16 results about "DNA Ligases" patented technology

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.

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 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.