24 results about "DNA Modification" patented technology

Provided herein are compositions and methods for the multiplexed profiling of RNA and DNA modifications across transcriptomes and genomes, respectively. The methods combine molecular recognition of non-canonical features (e.g., base modifications, backbone modifications, lesions, and / or structural elements) of a target nucleic acid with a step of writing the information from this recognition event into the neighboring genetic sequence of the target nucleic acid using a barcode. The resultant barcoded nucleic acids are then converted into sequencing libraries and read by DNA / RNA sequencing methods. This step reveals the sequence of the barcode, which is correlated with the non-canonical feature in the target nucleic acid(s). The high throughput profiling methods described herein allow for localization of one or more modifications in a target nucleic acid. The methods also allow for identification of the nature and location of several or all DNA / RNA modifications in parallel.

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.

This invention relates to the field of biotechnology, and more particularly to a high-density oligoA-tailed DNA-gold nanomaterial and its preparation method. The oligoA-tailed DNA-gold nanomaterial comprises AuNPs, and / or AuNRs, and modified oligoA-tailed DNA; the oligoA-tailed DNA sequence is shown in SEQ ID No. 1. This preparation method is low-cost, easy to operate, and can achieve high-density oligoA-tailed DNA modification on the surface of gold nanoparticles with different morphologies, while exhibiting high stability.

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.

This invention relates to a method for preparing a functional DNA gel and its applications. Addressing the problems of existing methods for immobilizing functional nucleic acids requiring DNA modification, which increases immobilization costs, and the reduction of functionality due to chemical fixation, this invention mixes functional circular DNA with a gel monomer solution, and adds an initiator and a coagulant to initiate a cross-linking polymerization reaction. During the reaction, the gel monomers pass through the functional circular DNA, polymerizing into long chains and then cross-linking into a network structure. This allows the functional circular DNA to be enriched on the network structure in a suspended, fixed form, resulting in a functional DNA gel. The functional circular DNA is circular DNA containing functional nucleic acid sequences. This invention immobilizes functional circular DNA on the gel network structure without chemical modification, and the preparation method is simple. It can be used for the efficient and specific removal of pollutants such as heavy metal ions, toxins, veterinary drugs, and pesticides from solutions, as well as the enrichment of small molecule nucleic acids in solutions.

Provided is a simple and efficient method of plant genome editing that does not involve the incorporation of a gene cassette for the expression system of a site-specific DNA modification protein. The method of the present invention comprises: (i) a step for mixing plant cells or a tissue containing plant cells together with a site-specific DNA modification protein and a needle-shaped inorganic compound in a liquid medium and making a disturbance to perforate the cells with the needle-shaped inorganic compound, thereby introducing the site-specific DNA modification protein into the cells; and (ii) a step for culturing the plant cells or the tissue containing the plant cells, into which the site-specific DNA modification protein has been introduced in step (i), thereby causing a DNA mutation specific to a target site in the genome of the plant cells.

The invention relates to a method for detecting DNA modification, in particular to a construction method of a DNA library with modified nucleotide, a detection method, a kit set and application. According to the scheme provided by the invention, the necessity of designing degenerate primers is avoided, the design difficulty of multiple primers is greatly reduced, the specificity of multiple PCR amplification is improved, and the formation of primer dimers is reduced.

The invention discloses a DNA modification and positioning method based on quantum dots, and relates to the technical field of DNA sequencing, and the method comprises the following steps: S1, combining CdSe quantum dots at the tail end of DNA to form a DNA and quantum dot compound; s2, enabling the DNA and quantum dot compound to pass through a nanopore of a nanopore detection system under the driving of an electric field, and collecting a tunneling current signal in real time by utilizing a current amplifier of the nanopore detection system; s3, performing filtering and noise reduction processing on the acquired tunneling current signal to obtain a processed current signal; s4, the processed current signals are subjected to feature recognition, the current base type and a recognition result are obtained, and the recognition result is recognition accuracy or recognition inaccuracy. According to the invention, the signal-to-noise ratio of single-base identification is improved, and the accuracy and stability of base identification are improved.

The present invention relates to a method of profiling covalent DNA modification(s) at a cellular or nuclei level comprising the steps of: providing a sample solution, comprising cell(s) comprising a nucleus comprising genomic DNA; adding a buffer comprising a nucleosome depletion agent to expose the genomic DNA; adding a fusion protein comprising a DNA modification binding domain and a nuclease part; incubating the fusion protein together with the exposed genomic DNA under conditions allowing the fusion protein to bind to the genomic DNA; washing the solution of step (d) with buffer to remove any excess fusion protein; incubating the solution of step (e) under conditions allowing the fusion protein to cut the genomic DNA in proximity of the DNA modification(s); and determining the sequence of the fusion protein incubated DNA of step (e) by high-throughput sequencing, thereby identifying the position(s) of the DNA modification(s) of the genomic DNA that have been sequenced. In other aspects, the invention also relates to a kit for use in performing the method.

Provided herein are methods and compositions for use in detecting on-target and / or off-target cleavage of genomic DNA in a cell by a nuclease. The on-target and off-target cleavage is detected by using oligonucleotides that bind to the single-stranded 3′-overhang created at the cleavage site. The nuclease may be an engineered nuclease comprising a nucleic acid binding domain that binds to a target nucleic acid of interest.

The present invention relates to a composition comprising a modified DNA identifier sequence (MoDIS) comprising (i) a verification code nucleotide sequence (VC), (ii) a random index code nucleotide sequence (RIC) and (iii) an annealing site nucleotide sequence (AS) for specific binding to a primer for PCR amplification, wherein (a) AS is linked to an affinity tag or label and / or affinity tag compound for identifying, enriching and / or purifying MoDIS-bound nucleotide sequences; and (b) the nucleotide at the 5 '-terminus of the MoDIS comprises a 5'-azido-modified moiety or a 5 '-alkynyl-modified moiety.

The invention belongs to the technical field of DNA modification site detection, and particularly discloses a sequencing method for identifying a zooxanthellae genome 5-hydroxymethyl uracil based on double-enzyme cascade. The method comprises the following steps: specifically recognizing and cutting 5-hydroxymethyl uracil through human single-chain selective single-function uracil glycosylase to generate a base removal site, efficiently cutting an AP site by combining with cutting mismatched nuclease optimized by a catalytic structural domain, and directionally generating a 200-300bp DNA fragment containing an adhesive tail end; and analyzing the cleavage site by using next-generation sequencing to realize single-base resolution positioning. According to the method, cross interference and DNA damage of a traditional chemical oxidation method to 5hmC / 5fC are avoided through a double-enzyme cascade reaction, and single-base resolution positioning can be achieved; meanwhile, according to the method, the experimental process is simplified, 5hmU recognition, AP site generation and cutting are integrated into one-step reaction, and the library building period is shortened. Accurate positioning and quantitative analysis of the single base level of 5hmU are realized in a zooxanthellae genome, and epigenetic regulation and environmental stress response of the zooxanthellae are disclosed.

The DNA modification kit comprises a kit body, one side of the inner wall of the kit body is fixedly connected with a fixing plate, circular grooves are formed in the top surface of the fixing plate at equal intervals, test tubes are inserted into the circular grooves, a sliding rod is fixedly installed in the middle of the inner bottom wall of each circular groove, and one side of the surface of each sliding rod is fixedly connected with a circular piece. A spring is fixedly connected to the top of the circular piece, a sliding block is slidably connected to the surface of the sliding rod, a special-shaped sliding groove is formed in the front face of the sliding block, and a connecting block is fixedly connected to one side of the sliding rod. The fixing plate, the test tubes, the springs, the driving assemblies and the special-shaped sliding grooves are used in cooperation, when medical staff need to fix the test tubes, the staff can place the specified test tubes in the circular grooves, the bottoms of the test tubes abut against the top of a circular disc, sliding blocks slide downwards along sliding rods, and the test tubes are fixed through the sliding rods. And then when the spring contracts, the driving rod can be matched with the sliding block to slide from the bottommost part of the special-shaped sliding groove to the topmost part for clamping.

This invention provides a DNA modification detection method using gel electrophoresis coupled with liquid chromatography-mass spectrometry (LC-MS / MS), comprising the following steps: separating a DNA sample to be tested by gel electrophoresis to obtain a gel strip containing the target DNA, wherein the electrophoresis buffer used in the gel electrophoresis method does not contain EDTA; enzymatically digesting the target DNA in the gel strip into single deoxynucleosides / deoxynucleotides by in-gel DNA digestion to obtain enzymatic digestion products; and detecting the enzymatic digestion products by liquid chromatography-mass spectrometry to obtain the results of DNA chemical modifications (including epigenetic modifications and DNA damage adducts). The detection method of this invention can accurately quantify the chemical modification information in different DNA components separated by gel electrophoresis.

To provide a DNA-targeting RNA that comprises a targeting sequence and provides, together with a modifying polypeptide, site-specific modification of a target DNA and / or a polypeptide associated with the target DNA.SOLUTION: Provided is a DNA-targeting RNA comprising: (i) a first segment comprising a nucleotide sequence complementary to a sequence in the target DNA; and (ii) a second segment that interacts with a site-directed modifying polypeptide, where, as an aspect, the first segment comprises 8 nucleotides having 100% complementarity to a sequence in the target DNA.SELECTED DRAWING: None

Aspects of the application relate to template nucleic acids capable of introducing edits into DNA targets. Templates of the application comprise two homology arms flanking a heterologous nucleic acid comprising nucleotides that do not base pair with a sequence in a DNA target. The nucleotides that do not base pair with the DNA target can be utilized to, for example, reduce recombination between the heterologous nucleic acid and the DNA target relative to recombination between the homology arms and the DNA target, which allows for site-specific insertion of one or more genetic edits. In some aspects, templates are described that can be used to introduce edits at a site that is separated from a DNA break by tens, hundreds, thousands, or more than thousands of nucleotides. Further aspects of the application relate to recombinant adeno-associated virus particles and methods related to template nucleic acids described herein.

The invention provides crRNA, a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system and a kit for SMN1 gene mutation detection. The nucleotide sequence of the crRNA is as shown in SEQ ID NO: 31. The crRNA is very suitable for detecting SMN1 gene mutation through an RPA-Cas12a one-step method, the tolerance of the crRNA to single base mismatch in a target is low, and the SMN1 gene recognition specificity is obviously enhanced; moreover, 9-nt single-stranded DNA modification is added to the 3'terminal of the RPA-Cas12a, so that the cutting efficiency of Cas12a to a substrate in RPA-Cas12a one-step detection can be effectively reduced, amplification and accumulation of target DNA are promoted, the detection sensitivity is effectively improved (about 100 times), and the detection performance is improved. Furthermore, a lateral flow chromatography test strip is developed on the basis of the crRNA, non-SMA patients and SMA patients can be rapidly and visually distinguished, large expensive equipment and skilled operators are not needed for detection, and the lateral flow chromatography test strip has the advantages of high sensitivity, high specificity, low cost and the like, is suitable for large-scale screening, is high in universality and can be widely applied to the field of clinical detection. And a new means is provided for early screening and prevention of SMA.

The present invention provides a synthetic, modular system for DNA modification as may be employed for genome editing comprising a targeting nucleic acid possessing both DNA targeting ability and ability to bind a recognition module of a modular polypeptide component, where the modular polypeptide component also includes an effector component and a short peptide DNA-binding sequence (DBD) which binds a pre-determined sequence (PBS) in the target. The DBD may preferably be no more than a 15 mer and serves to destabilize the structure of a targeted dsDNA upon binding thereby facilitating the desired DNA modification. The effector component may, for example, be an artificial nickase comprising linked self-assembling peptides thereby providing a complete genome-editing system of advantageous small size for vector delivery to cells.

The invention relates to the field of biotechnology and biomedicine, in particular to a trace DNA modified high-throughput sequencing library, a construction method thereof and a method for detecting trace DNA modification in a to-be-detected sample. Comprising the following steps: extracting a DNA sample from a to-be-detected sample; carrying out a transposition reaction on the mixed transposase complex and a DNA sample to prepare a transposition product; a DNA fragment is prepared from a transposon product through notch chain supplementation; mixing a plurality of DNA fragments with different sample tags with quality control DNA to prepare a multi-sample DNA fragment mixture; carrying out denaturation treatment on the multi-sample DNA fragment mixture to prepare a single-chain mixture; carrying out binding reaction on the mixed binding molecule-solid phase conjugate and the single-chain mixture to prepare a target chain-binding molecule-solid phase conjugate; collecting a target chain on the target chain-binding molecule-solid phase conjugate, and performing PCR amplification by taking the target chain as a template to construct a high-throughput sequencing library. The method is high in sensitivity and specificity, extremely low in sample demand quantity and beneficial to high throughput and automation.

The present disclosure relates to methods and systems for improved specificity and / or efficiency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) system. In particular, the present disclosure provides systems and methods for increasing specificity and efficiency of CAST system by: modulating TnsC function and abundance; modulating TnsB function and abundance; and influencing CAST target preference.

The present invention provides a novel protein that can be used in modifying target DNA. A protein according to the present disclosure is a protein (a1), a protein (a2), or a protein (a3). (a1) is a protein that comprises the amino acid sequence of any one of SEQ ID NO: 1-19. (a2) is a protein that comprises an amino acid sequence obtained by deleting, inserting, substituting, or adding one or more amino acids in the amino acid sequence of any one of SEQ ID NO: 1-19, and that has nuclease activity. (a3) is a protein that comprises an amino acid sequence having not less than 80% identity with the amino acid sequence of any one of SEQ ID NO: 1-19, and that has nuclease activity.

The present disclosure provides methods and systems for DNA modification and gene targeting comprising an engineered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated transposon (CAST) systems. More particularly, the present disclosure provides systems comprising: an engineered CAST system or one or more nucleic acids encoding the engineered CAST system, wherein the CAST system comprises at least one or both of: a) at least one Cas protein (e.g., Cas6, Cas7, Cas5, and / or Cas8) and b) one or more transposon-associated proteins (e.g., TnsA, TnsB, TnsC, TnsD, and / or TniQ), and at least one unfoldase protein (e.g., ClpX), or a nucleic acid encoding thereof. The present disclosure also provides systems, kits, and methods for nucleic acid modification in a cell.

Provided herein are compositions, methods, and systems for DNA modification. In particular, provided herein are compositions, and systems comprising TnpB-like nuclease-dead repressors (dTnpB / TldRs), dCas12f or dCas12f-like proteins, and / or a TnpB-transposase fusion proteins and methods using thereof.

The invention discloses a DNA modification and sequencing method based on quantum dots and charge-rich groups, and relates to the technical field of DNA sequencing, and the method comprises the following steps: S1, preparing a plurality of DNA modifiers, splitting the plurality of DNA modifiers into DNA modifiers for calibration and DNA modifiers to be sequenced in parallel, and obtaining characteristic parameters of the DNA modifiers by using the DNA modifiers for calibration; s2, after electric field parameter setting is carried out on the nanopore sequencing device, the to-be-sequenced DNA modifier is placed in the nanopore sequencing device, and the to-be-sequenced DNA modifier starts to move towards the nanopore under the driving of one end of a negative charge group of the nanopore sequencing device through an electric field; s3, after a current amplifier of the nanopore sequencing device is subjected to amplification parameter setting, the current amplifier collects a current signal generated in the process that the DNA modifier to be sequenced passes through the nanopore; and S4, processing and identifying the collected current signal to obtain a DNA sequence identification result. The sequencing accuracy and reliability can be improved.