38 results about "Cytosine" patented technology

本文描述了一种用于制备测序文库的方法。所述方法可包括：在连接缓冲液中使一种或多种衔接子连接至核酸分子（例如，DNA 或 RNA）；在所述连接缓冲液中使所述核酸分子与多个珠结合；从所述连接缓冲液中分离与所述核酸分子结合的所述多个珠；将所述多个珠悬浮在包含醇的溶液中；以及使用亚硫酸氢盐反应，使所述核酸分子中的未甲基化胞嘧啶转化为尿嘧啶，以生成转化的核酸分子。

The methods, compositions, and kits of the disclosure provide a novel approach for a whole genome, unbiased DNA analysis method that can be performed on limited amounts of DNA can be used to analyze DNA to determine its modification status. Aspects of the disclosure relate to a method for amplifying bisulfite-treated deoxyribonucleic acid (DNA) molecules comprising: (a) ligating an adaptor to the DNA molecules, wherein the adaptor comprises a RNA polymerase promoter comprising bisulfite-protected cytosines; (b) treating the ligated DNA molecules with bisulfite; (c) hybridizing the bisulfite-treated DNA molecules with a primer; (d) extending the hybridized primer to make double stranded DNA; and (e) in vitro transcribing the double-stranded DNA to make RNA.

The application provides a bovine cell gene editing reagent and a construction method and application of a BCO2 gene mutant cell strain, and belongs to the technical field of gene editing. The application provides a bovine cell gene editing reagent, which comprises BE4max SpCas9 V922 Δ mRNA and mRNA containing a target gene target point sequence and gRNA. When the BE4max-SpCas9 V922 Δ mRNA is combined with the gBCO2-1 mRNA to transfect target cells in bovine mammary epithelial cells (Mac-T), the cytosine base editing function in the cells can be normally exerted, and the editing of the bovine BCO2 gene target point is successfully realized.

Aspects of the present disclosure are directed to methods, compositions, and kits for detection and analysis of DNA and RNA cytosine methylation and / or RNA pseudouridylation. Certain aspects include methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids, including methylated and / or pseudouridinylated nucleic acids from low-input samples such as cell-free DNA (cfDNA) and cell-free RNA, long fragment polynucleotides, and / or cfDNA and DNA from formalin-fixed paraffin-embedded (FFPE) samples. Also disclosed herein are methods, compositions and kits useful in bisulfite sequencing of methylated and / or pseudouridinylated nucleic acids with low nucleic acid damage, higher true positive results, and / or lower false negative results.

The present application belongs to the field of biology, and specifically provides an engineered cytosine deaminase, a preparation method and use thereof, the engineered cytosine deaminase comprising a skeleton composed of an N-terminal domain and a C-terminal domain derived from a cytosine deaminase protein and a deamination domain derived from another cytosine deaminase protein. The engineered cytosine deaminase can be used in an RNA and DNA base editing system, wherein an RNA base editing system does not require gRNA, is simple and efficient, has low off-target rate, can be stably expressed in cells and organisms, and meets the efficient delivery requirements of late clinical applications; typical applications include gene editing at the RNA level, thereby eliminating pathogenic RNA to correct pathogenic error point mutations; or editing of expressed RNA molecules to specifically manipulate gene functions.

The application discloses a whole-genome DNA cytosine hydroxymethylation modification single-base precision library construction method, relates to the technical field of genomics, epigenetics and molecular biology, and contains the following steps: 5hmC labeling reaction, click chemistry reaction, sample DNA fragmentation, purification of the fragmentation product through magnetic beads, 5hmC fragmented DNA capture, washing of the capture product, reduction reaction, APOBEC enzyme deamination reaction, purification of the deamination product, double-strand conversion reaction, end repair and 3' end A addition reaction, linker ligation, purification of the ligation product and double sorting, library amplification and library purification. The library construction method integrates low data volume, single-base precision and conventional double-strand library construction kit in one under the premise of ensuring data authenticity, and has the technical advantages of high universality and highly repeatable data.

The present application relates to the field of second-generation sequencing, in particular to the field of methylation sequencing, and more particularly to a marker sequence, a linker comprising the marker sequence, a kit comprising the sequence or the linker, and a DNA methylation sequencing method using the linker or the kit. The marker sequence comprises a polycytosine sequence on one side or both sides of an insert, wherein each cytosine at each site on each side has two states of modification or no modification. The present application can reduce cross-contamination between sequencing samples, and can be applied to DNA methylation detection.

This invention discloses a base editor, a base editing method, and its applications. First, the invention constructs highly efficient targeting sites unrestricted by PAM sequences through point mutation. A random insertion library is constructed using a transposase system, and proteins capable of cytosine base editing in mammals are screened using a BFP-GFP reporter system. Further point mutations are performed on deaminases to screen for highly efficient cytosine base editors unrestricted by PAM. The optimized base editing method of this invention achieves an efficiency of over 70%, with some sites reaching over 90%, demonstrating significant application value in basic life sciences, medicine, and other fields.

The application belongs to the field of aquatic molecular breeding and epigenetic marker development, and particularly relates to a group of DNA methylation markers related to loss of Chinese mitten crab in summer extreme high temperature and application thereof in breeding. The methylation markers include 8 differentially methylated cytosine sites located on the reference genome ASM2467909v1, adjacent to LOC126983793 (ADCY9), LOC126997354 (UNC79), LOC126998428 (UBN1), LOC126997943 (IFT52), LOC126991093 (ACO2), LOC126986070, LOC127001126 and LOC126997895 respectively. Among them, the high temperature damage group shows high methylation at part of the sites, low methylation at part of the sites, and abnormal expression of the corresponding adjacent genes. The above markers can be used for molecular detection and assisted selection of heat tolerance, survival rate maintenance ability, large-size crab output stability and high temperature damage risk of Chinese mitten crab.

Aspects of the disclosure relate to mRNAs comprising a relatively low abundance of cytidine: adenosine (CA) dinucleotides that benefit from increased stability relative to mRNAs containing more CpA dinucleotides. The disclosure also relates to methods of modifying an mRNA sequence to improve stability. In some aspects, the disclosure relates to mRNAs comprising modified mRNA sequences with relatively reduced numbers of CpA dinucleotides, and compositions comprising mRNAs with relatively reduced numbers of CpA dinucleotides.

The present application relates to the field of biological medicine, and particularly relates to a universal T cell and a preparation method and application thereof. The universal T cell provided by the present application is edited by base editing on genes affecting graft versus host disease, host versus graft reaction, immune checkpoint or genes encoding immune checkpoint specific receptors, wherein the base editing is deamination of at least part of cytosine bases and / or part of adenine bases on polynucleotides of genes related to graft versus host disease, host versus graft reaction, immune checkpoint or genes encoding immune checkpoint specific receptors by using a base editing system. The universal T cell obtained by using the efficient and safe base editing technology overcomes the harmful effects caused by DNA double strands of CRISPR / Cas technology, and has obvious application prospect and clinical application value.

This invention belongs to the field of agricultural biotechnology and molecular detection technology, and relates to a mutation site in the SdhB gene related to fluopyram resistance in the southern root-knot nematode, along with detection primers and applications. The mutation site involves a change in nucleotide position 1108 from cytosine (C) to thymine (T) at the 5' end of the SdhB gene coding sequence, resulting in a change in amino acid position 190 from proline (P) to leucine (L) at the N-terminus of the SdhB subunit protein encoded by this gene. By introducing a base mismatch, the invention effectively overcomes the problem of false positives in SNP detection using LAMP technology, exhibiting high specificity. No PCR instrument is required; only a constant temperature water bath is needed, significantly shortening the detection cycle. It is simple, rapid, and suitable for field use. Results can be determined visually based on color changes, eliminating the need for electrophoresis, making it suitable for grassroots promotion and large-scale application.

A PARP inhibitor-tolerant CAR-T cell and its construction method are provided. A sequence of sgRNA for targeted editing PARP1 gene in CAR-T cells includes sg-D45, sg-S588, or sg-G745. The construction method includes using a cytosine base editor to introduce S588F, G745K and / or D45N mutations into the PARP1 proteins in CAR-T cells with the sgRNA, significantly increasing the tolerance of the CAR-T cells to PARP inhibitors and allowing the CAR-T cells to work synergistically with the PARP inhibitors, thereby maximizing the anti-tumor effect.

The application belongs to the technical field of biological medicine, and particularly relates to a double-network hybrid cross-linked hydrogel, a preparation method and application in 3D organoid culture. The double-network hybrid cross-linked hydrogel is formed by natural cross-linking of a hydrogel precursor material at room temperature, and the double-network hybrid cross-linking includes covalent cross-linking and hydrogen bond cross-linking formed between the hydrogel precursor materials; the hydrogel precursor material includes mercapto-hyaluronic acid-hexanediamine-cytosine, mercapto-hyaluronic acid-hexanediamine-guanine and a polyethylene glycol compound with a multi-arm star structure. The hydrogel has both the mechanical stability of covalent cross-linking and the dynamic responsiveness of hydrogen bond cross-linking, and can be quickly formed under physiological conditions, thereby providing a new matrix material for organoid culture, which is safe and stable, has clear components and can dynamically simulate a tumor microenvironment.

The utility model relates to cytosine centrifugal technical field discloses a centrifugal device for cytosine preparation, including support, one end fixedly connected with base of support, the top of base is provided with protection cover, the top fixedly connected with drive arrangement of base, drive arrangement output fixedly connected with rotating wheel, the lateral wall of base is provided with cleaning component, the inside of rotating wheel is provided with separation component, the lateral wall of base is provided with connecting component, the cleaning component includes first connecting pipe, one end fixedly connected in the lateral wall of base of first connecting pipe, in the utility model, liquid is connected in one end of first connecting pipe, and liquid flows into the inside of protection cover, at the same time, the flow of liquid is controlled through the setting of solid outflow pipe and second valve, the problem that the internal impurity mixes in cytosine product in centrifugal process, causes the product quality to drop is solved, and the practicability of centrifugal device for cytosine preparation is improved.

Provided herein is a method for analyzing genomic DNA. In some embodiments, the method may comprise labeling a genomic sample by adding a capture tag to the ends of the DNA molecules in the sample and labeling molecules that comprise hydroxymethylcytosine with a first fluorophore, immobilizing the labeled DNA molecules on a support, and imaging individual molecules of hydroxymethylated genomic DNA on the support.

The application discloses a kind of for sheep MSTN And SOCS2 The sgRNA of gene cytosine base editing and application thereof, the sgRNA includes nucleotide such as any one or several of SEQ ID NO.1~4.This application is respectively designed sgRNA guide sequence for sheep MSTN And SOCS2 Gene, introduce stop codon in target site using CBE system, select sgRNA guide sequence and its combination from which can efficiently target sheep MSTN And SOCS2 Gene, carry out gene editing efficiency verification on cell, and successfully obtain MSTN And SOCS2 Double gene editing goat by prokaryotic embryo microinjection technology, and gene editing sheep shows muscle fiber thickening, and fast growth rate.This application establishes a set of efficient, precise double gene editing technology system, provides key breeding material and technical support for quickly cultivating muscle developed, fast growth meat sheep new variety.

This invention discloses a method for detecting 5-hydroxymethylcytosine based on triple cascade signal amplification, belonging to the field of biochemical detection technology. The method includes the following steps: 1) preparation of 5hmC-specific glycosylation and azide labeling; 2) magnetic bead capture and enrichment separation of target DNA; 3) TdT-mediated nucleic acid end polymerization and extension; 4) APE1-triggered primer release and rolling circle amplification; 5) DNase-catalyzed cleavage and fluorescence signal detection. This invention is bisulfite-free and sequence-independent, achieving extremely low background and ultra-high sensitivity detection with a detection limit of 4.41 fM. It can be widely applied to early clinical cancer screening, biomarker detection for neurological or cardiovascular diseases, and provides a powerful platform for low-abundance epigenetic analysis in clinical diagnosis and biomedical research.

The application provides a high-efficiency and high-precision base editor for cytosine C to guanine G conversion. The application establishes a novel C-to-G base editor, compared with the existing C-to-G base editor, the targeting editing efficiency and editing precision of the editor of the application are significantly improved, and the editor product has the characteristics of high product purity, narrow editing window and low off-target effect.

This invention relates to the field of genetic engineering. Specifically, it relates to cytosine deaminases and their use in base editing. More specifically, it relates to a method for screening and identifying deaminases, a base editing system based on newly identified cytosine deaminases, a method for base editing a target sequence in the genome of an organism (e.g., a plant) using this base editing system, and genetically modified organisms (e.g., plants) and their offspring produced by said method.

The technical field relates to methods for promoting gut microbiome health using low G+C Directed (LDN) analogs. More particularly, the technical field relates to methods of using LDN analogs as inhibitors of DNA Polymerase IIIC (DNA pol IIIC) enzyme to selectively reduce physiologically harmful Gram-positive bacteria with a genome having low amounts of Guanine and Cytosine (rather the genomes comprise a greater amount of adenine and thymine / uracil nucleotides) in the gut microbiome while simultaneously maintaining or increasing the proportions of beneficial microflora.

A method for producing a 2′-carbamoylalkyl-modified uridine compound characterized by reacting an anhydrouridine compound and an amide compound having a protected or unprotected hydroxy group in a solvent such as an ether solvent in the presence of a boron reagent, and a method for producing a 2′-carbamoylalkyl-modified cytidine compound including a step of converting a uracil base of a 2′-carbamoylalkyl-modified uridine compound into a cytosine base.

This invention discloses a CoolMPS antibody mutant that specifically binds to a blocking-modified cytosine deoxynucleotide and its applications. The invention provides an antibody (referred to as an antibody mutant or modified antibody) that binds to a target, obtained by mutating the variable region of the heavy chain of the antibody to be modified; the mutation is located at: amino acid residue 2 of CDR1; amino acid residue 2 of CDR2; amino acid residue 15 of FR1; amino acid residue 6 of FR3; amino acid residue 21 of FR3; amino acid residue 30 of FR3; amino acid residue 38 of FR3; the target is a blocking-modified cytosine deoxynucleotide. The antibody provided by this invention can be used in basic scientific research, biosensor technology, gene sequencing, and other fields, and has broad application prospects.

Provided are a fusion protein that improves gene editing efficiency and an application thereof. The fusion protein comprises a single-stranded DNA binding protein functional domain, nucleoside deaminase and nuclease. According to CBEs, when carrying our base conversion from C-G to T-A, nucleoside deaminase such as cytosine deaminase carries out deamination by using single-stranded DNA as a substrate, and by re-fusing the single-stranded DNA binding protein functional domain on the fusion protein of the nucleoside deaminase and nuclease, the chance of single-stranded DNA being exposed to the nucleoside deaminase is greatly increased, thereby significantly improving base editing efficiency. The present disclosure provides a breakthrough improvement of single-base gene editing technology and can greatly promote the application thereof in aspects such as gene editing, gene therapy, cell therapy, animal model making, and crop genetic breeding.

The application relates to the technical field of medicines, and particularly discloses a novel immune stimulant, a preparation method and application thereof. The immune stimulant comprises an alpha-mannose modified imidazoquinoline derivative, an N-acetyl cysteine modified cytosine-guanine dinucleotide, a glycyrrhetinic acid-polyethylene glycol derivative, a phytic acid-glycine oligomer, a medicinal excipient and water for injection, three modified active ingredients are bridged by the phytic acid-glycine oligomer as a bridging unit, and a ternary coordination complex is formed through dynamic non-covalent coordination. The preparation method comprises the following steps: after raw materials are pretreated, the active ingredients are premixed, dissolved and solvent is recovered, then dynamic coordination reaction, ultrafiltration purification and concentration are carried out, and finally, a finished product is obtained through double-cavity preparation forming. The immune stimulant can be used for preparing a tumor immunotherapy, an anti-infection treatment, an immune deficiency disease auxiliary treatment drug or a vaccine adjuvant, and has the advantages of high active ingredient stability, good in-vivo delivery efficiency and outstanding synergistic effect.

The present application provides a RNA base editor with controllable editing window (e.g., adenine base editor, cytosine base editor, adenine / cytosine dual base editor) and its application. In addition, the present application also provides a series of novel TadA mutants for RNA base editing, and base editing tools based on the TadA mutants.

The present invention addresses the problem of providing a method for producing, by single primer PCR, a DNA fragment having ITR sequences on both ends. In the present invention, a DNA fragment including a transgene is produced by single primer PCR with a DNA having ITR sequences serving as a template DNA, using: (A-i) a polynucleotide A comprising an artificially designed polynucleotide a1 comprising at least 5 bases which are any of the bases selected from adenine, guanine, cytosine, and thymine and a polynucleotide a2 linked to the 3' end of the polynucleotide a1, the polynucleotide a2 comprising the full length sequence from 1st to 54th of the base sequence represented by SEQ ID NO: 1 or comprising a consecutive partial sequence of at least 16 consecutive bases in the base sequence from the 12th to 39th of the base sequence represented by SEQ ID NO: 1; (A-ii) the polynucleotide A of (A-i) with one or more bases deleted, substituted, added or inserted; or the like.

The invention discloses engineered RNA-guided DNA-binding (RGDB) variants, especially of the Lachnospiraceae bacterium CRISPR-Casl2a (LbCasl2a) that exhibit superior performance, like enhanced specific binding to target DNA sequences, reduced non-specific DNA binding, or reduced catalytic activity. By introducing numerous substitution mutations across distinct structural blocks, the variants achieve stronger, more specific binding to target DNA while markedly reducing off-target or non-specific interactions. A second tier of mutations targets catalytic residues and other functional sites to generate catalytically inactive forms (dRGDP) that retain DNA-binding capability. These RGDB scaffolds can be fused to diverse effector domains, including transcriptional regulators, epigenetic modifiers, or nucleases, to modulate gene expression or repair defective genes in mammalian or plant cells. When coupled with base-editing enzymes such as adenosine or cytosine deaminases, the resulting adenine or cytosine base editors display enhanced editing efficiency and altered editing windows, enabling precise conversion of additional nucleobases.

This invention relates to the field of next-generation sequencing, specifically to a method for preparing methylated sequencing libraries. The method includes the following steps: a. pretreating a DNA sample by converting methylated cytosine (C) or unmethylated C into non-C bases; b. denaturing the nucleic acid product obtained in step a; c. ligating adapters to the denatured nucleic acid, wherein in step c, the denatured single-stranded nucleic acid is ligated with paired-end adapters. The 5' adapter includes a random sequence complementary to the 5' end of the single-stranded nucleic acid, and the 3' adapter includes a random sequence complementary to the 3' end of the single-stranded nucleic acid. In the random sequence, the content of at least one of A, T, C, and G is not higher than 24% or not lower than 26%. This invention effectively improves the library yield, quality, complexity, uniformity, methylation quantification accuracy, and methylation site detection sensitivity. Furthermore, this invention enables "one-tube" library construction, simplifying the operation, avoiding sample loss, saving costs, and shortening the library construction time.

The present application relates to the field of biotechnology and molecular diagnosis, and particularly relates to a methylation detection pretreatment kit and method for protecting DNA integrity, which can solve the problem that DNA degradation is serious in traditional bisulfite conversion technology, and conversion efficiency and DNA integrity are difficult to be obtained simultaneously, the kit comprises a conversion solution, a protection solution, a combination solution, a desulfonation solution, a washing solution and an elution solution, the conversion solution contains 2.5-4 mol / L bisulfite and linear polyethylene polyamine, the protection solution contains an organic denaturant and a polyphenol antioxidant, the DNA melting temperature is reduced by the organic denaturant, the linear polyethylene polyamine accelerates the conversion reaction, and the desulfonation system with low water activity is combined to complete the bisulfite conversion under mild conditions of 50-70 DEG C; the present application can realize the conversion efficiency of greater than 99.5% of unmethylated cytosine, greatly reduce DNA degradation, significantly improve the long fragment DNA recovery rate and the detection sensitivity of trace samples, and is suitable for the methylation detection of three generations of long read sequencing and clinical trace samples.