73 results about "Circular DNA" patented technology

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The invention relates to the technical field of biological detection, and discloses an in-situ detection method for the tissue level of a protein and phosphoinositide compound, which comprises the following steps: providing a to-be-detected tissue sample containing the protein and phosphoinositide compound; incubating the sample to be detected by using a primary antibody mixture; incubating a product obtained in the previous step by using a second antibody mixture containing a PLA probe, and reacting to obtain circular DNA (Deoxyribose Nucleic Acid); carrying out in-situ amplification by taking the circular DNA as a template; and acquiring and analyzing a signal of the detection probe. According to the method, the interaction between the protein and the phosphoinositide compound can be specifically detected in situ at the tissue level, the actual state of the protein and the phosphoinositide compound in the physiological and pathological processes of cells can be more truly reflected by the technology, and more accurate information is provided for researching the function and regulation mechanism of the protein and the phosphoinositide compound; wide application prospects are realized.

The invention relates to the technical field of food safety detection, in particular to a food crop pathogenic mycotoxin detection method based on double signal amplification. The core of the method is a section of linear lock-type probe containing a mycotoxin specific nucleic acid aptamer, under the condition that target mycotoxin exists in a sample, the aptamer is combined with the mycotoxin, the probe is induced to generate conformational change, and the probe is closed into annular DNA under the catalysis of DNA ligase; then, the circular DNA is used as a template, isothermal rolling circle amplification is carried out through Phi29 DNA polymerase, and a long-chain DNA product containing a large number of repetitive sequences is generated; subsequently, the long-chain product is used as a molecular scaffold, and two kinds of gold nanoparticles of which the surfaces are modified with different complementary probes are cross-linked at the same time, so that the nanoparticles are quickly gathered, the color of the solution is changed from wine red to blue or purple, and convenient visual detection is realized. The method has the outstanding advantages of ultrahigh sensitivity, rapid detection and the like, and is suitable for on-site rapid screening of mycotoxin pollution.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The invention discloses an early-stage lung cancer prediction method based on a multi-modal eccDNA marker, and relates to the technical field of liquid biopsy, and the method comprises the following steps: collecting a peripheral blood sample, carrying out differential centrifugal separation on plasma, constructing a cfDNA library, and carrying out double-end sequencing to obtain original sequencing data; extracting structural features through an eccDNA model, and inputting the structural features into a first machine learning model to generate a circular DNA score; extracting variation features through an SNV model, and inputting the variation features into a second machine learning model to generate a spectrum feature score; extracting copy number features through a CNV model, and inputting the copy number features into a third machine learning model to generate copy number scores; carrying out probability distribution calibration on the circular DNA score, the spectrum feature score and the copy number score; inputting the three types of molecular features into a deep neural network to generate a first fusion score; inputting the calibrated score into a logistic regression model to generate a second fusion score; and generating a final lung cancer risk probability according to the first fusion score and the second fusion score.

The invention belongs to the technical field of DNA amplification, and relates to a mixed enzyme system capable of amplifying circular DNA as well as application and a product of the mixed enzyme system. The invention provides a mixed enzyme system capable of amplifying circular DNA (deoxyribonucleic acid). The mixed enzyme system comprises a polymerase III tau clip loader and a catalytic core compound. The mixed enzyme system provided by the invention is remarkably superior to a traditional method in the aspects of efficiency, cost, safety, applicability, sensitivity and the like, provides a brand new solution for efficient preparation of the circular DNA, and has a wide application prospect in the field of biotechnology.

The invention belongs to the technical field of detection, and particularly relates to a nucleic acid detection method based on triple amplification and application of the nucleic acid detection method in ginseng identification. Mixing the liquid to be detected with the hairpin DNA, and assembling to form DNA enzyme; a hairpin substrate is added, enzyme digestion reaction is completed, and a trigger chain is generated; adding a circular DNA probe, carrying out rolling circle amplification, and collecting generated long single-stranded DNA; a G quadruplex structure in the long single-stranded DNA is utilized to catalyze a chromogenic reaction, and the concentration of miRNA in the to-be-detected liquid is analyzed through the RGB value of the color. According to the method disclosed by the invention, the low detection limit of the 651 fM is realized. The analysis of ginseng samples and related products verifies the effectiveness of the triple amplification platform, and a portable and reliable solution is provided for food safety supervision and quality control.

Methods are provided for the isolation and analysis of circular DNA from complex samples, based on the topology of the DNA molecule. A sample comprising DNA species is combined with a chaotropic dense salt solution. A fraction containing the circular DNA of interest is isolated and dialyzed to remove excess salt. In some embodiments salt gradients are generated by ultracentrifugation in the absence of intercalating dyes, e.g. ethidium bromide; and in the absence of protease digestion. The circular DNA thus isolated is substantially pure, e.g. greater than about 75%, greater than about 80%, greater than about 90%, greater than about 95% of DNA in the isolated fraction is comprised of circular DNA.

Provided are a circular functional nucleic acid, a preparation method therefor and a use thereof. The method involves combining a functional nucleic acid with a circular nucleic acid, using a method combining DNA template-directed assembly with enzymatic conjugation to first prepare a single-stranded circular DNA, synthesize a corresponding outer short strand, and conjugate a functional nucleic acid drug onto the outer short strand, and optimizing and screening reaction conditions, thereby preparing a circular functional nucleic acid. The circular functional nucleic acid has the characteristics of adjustable size, adjustable valence state (multivalent or polyvalent), high stability, and the capability for chemical conjugation with drugs or drug incorporation, etc.; a novel circular multivalent functional nucleic acid technology is created, which significantly enhances the targeting ability and stability of nucleic acid drugs; thus, a nucleic acid drug development platform o-FLARE having efficient targeting, immune agonism, and protein degradation functions can be established.

The application discloses an ecDNA recognition method based on scATAC-seq data. The ecDNA recognition method provided by the application can accurately detect copy number amplification, breakpoint quantity and ecDNA feature quantity on the whole cell level. The method provided by the application can recognize specific circular oncogenes carried in tumors, analyze the distribution characteristics of the circular oncogenes in the genome, and reveal the overall composition of all circular DNAs in a single cell and the specific composition of a single circular DNA. In addition, the method can also verify the existence of the circular DNA through a staining experiment, so that the reliability of the detection result is ensured.

The application discloses a double-target miRNA detection method based on CRISPR and rolling circle amplification, which comprises the following steps: (1) mixing a padlock probe, a connecting DNA and miRNA-21 and miRNA-155, carrying out annealing treatment, incubating the mixture in a DNA ligase reaction buffer system by using a DNA ligase, then terminating the reaction by heat treatment, and obtaining a circular DNA template; (2) incubating the circular DNA template with a DNA polymerase, dNTPs, recombinant albumin, a primer and a polymerase reaction buffer, then terminating the reaction by heat treatment, and obtaining an RCA product; (3) adding pre-assembled Cas12a-crRNA, the RCA product and FQ fluorescent probes into a buffer, and incubating; and (4) measuring a fluorescent signal. Through optimization of a detection system, the application successfully realizes detection of double-target miRNA, and has high detection sensitivity.

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.

This invention discloses a method and kit for detecting RNA using a three-stage amplification melting curve based on isothermal self-circulation. The method includes designing a first primer, a second primer, and a circular amplification probe targeting the target nucleic acid sequence. The first primer consists of an RNA polymerase promoter sequence and a target nucleic acid-specific binding sequence, sequentially from the 5' end to the 3' end. The circular amplification probe is a circular DNA segment whose sequence consists of three parts: the first part completely contains a continuous sequence composed of the tag sequence and the nicking enzyme-specific recognition sequence from the second primer; the second part is a self-cleaving DNase sequence. In the technical solution used in this invention, a cascaded nucleic acid amplification system is constructed, achieving exponential signal amplification of the target nucleic acid through the synergistic effect of rolling circle amplification and RNA polymerase-mediated transcriptional amplification.

The invention relates to circular DNA molecule for rescuing recombinant adenoviruses comprising a recombinant adenoviral genome with two inverted terminal repeats (ITRs) flanking the genome ends, wherein at least one of the ITRs is associated with a target sequence adjacent to a PAM sequence, wherein the target sequence is configured for generating an RNA-guided DNA endonuclease-mediated DNA double strand break at the external end of or in close proximity outside the external end of the respective ITR, preferably within less than about 15 nucleotides. The invention also relates to a kit and a method for rescuing recombinant adenoviruses comprising or using a circular DNA molecule as described herein.

The application belongs to the technical field of DNA amplification, and has a mixed enzyme system capable of amplifying circular DNA, application and product thereof. The application provides a mixed enzyme system capable of amplifying circular DNA, wherein the mixed enzyme system comprises a polymerase III tau clamp loader and a catalytic core complex. The mixed enzyme system provided by the application is significantly superior to traditional methods in terms of efficiency, cost, safety, applicability and sensitivity, and provides a brand-new solution for efficient preparation of circular DNA, and has a wide application prospect in the field of biotechnology.

Disclosed herein is a method of purifying circular DNA from a sample by exposure of the sample to a nanoclay that preferentially adsorbs multiple contaminants, including non-circular DNA, RNA, proteins, and endotoxins, while leaving circular DNA substantially unbound in solution. The elution-free method also includes a step of separating nanoclay-contaminant complexes from the enriched circular DNA fraction. Also disclosed herein are compositions and systems operable for purifying circular DNA from a sample. Also disclosed herein are methods of refining, formulating or modifying a nanoclay for use in purifying circular DNA from a sample.

This invention discloses a bisphosphonate-nucleic acid nanoparticle, its preparation method, and its applications. The preparation method involves adding divalent metal ions to a rolling circle amplification (RoBA) reaction solution, followed by the addition of bisphosphonate, to perform a RoBA reaction to obtain the bisphosphonate-nucleic acid nanoparticles. The RoBA reaction solution contains a circular DNA template. The preparation method of this invention has the advantages of high speed, precise synthesis, and simplicity, realizing multiple reactions—RoBA, ion coordination, crystallization, and co-assembly of DNA and bisphosphonate—in the same reaction system, greatly simplifying the reaction process and improving reaction efficiency. The obtained bisphosphonate-nucleic acid nanoparticles exhibit dual-pathway therapeutic effects and excellent ribozyme stability, showing promising application prospects.

The application discloses a kind of for sweat glucose concentration monitoring method, it is related to sweat monitoring field, comprising the following steps: step 1, enzyme-fixed chain conjugate synthesis: glucose dehydrogenase is reacted with bilirubin oxidase respectively with bifunctional crosslinking agent and is purified, obtain enzyme-crosslinking agent intermediate;It is clicked with azide modified DNA fixed chain chemistry reaction again, and after purification, obtain GDH-fixed chain and BOx-fixed chain conjugate;Step 2, preparation of circular DNA template: after annealing hybridization of phosphorylated template chain and primer chain, it is connected into ring by T4 DNA ligase;Step 3, rolling circle amplification self-assembly package enzyme DNA hydrogel;The present application uses super-flexible DNA hydrogel as flexible electrode material, realizes the efficient fixation and continuous stable catalysis of GDH and BOx, and constructs flexible sweat-based EBFC self-powered sensor as flexible bioelectrode, to realize continuous non-invasive blood glucose monitoring.

The present invention is in the field of biomedicine and drug delivery as well as pest and vector control. The present invention relates to a novel family of silicon-introduced ionizable cationic lipids belonging to the trademark LipexSill second generation lipids wherein the tail is linked to the head group via a biodegradable silicon-based acetal linker. Lipids containing a silicon-based acetal linker are the most advanced, and are effective as ionizable cationic lipids in formulating empty or loaded lipid nanoparticles (LNPs). The novel linker according to the present invention is [WO2022129966] designed using a proprietary borane catalyst. The present invention describes the synthesis of lipids of formula (I), the formation and characterization of nanoparticles, and biological experiments that prove that lipid nanoparticles prepared with these novel lipids can effectively deliver their cargo receptors (cargo) (e.g., RNA, DNA, mRNA, siRNA, dsRNA, pDNA, microRNA, circular DNA, small bioactive molecules) into cells.

Provided herein are improved DNA vectors (e.g., circular DNA vectors and / or non-viral DNA vectors) that can provide features including enhanced expression, persistence, safety, and manufacturability. Also provided herein are methods of producing such DNA vectors, host cells (e.g., engineered bacterial cells useful for producing such DNA vectors) containing such DNA vectors, methods of using such DNA vectors (e.g., engineered bacterial cells useful for producing such DNA vectors), and methods of using such DNA vectors. Methods of expressing therapeutic sequences in target cells by administering such DNA vectors and methods of treating diseases or conditions by administering such DNA vectors), as well as pharmaceutical compositions containing such DNA vectors.

A method for producing deoxyribonucleic acid (DNA) includes providing a circular DNA template. The method also includes conducting in vitro an amplification reaction on the circular DNA template to generate a DNA product that is rolling circle amplified, wherein a reaction fold-amplification is minimized to enhance protein expression from the DNA product, and wherein a rolling circle amplification reaction is conducted with at least one of (i) the rolling circle amplification reaction being conducted at 15 degrees Celsius to less than 25 degrees Celsius to minimize the reaction fold-amplification, (ii) the rolling circle amplification reaction being conducted with an input concentration of the circular DNA template being greater than 1 nanogram per milliliter to minimize the reaction-fold amplification, or (iii) the rolling circle amplification reaction being conducted with input concentrations of deoxyribonucleoside triphosphates being titrated to 1.2 millimolar or less per deoxynucleotide to minimize the reaction-fold amplification.

The invention relates to a method and a kit for generating nucleic acid for target capture. The method involves binding of a circ-olio sequence to at least one target oligonucleotide to generate a single stranded circular DNA. The single stranded circular DNA is bidirectionally amplified with the sense and antisense oligonucleotides, followed by biotinylation to obtain for a double-stranded concatemer of linear sequence. The double-stranded concatemer of linear sequence with the restriction endonucleases produces a target capture nucleic acid. The method offers a scalable targeted capture of pr eselected genomic regions, and is cost effective. The method of generating nucleic acid for target capture has application in the field of oncology hematology, inherited malignancies, germline disorders, microbiology and prenatal diagnosis. The invention generates baits over high GC-rich regions of the genome.

The disclosure provides, for example, double stranded DNA (dsDNA) molecules comprising one or more chemically modified nucleobases. In some embodiments, the dsDNA molecule is circular and comprises a first strand and a second strand, wherein the first strand comprises one or more chemically modified nucleobases, and the second strand is free of chemically modified nucleobases. In some embodiments, the dsDNA molecule comprises a promoter sequence and an effector sequence that encodes an effector.

The present invention belongs to the field of biomedicine and drug delivery as well as pest and vector controls. The invention relates to a novel ionizable cationic lipid family incorporating silicon, which belongs to the trademark LipexSil® second generation lipids, wherein the tail is connected to the headgroup with biodegradable silyl acetal linker. Lipids containing silyl acetal linker(s) are state-of-the-art and are effective as ionizable cationic lipids in the formulation of empty or loaded lipid nanoparticles (LNPs). The novel linkers according to the invention are designed by means of proprietary borane catalysts [WO2022129966]. The invention describes the synthesis of the lipids of formula (I), formation and characterization of nanoparticles and biological experiments demonstrating that the lipid nanoparticles prepared with these novel lipids can efficiently deliver their cargo (e.g. RNA, DNA, mRNA, siRNA, dsRNA, pDNA, micro RNA, circular DNA, small biologically active molecules) into the cells.

The present invention relates to methods of generating one or more transgenic cells using circular single stranded DNA (CiSSD) as a donor template, as well as target genome modification. The methods include transferring one or more DNA polynucleotides into a cell for site-specific nuclease-mediated DNA repair, and selecting one or more cells into which the transferred DNA is introduced in the genome of the cell.

The application discloses a single-chain circular DNA virus GyH1 recombinant polyepitope adenovirus vector vaccine and application thereof, and belongs to the technical field of vaccines.The antigen epitopes of GyH1 VP1, VP2 and VP3 proteins are screened through an online bioinformation analysis server, the antigen epitopes are connected by using a linker to construct a polyepitope vaccine GyMEV, and the nucleotide sequence of the GyMEV is obtained through reverse translation and codon optimization.The antigen polypeptide GyMEV nucleotide sequence is cloned into a plasmid vector pMD19-T through artificial synthesis, the GyMEV is directionally cloned into a linearized adenovirus vector by using seamless cloning technology to obtain a recombinant adenovirus plasmid rAd5-GyMEV, and the recombinant plasmid is transfected into HEK 293 cells to obtain the recombinant polyepitope adenovirus vector vaccine GyMEV.The results show that the GyMEV can more effectively stimulate the immune response of the body and has stronger protection.The vaccine also has the safety of a subunit vaccine and can adopt various immunization modes, can stimulate the immune response of the body in all directions, and thus has a wide development and application prospect.

The present invention relates to a method for producing a desired linear combined DNA molecule, linear or circular DNA plasmid using at least one genetically altered E. coli strain, wherein linear DNA fragments are converted into a linear combined DNA molecule or circular DNA plasmid. The method can be performed in vivo or in vitro, in particular as a single step method. The invention further relates to a method for cloning a nucleic acid fragment of interest, comprising the method according to the present invention, specific genetically altered E. coli strains, a kit for performing the method according to the present invention, and the use of the genetically altered E. coli strains, or the kit for producing a linear combined DNA molecule or circular DNA plasmid or cloning a nucleic acid fragment of interest.

The invention discloses a single-stranded circular DNA Marker as well as a preparation method and application thereof, and relates to the technical field of biology. The DNA Marker prepared by the invention has the characteristics of uniform and sharp bands and obvious brightness among the bands during use, can be widely applied to molecular weight indication in a CssDNA electrophoresis process, can be stored at room temperature for a long time, and is convenient to use. The preparation method of the CssDNA Marker is high in stability, repeated production between batches can be guaranteed, and therefore large-scale production is achieved.

The application discloses a bovine viral diarrhea virus chimeric virus expressing porcine circovirus type 3 capsid protein and a preparation method thereof. In the experiment, a gene sequence for encoding a porcine circovirus type 3 capsid protein and a His tag fused with the C terminal of the protein is inserted into different sites of a bovine viral diarrhea virus genome through a circular polymerase extension reaction, and the chimeric virus is prepared through virus rescue of the obtained circular DNA. The His tag sequence in the gene sequence and the bovine viral diarrhea virus genome are connected through a 2A peptide sequence, so as to realize the target of effectively releasing the antigen through the co-expression of the heterologous protein. The chimeric virus prepared by the application retains the infectivity and replication capacity of the parent virus, can stably replicate in cells and efficiently express the target antigen, and provides an efficient and flexible virus carrier platform for the research and development of a porcine circovirus type 3 vaccine.

To provide a novel method that forms a complex linking a peptide with DNA encoding the peptide to establish correspondence therebetween, and to provide a method for readily selecting DNA encoding a desired peptide from a complex library in which peptides correspond to DNA encoding the peptides.SOLUTION: A method for creating a complex in which a peptide is linked to a nucleotide sequence encoding the peptide, the method comprising: (1) preparing circular DNA having a nucleotide sequence encoding a peptide; and (2) transcribing and translating the nucleotide sequence from the circular DNA using a cell-free transcription-translation reaction to express a peptide, wherein the expressed peptide forms a complex linked to the circular DNA.SELECTED DRAWING: None