25 results about "Uracil in DNA" patented technology

Provided in the present invention are a method for preparing a strand-specific library for the rapid detection of various types of RNAs, and a high-throughput sequencing method. A polyadenylic acid (poly A) tail is artificially added at the 3' terminal of various RNAs by using a poly A polymerase, and a single-stranded cDNA is synthesized using a poly-deoxythymidine ribonucleotide primer with deoxyuridine under the action of a reverse transcriptase. The obtained single-stranded cDNA molecule is subjected to a series of reactions, and the reaction product is finally amplified by means of PCR to obtain a strand-specific library of various types of RNAs. The library sample can be subjected to on-machine sequencing.

The invention belongs to the technical field of biological detection, and particularly relates to a bisulfite-free detection kit for specific site 5-methylcytosine and a detection method thereof. According to the method, a TAPbeta treatment method is adopted, 5hmC is sealed through beta-GT, 5mC oxidation is carried out through TET, and 5mC is reduced into dihydrouracil (DHU) through pyridine borane, so that the apparent difference between 5mC and C and 5hmC is converted into single nucleotide polymorphism difference. In addition, a flow microsphere technology based on PNA-assisted click chemical connection starting is established to detect DNA methylation specific sites. A PNA clip is innovatively introduced into click chemical connection, and non-specific connection is inhibited. Target sequence enrichment, signal amplification and signal acquisition are carried out through surface-functionalized magnetic nanoparticles, and the detection sensitivity is improved to fM. The invention provides a new tool for high-specificity and high-sensitivity epigenetic marker detection.

To provide an antisense oligonucleotide for preventing, delaying, and / or treating a human cis-element repeat instability-associated genetic disorder.SOLUTION: A medicament comprises, as an active ingredient, an oligonucleotide, wherein the oligonucleotide comprises 2'-O-methyl RNA nucleotide residues, has a backbone with at least one phosphate moiety being replaced by a phosphorothioate moiety, and comprises or consists of a repetitive nucleotide unit (XYG)m, wherein m is an integer from 4 to 12, each X is C or 5-methylcytosine, each Y is U or 5-methyluracil, and at least one X is 5-methylcytosine.SELECTED DRAWING: None

This disclosure provides methods for cost-effective bisulfite-free identification in DNA, including whole genomic DNA, of the locations of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and 5-formylcytosine. The methods described herein are based on the conversion of modified cytosine (5mC, 5hmC, 5fC, 5caC) to dihydrouracil (DHU), for example by TET-assisted pyridine borane treatment, followed by endonuclease cleavage of the DHU, and identification of the cleavage site, which corresponds to the location of the modified cytosine.

The invention provides a chain-specific library preparation method and a high-throughput sequencing method for rapidly detecting multiple types of RNAs (Ribonucleic Acid). The method comprises the following steps: artificially adding poly A tails at 3'tail ends of multiple RNAs by utilizing poly A polymerase; meanwhile, a polydeoxythymine ribonucleotide primer with deoxyuracil is used for synthesizing single-stranded cDNA under the action of reverse transcriptase, obtained single-stranded cDNA molecules are subjected to a series of reactions and finally subjected to PCR amplification to obtain strand specific libraries of multiple types of RNA, and samples of the libraries can be subjected to computer sequencing.

The invention discloses a lung nodule diagnosis method based on plasma DNA methylation characteristics and application, and the method comprises the following steps: S1, collecting peripheral blood of a subject in a special cfDNA preservation tube, carrying out centrifugal separation to obtain plasma, extracting cfDNA by adopting an optimized paramagnetic particle method or column extraction method, carrying out quality control on the cfDNA, then carrying out thorough hydrosulphite conversion treatment on the cfDNA, and carrying out purification treatment on the cfDNA to obtain the plasma; unmethylated cytosine is converted into uracil, and methylated cytosine is kept unchanged. The invention is derived from the selection of an early and stable epigenetic marker of DNA methylation, and replaces traditional dependent clinical parameters or mutation signals appearing at a late stage. According to the method, the problem of insufficient discrimination capability (AUClt; 0.60) caused by limited input information of an existing clinical prediction model (such as a MayoClinic model) is directly solved.

The invention discloses a diagnosis and treatment integrated nano antibody for targeting shrimp iridovirus deoxyuracil nucleoside triphosphate hydrolase as well as a preparation method and application of the diagnosis and treatment integrated nano antibody. According to the invention, a nano-antibody library is constructed on the basis of a squalus leukoderma new antigen receptor variable fragment VNAR, a nano-antibody 4A12-coated VNAR targeting shrimp iridovirus deoxyuracil nucleoside triphosphate hydrolase dUTPase protein is screened from the nano-antibody library, the nucleotide sequence of the nano-antibody 4A12-coated VNAR is shown as SEQ ID No.5, and the amino acid sequence of the nano-antibody 4A12-coated VNAR is shown as SEQ ID No.6. The invention further discloses a preparation method of the nano-antibody 4A12-coated VNAR. The molecular weight of the nano antibody 4A12atVNAR is much smaller than that of a conventional full-length antibody, and the nano antibody 4A12atVNAR has better tissue permeability and capability of being combined with an epitope with a steric hindrance effect, can be massively expressed in a prokaryotic expression system, and is short in period and low in production cost. The nano antibody 4A12-coated VNAR can be used for detection, diagnosis and treatment of shrimp iridovirus.

The present invention relates to base editing using dead uracil DNA glycosylase (dUDG). The present invention is useful for C-to-T base editing in nuclear DNA or organelle DNA, and in particular, is useful for C-to-T base editing in organelle DNA such as chloroplasts or mitochondria. The present invention also relates to a UDG variant and a novel DNA base editing use thereof.

The invention discloses a method for rapidly detecting MTHFR (Methylene Tetrahydrofolate Reductase) gene polymorphism based on a complementary probe technology, which comprises the following steps: S1, acquiring a sample: extracting genome DNA (Deoxyribose Nucleic Acid) from a sample to be detected as an amplification template; s2, preparing a reaction system: adding the genome DNA, the upstream primer, the downstream primer, the C probe, the T probe, uracil-N-glycosylase, DNA polymerase, deoxyribonucleoside triphosphate and deoxyuridine triphosphate of the sample to be detected into the reaction system; s3, carrying out PCR (Polymerase Chain Reaction) amplification reaction under the conditions that the temperature is 90-98 DEG C, and the time is 5-20 minutes; performing denaturation at the temperature of 90-98 DEG C for 10-50 seconds; the temperature is 55-69 DEG C, and annealing is conducted for 60-120 s; the temperature is 68-72 DEG C, and extension is carried out for 15-300 s; carrying out 35 to 40 cycles; and S, genotype judgment. A test result is the same as a first-generation sequencing test result, which shows that the detection method of the scheme has relatively high accuracy. Meanwhile, compared with a sequencing method, the method has the advantages that a series of complex follow-up treatment does not need to be carried out on a PCR product, PCR amplification and detection are synchronously carried out, the detection time is greatly shortened, and the detection cost is reduced.

The invention discloses a DNA and RNA methylation analysis method for a single cell or a small amount of cells. The method comprises the following steps: A, directly using a single cell or a small amount of mammalian cells; b, the purpose of breaking cells is achieved by directly using high temperature or protease treatment; c, treating the non-methylated DNA by using a methylation-sensitive incision enzyme; e, directly treating the sample by using hydrosulfite to convert unmethylated cytosine (C) in the DNA and RNA of the sample into uracil (U); f, simultaneously carrying out methylation analysis on DNA and RNA from the same cell source; g, DNA and RNA methylation results of a single cell source can be obtained at the same time. According to the method, DNA and RNA in a single cell are effectively utilized, the method is easy to implement, stable in result, high in detection sensitivity, more sufficient in information and simple and convenient to operate, and the limitation of the DNA amount of the single cell in DNA methylation analysis is greatly improved.

The invention discloses a construction method and application of a single-cell complete transcriptome library. The method comprises the steps that a universal primer is used as a cDNA synthesis primer and template conversion oligonucleotide at the same time, an amplifiable cDNA product is synthesized through a reverse transcription reaction, the universal primer is composed of a fixed sequence composed of three basic groups G, A and T, and the length of the universal primer is 10-30 nt; at least one thymine in the fixed sequence of the universal primer is replaced by deoxyuracil; the universal primer is partially degraded through special modification on the universal primer by utilizing a specific enzyme comprising reverse transcriptase MMLV with template conversion activity and a mutant of the reverse transcriptase MMLV, or TGIRT-III and a mutant of the TGIRT-III; a cDNA amplification product with chain specificity is obtained by using a specific amplification primer and method. By utilizing the characteristic that a universal primer is randomly and efficiently combined with RNA molecules at low temperature, a barcode is added to a single cell in combination with a specific coding microsphere, so that high-sensitivity and whole transcriptome amplification sequencing of the high-throughput single cell is realized.

The invention provides a gene editing system for editing a target gene in the genome of a cell. The gene editing system comprises CRISPR nuclease, cytosine deaminase, AP lyase, guide RNA and optional uracil-DNA glycosylase. The invention also provides a method for producing a genetically modified cell, and a kit comprising the gene editing system.

The present invention discloses a preparation method and application research of uracil-DNA glycosylase electrochemical sensor based on Ag@Cu-MOF. Compared with electrochemical sensors without HCR reaction, the present invention first synthesizes DNA nanotriangular prisms carrying UDG shearing sites, and uracil bases are recognized and removed by UDG, so that hairpin DNA becomes straight-chain DNA to start hybridization chain reaction to achieve alternating hybridization of hairpin H1 and H2. This new and powerful DNA nanobody can "grasp" Ag@Cu-MOF in multiple dimensions through π-π conjugation, build a balanced homeostatic environment, better catalyze 3,3,5',5'-tetramethylbenzidine solution (TMB) reaction, facilitate electron transport and transfer during the reaction to achieve signal amplification, and realize UDG analysis and detection through current-time curve, while when UDG is not present, the electrochemical signal that can be monitored is relatively small, based on which a new electrochemical biosensor is constructed to achieve sensitive analysis of UDG activity and its small molecule inhibitors.

To provide methods for cost-effective bisulfite-free identification in DNA, including whole genomic DNA, of the locations of one or more of 5-methylcytosine, 5-hydroxymethylcytosine, 5-carboxylcytosine and 5-formylcytosine.SOLUTION: The methods described herein are based on the conversion of modified cytosine (5mC, 5hmC, 5fC, 5caC) to dihydrouracil (DHU), for example by Tet-assisted pyridine borane treatment, followed by endonuclease cleavage of the DHU, and identification of the cleavage site, which corresponds to the location of the modified cytosine.SELECTED DRAWING: Figure 29A

The present invention provides a method for preparing a 5-fluorouracil-quercetin-DNA hydrogel, comprising mixing a first DNA primer and a linear template, heating and annealing for assembly, adding a T4 ligase reaction to connect, and forming a circular DNA template; adding dATP, dCTP, dGTP, 5F-dUTP, DTT, DNA polymerase and DNA polymerase buffer to the circular DNA template obtained above, and performing an RCA reaction; adding a second DNA primer and a third DNA primer to the above RCA product, performing a multi-primer chain amplification reaction, and obtaining a 5-fluorouracil-DNA hydrogel; fully mixing and incubating a quercetin solution with the above-mentioned 5-fluorouracil-DNA hydrogel to obtain a 5-fluorouracil-quercetin-DNA hydrogel. The present invention also provides the use of the above-mentioned 5-fluorouracil-quercetin-DNA hydrogel in the preparation of anti-tumor drugs. The present invention solves the problems of severe toxic and side effects of chemotherapeutic drugs and poor immunotherapy effects caused by immunosuppressive tumor microenvironment.

The invention discloses a labeling method and application of new RNA in exosomes, and the labeling method comprises the following steps: S1, constructing an animal model of tissue specific expression uracil phosphoribosyltransferase, and then carrying out 4-thiouracil solution administration treatment; s2, collecting plasma exosomes, extracting exosome RNA (Ribonucleic Acid), and then removing ribosome RNA in the exosome RNA to obtain transcriptome RNA; and S3, carrying out sulfydryl-specific biotinylation treatment on the transcriptome RNA, then collecting the biotinylated RNA, and carrying out RNA alkylation treatment on the biotinylated RNA. By adopting the method, the new RNA in the exosome of the liver or fat source in circulation can be marked, the biological information exchange effect between the RNA of the liver or fat source and other organs can be researched, and the method has important significance for promoting the early diagnosis and treatment of metabolic diseases.

The invention provides a DNA molecular lock for DNA fragment methylation detection, the DNA molecular lock and a sequence for replacing cytosine into uracil in a DNA fragment are complementary in the same direction, and cytosine basic groups in the DNA molecular lock are replaced into isocytosine basic groups. The invention further provides a simple and efficient qPCR method beneficial to detection of trace target gene methylation on the basis of the DNA molecular lock, stable homodromous parallel DNA double strands are formed by combining the DNA molecular lock with the non-methylated DNA fragment, amplification of the non-methylated DNA fragment is inhibited, specific amplification of the target methylated DNA fragment is helped, and the detection accuracy of the methylated target gene is improved. The methylation degree detection of trace DNA fragments is further improved.

The invention discloses a method for coupling polypeptide or protein and oligonucleotide promoted by visible light, which comprises the following steps: taking polypeptide or protein at least containing one free and solvent-exposed cysteine as a raw material, and oligonucleotide or 6-aza-uracil derivative containing 6-aza-uracil nucleoside as another raw material; a pyridinium salt is used as an activating reagent, a buffer solution is used as a reaction solvent, and polypeptide or protein and oligonucleotide are coupled to form a stereoscopically retained stable C-C bond under the participation of an additive and the irradiation of visible light. The configuration of the conjugate obtained by the invention at a modification site is maintained. Meanwhile, the method has excellent site selectivity, accurate modification of polypeptide or protein can be realized, the reaction is carried out under a biocompatible condition, a catalyst or a reducing agent does not need to be added, the reaction efficiency is high, biocompatibility is realized, and disulfide bonds in the polypeptide or the protein can be compatible.

Methods and compositions for a single- or multi-pot protocol for the efficient end to end capture of RNAs (inclusive of their poly-A tail or their 3′ end) is described. Capture oligonucleotides containing a 3′ non-extendable end and a selectively cleavable base upstream of an oligo-dT or oligo-dN and a 5′ sequence containing unique molecular identifiers, and 2) a deoxyuracil glycosylase that acts only on a deoxyuracil present in a DNA: DNA duplex or DNA / RNA heteroduplex are used. A dual template switching mechanism may be used.

The invention discloses a construction method of a high-throughput single-cell ATAC-seq sequencing library, and belongs to the technical field of single-cell chromatin accessibility sequencing. The method comprises the steps that a single nucleic acid specific sequence with uracil deoxyribonucleotide and Tn5 transposase are assembled into a Tn5 transposase complex, and the transposase complex can be randomly combined with a target DNA sequence, cut and inserted into a DNA fragment carried by the target DNA sequence. Carrying out transposition reaction on the permeabilized cell nucleus by using a Tn5 transposase complex; the method comprises the following steps: preparing single-cell liquid drops by adopting a micro-fluidic chip, supplementing the tail ends of DNA fragments generated by enzyme digestion through PCR (Polymerase Chain Reaction), and carrying out single-cell coding marking; and finally carrying out PCR amplification and library construction. A single nucleic acid sequence joint and Tn5 transposase assembly strategy is adopted, so that the problem of PCR amplification failure caused by insertion of same joints of double-end nucleic acid sequences when nucleic acid fragments are inserted in traditional Tn5 transposase assembled by two or more nucleic acid sequences is fundamentally solved, and the recovery rate and the detection number of effective DNA fragments are remarkably improved.

Methods are provided for the epigenetic analysis of cell-free DNA using organic boranes to convert oxidized 5-methylcytosine residues in the cell-free DNA to dihydrouracil (DHU) residues. Cell-free DNA is contacted with an organic borane selected to successively bring about reduction, deamination, and decarboxylation of oxidized 5-methylcytosine residues such as 5-carboxylcytosine and 5-formylcytosine, resulting in DHU residues in place thereof. Following amplification, the treated cell-free DNA is sequenced, with the DHU residues read as thymine residues. Reaction mixtures, kits and additional methods are also provided, as are related methods for the epigenetic analysis of DNA, including cell-free DNA.

The application discloses a nucleic acid detection method and kit for distinguishing pathogen DNA / RNA, and a section of artificial sequence is introduced at the 5' end of a primer to improve the specificity of the primer, the artificial sequence is not complementary to a template, and the artificial sequence is complementary to the 3' end sequence of the primer to form a stem loop structure. When the template is not completely complementary to the 3' end of the primer, the primer exists in the form of the stem loop structure, and when the template is completely complementary to the 3' end of the primer, the stem loop structure is unbound to perform reverse transcription. When RNA is amplified, the synthesized cDNA is provided with the artificial sequence, the primer is completely complementary to the cDNA during amplification, 7-11 bases at the 3' end of the primer are complementary to the template, and deoxyuracil is used to replace deoxythymine base at the 3' end of the primer. According to the application, the Tm value of the primer binding is relatively low when DNA is amplified under a certain annealing temperature, so that the DNA cannot be amplified, and when RNA is amplified, the 5' end of the reverse transcription primer is provided with the artificial sequence, so that the synthesized cDNA is provided with the artificial sequence, the primer can be completely complementary to the cDNA during amplification, and the RNA can be accurately detected.

The present invention relates to a self-replicating RNA vector, a preparation method therefor, and a use thereof. The sequence of the self-replicating RNA vector sequentially comprises in the direction from a 5' end to a 3 'end: a T7 promoter sequence, an ATG sequence, an SINV RNA replicase coding sequence, a target gene sequence, and a signal sequence containing poly (A). The preparation method comprises: inserting an ATG sequence at an insertion sequence position of a pKGCT7-SINV-GFP backbone to obtain pKGCT7-SINV-GFP, carrying out AscI-NotI double enzyme digestion to remove a GFP sequence, and then using a recombinase to insert a target gene sequence between AscI and NotI, so as to obtain the self-replicating RNA vector. The use is to prepare self-replicating RNA. The self-replicating RNA vector can use pseudouridine or N1-methyl-pseudouridine to achieve low immunogenicity without reducing expression efficiency.

The invention provides a primer and a kit for single cell whole genome amplification, and belongs to the technical field of molecular biology. According to the invention, dUTP deoxyuracil nucleotide is used for replacing conventional dTTP for the last basic group of the fixed sequence at the 5'end of the primer, and the primer can be specifically cut off subsequently according to the site, so that the interference of the primer sequence on sequencing is reduced. Meanwhile, compared with commercially available products, the kit comprising the primer is designed, the CV value of the kit is reduced by 63.60%, the kit has good amplification product uniformity and low base preference, the comparison rate in an SE50 sequencing mode is as high as 97.25%, no primer residue interference exists in the amplification process, and the kit is suitable for high-precision single cell sequencing analysis.

To provide a means to rapidly and efficiently inhibit the activity of DNA aptamers and avoid side effects caused by aptamer drugs. [Solution] An inhibitor of a DNA aptamer or a DNA aptamer in which one or more thymines in the base sequence of the DNA aptamer are replaced with DNA uracil, comprising uracil DNA glycosylase (UdgX) as the active ingredient.