing methods that combine DNA nicking enzymes, including but not limited to RNA programmable CRISPR nicking enzymes, with DNA ligases or DNA polymerases to perform many different genome edits. Edits of interest are encoded on nucleic acid templates or substrates (optionally'click nucleic acids' (clkNA) or'click DNA '(clkDNA) as described herein) that can be provided in trans form or recruited preferentially to a target site via a variety of nucleic acid telomere methods (optionally HUH family endonucleases (HUHe), telomere binding proteins (TBP), or DNA binding proteins or RNA binding proteins).

The present disclosure provides novel mirror image CRISPR compositions that include components such as mirror image CRISPR-associated endonucleases and mirror image guide RNA for processing of mirror image nucleic acids. In some embodiments, the composition is a mirror image DNA-cleaving and / or modifying composition including a D-form DNA endonuclease (e.g., a D-form Cas protein) and a L-form guide RNA that includes a sequence complementary to a target sequence of a target L-DNA. In some embodiments, the composition further comprises a D-form DNA ligase (e.g., a D-form T4 ligase protein). Also provided are D-form Cas protein and D-form T4 ligase protein compositions, synthetic precursors thereof, and methods of preparing the same.

The invention discloses a bar code sequence for nanopore sequencing, ligase and application of the bar code sequence and the ligase. Firstly, a bar code sequence for nanopore sequencing is disclosed, the bar code sequence is composed of an internal bar code sequence and an external bar code sequence which have complementary replaceable areas, and the internal bar code sequence corresponds to SEQ ID NO.1 and SEQ ID NO.2; and an external bar code sequence corresponds to SEQ ID NO.3 and SEQ ID NO.4. The invention further discloses a modified T4 DNA ligase with a sequence as shown in SEQ ID NO.194 and application of the modified T4 DNA ligase and a bar code sequence in nanopore sequencing and library building. According to the bar code sequence, bar code combination is achieved through the pre-ligation process to obtain a double bar code product, the variety of bar codes is greatly expanded, meanwhile, the connection efficiency of the double bar code product and a substrate is improved through the modified T4 DNA ligase, the sequencing cost is remarkably reduced, and the sequencing efficiency is improved.

The present application relates to a kind of genetic code NAD+ protein probe based on resonance energy transfer and its preparation method and application, specifically disclose a kind of genetic code NAD+ protein probe, it is formed by resonance energy transfer donor, NAD+ response protein and resonance energy transfer acceptor series connection;Wherein NAD+ response protein is the mutant of DNA ligase, the sequence of the mutant of DNA ligase is as shown in SEQ ID NO.3, or SEQ ID NO.6;The resonance energy transfer donor is selected from luciferase or fluorescent protein;The resonance energy transfer acceptor is selected from fluorescent protein and the fluorescent protein as resonance energy transfer acceptor is different from the fluorescent protein of resonance energy transfer donor.The protein probe of the present application can be synthesized in living cell and be used to detect NAD+ concentration in living cell.

This invention discloses a DNA-encoded multiplex SNV detection system, detection method, and application, belonging to the field of molecular diagnostics and gene detection technology. The system includes an SNV recognition and amplification module, a signal amplification and encoding module, and a product enrichment module. The detection method involves designing CP and TP probes targeting different SNV sites; CP and TP hybridize with target DNA in the sample; a thermostable DNA ligase catalyzes the formation of a ligation product between CP and TP under perfectly matched conditions; complementary probes HT1 and HT2 are introduced for thermal cycling amplification; fluorescently labeled hairpin probes H1 and H2 are added, triggering the HCR self-assembly to form a long-chain DNA polymer, outputting a detectable fluorescent signal. This invention achieves highly sensitive and specific detection of multiplex SNVs within a single tube through different fluorescent combinations, and is suitable for fields such as genetic disease screening and tumor mutation detection.

The invention discloses an oligonucleotide composition, a kit and a method for constructing a single-cell multiomics library, and the construction method comprises the following steps: in a single-cell same drop, carrying out short-promoting PCR (Polymerase Chain Reaction) by using a long primer with T1 as a 3'anchor point, writing AA / AB / BB oriented transposition fragments into a bar code and a sequencing handle, carrying out parallel PCR amplification after demulsification and purification, and carrying out sequencing by using a sequencing handle to obtain the single-cell multiomics library. Only the AB-oriented transposition fragment is subjected to exponential amplification; dividing an amplification product into two parts, and respectively carrying out cDNA and ATAC library establishment, so as to obtain the gene. According to the construction method of the single-cell multi-omics library, DNA ligase or bridging oligonucleotide does not need to be used, the substrate utilization rate, molecular diversity and process robustness are remarkably improved, and the construction method is compatible with a mainstream single-cell multi-omics work process.

The invention relates to the technical field of anti-tumor drugs, in particular to a drug targeting exosomal DNA, a preparation method and application of the drug in treating urothelial carcinoma, the drug is an antibody drug conjugate and comprises a bispecific antibody and a cytotoxic drug coupled with the bispecific antibody, the bispecific antibody can be combined with a DNA ligase III and a programmed death ligand 1 at the same time, the DNA ligase III is a nuclear DNA ligase III, an extrachromosomal DNA maintenance mechanism is used as a treatment target for the first time, cyclization and maintenance of extrachromosomal DNA are destroyed by targeting the nuclear DNA ligase III, and the driving effect of oncogene amplification is weakened fundamentally.

The present invention provides a novel DNA ligase, comprising: an amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4; or, compared with the amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4, an amino acid sequence obtained after substitution and / or deletion and / or addition of one or more amino acid residues and retaining DNA ligation activity; or an amino acid sequence having at least 70% identity with the amino acid sequence as shown in SEQ ID NO: 2 or SEQ ID NO: 4 and retaining DNA ligation activity. The novel DNA ligase of the present invention has good ligation activity, and can be applied to a variety of scenarios.

One or more enzymes are used to repair damage in synthetic DNA molecules that encode digital information. The enzymes are included in a repair mixture containing one or more of DNA polymerase, DNA ligase, T4 Endonuclease, Endonuclease IV, Endonuclease VIII, and uracil glycosylase. The repair mixture may also contain one or more of a buffering solution, oxidized nicotinamide adenine dinucleotide (NAD+), and deoxyribose nucleoside triphosphates (dNTPs). The synthetic DNA molecules are incubated with the repair mixture for approximately four hours. Use of the repair solution allows recovery of the digital information from damaged DNA molecules.

The invention provides a ligase buffer solution, a product thereof and application of the ligase buffer solution in mRNA tailing, and relates to the technical field of nucleic acid synthesis. The invention provides a T4 DNA (deoxyribonucleic acid) ligase buffer solution. The T4 DNA ligase buffer solution is prepared from the following components: Tris-HCl, ATP (adenosine triphosphate), MgCl2, reduced glutathione (GSH), polyvinylpyrrolidone K30 (PVP K30) and Triton X-100; the buffer solution remarkably improves the connection efficiency of T4 DNA ligase, and can be applied to mRNA tailing based on enzyme digestion and connection.

The invention discloses a kit for rapidly constructing an endothelin converting enzyme ECE1 knock-down plasmid and application of the kit, and relates to the technical field of molecular biology and genetic engineering. Comprising the following components: a high fidelity PCR reagent which comprises # imgabs0 # High-Feliability DNA (deoxyribonucleic acid) Polymerase, a buffer solution and dNTPs (deoxyribonucleoside triphosphates) and is used for amplifying an ECE1 gene; the double enzyme digestion reagent comprises AgeI, EcoRI and a buffer solution thereof, and is used for linearizing the carrier; and the T4 DNA ligase and the buffer solution are used for connecting the ECE1 gene and the vector. By adopting high-fidelity PCR and seamless cloning technologies, the plasmid construction time is remarkably shortened, and the experiment efficiency is improved; according to the method, PCR (Polymerase Chain Reaction) amplification is carried out by using # imgabs1 # High-Felidity DNA (Deoxyribose Nucleic Acid) Polymerase, so that the high fidelity and the specificity of an amplified product are ensured.

According to the targeted anti-aging nutrition capsule and the preparation method thereof provided by the invention, the ergothioneine and the nervonic acid are formed by cooperatively regulating and controlling a PARP1-XRCC1-DNA ligase III complex, so that the DNA fracture repair efficiency is remarkably improved; a sodium hyaluronate-spermidine cationic liposome carrier is developed, and efficient co-delivery of water / fat double-soluble components is achieved; through clinical verification, triple anti-aging pathways of telomere maintenance, mitochondrial optimization and skin regeneration can be synchronously activated. The compound benefits of reducing DNA damage, improving skin elasticity and increasing NK cell activity can be realized.

The invention is related to methods for sequencing nucleic acid molecules and kits useful in such methods. The invention is in one embodiment related to a method for sequencing nucleic acid molecules in a sample comprising a plurality of linear DNA molecules; contacting said sample with a terminal deoxynucleotidyl transferase (TdT) and a mixture of two or more different nucleotides under conditions in which nucleotides in the mixture are sequentially and randomly added to the 3' termini of the linear DNA molecules by the TdT and denaturing, when present, double stranded DNA in said sample, thereby generating linear single-stranded DNA molecules comprising sequence identifiers at their 3' termini. The linear DNA molecules comprising the sequence identifiers a circularized with a single-stranded DNA ligase and the circles in the sample are subjected to rolling circle amplification. The amplification products are subsequently sequenced whereby sequence identifiers are used to assemble sequences from the same original nucleic molecule in the sample. The invention also relates to kits comprising materials that are useful in the performing the methods. In another embodiment the invention relates to methods for detecting methylation in a plurality of DNA molecules.

The invention provides a non-traditional base DNA storage method and device based on enzyme catalysis, and the method comprises the steps: obtaining to-be-stored data, and converting the to-be-stored data into binary data; on the basis of binary data, encoding to obtain a specific chemical modification group on the DNA short chain; a DNA long chain is used as a template, a DNA short chain complementary with a base sequence of each region on the DNA long chain is introduced, the DNA long chain is divided into a plurality of regions, each region has a different specific base sequence, and the DNA short chain is provided with a specific chemical modification group used for representing a value of an information bit; the DNA short chain and the DNA long chain are hybridized to form a hybrid complex; carrying out enzyme catalysis connection reaction on the DNA hybrid complex by utilizing DNA ligase, and connecting the DNA short chains with specific chemical modification groups into a DNA long chain containing modification information; at the moment, the long-chain DNA connected and spliced by the short-chain DNA carries storage information in a molecular modification manner. According to the method, DNA storage is achieved through enzyme catalysis connection, modification transfer reaction does not need to be conducted, the data writing process is simplified, the storage cost is reduced, and due to different modification types, the method has wide application potential in related fields.

The present disclosure provides novel mirror image CRISPR compositions that include components such as mirror image CRISPR-associated endonucleases and mirror image guide RNA for processing of mirror image nucleic acids. In some embodiments, the composition is a mirror image DNA-cleaving and / or modifying composition including a D-form DNA endonuclease (e.g., a D-form Cas protein) and a L-form guide RNA that includes a sequence complementary to a target sequence of a target L-DNA. In some embodiments, the composition further comprises a D-form DNA ligase (e.g., a D-form T4 ligase protein). Also provided are D-form Cas protein and D-form T4 ligase protein compositions, synthetic precursors thereof, and methods of preparing the same.

This invention provides a ligase buffer and its product, and its application in mRNA tailing, relating to the field of nucleic acid synthesis technology. The invention provides a T4 DNA ligase buffer comprising Tris-HCl, ATP, MgCl2, reduced glutathione (GSH), polyvinylpyrrolidone K30 (PVP K30), and Triton X-100; this buffer significantly improves the ligation efficiency of T4 DNA ligase and can be applied to mRNA tailing based on enzyme digestion and ligation.

The application discloses a cell-free cloning reaction system, a kit and application thereof. The system comprises a cell lysate, an inserted DNA fragment and a linear carrier. The cell lysate is obtained from induced and lysed Escherichia coli, wherein a lambda Red system and a ligase system are expressed. The lambda Red system comprises Exo protein, Beta protein and Gam protein. The ligase system comprises DNA ligase. The optimized lambda Red system and the high-efficiency DNA ligase system can complete high-efficiency splicing of multi-fragment DNA within 15 minutes at 37 DEG C, and still maintain a high positive rate in a multi-fragment assembly system, which is superior to Gibson cloning and expands the applicability in complex pathway synthesis and gene circuit reconstruction.