165 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 discloses an ecDNA (ectodeoxyribonucleic acid) identification method based on scATAC-seq (scATAC-Seq) data. The ecDNA recognition method provided by the invention can be used for accurately detecting copy number amplification, breakpoint quantity and ecDNA feature quantity on the whole cell level. According to the method provided by the invention, the specific carried annular oncogene in the tumor can be identified, the distribution characteristics of the annular oncogene in the genome can be analyzed, and the overall composition of all annular DNAs in a single cell and the specific composition of the single annular DNA can be disclosed. In addition, according to the method, the existence of the circular DNA can be verified through a dyeing experiment, and the reliability of a detection result is ensured.

The invention provides an intratumoral bacterium detection and analysis method based on circular DNA enrichment sequencing and related model construction, and belongs to the field of molecular biology. The intratumoral bacterium detection method provided by the invention comprises the following steps: extracting total DNA of cancer tissue or para-carcinoma tissue, carrying out linear digestion, purifying a linear digestion product, and enriching to obtain circular DNA; the method comprises the following steps: amplifying circular DNA, and breaking a DNA amplification product to 300-500bp; purifying DNA fragments, constructing a library, and sequencing; and performing genome comparison according to a sequencing result to obtain information of the intracellular bacteria. According to the method, complete bacterial genome and plasmid genome information can be captured, and the detection sensitivity of the intracellular bacteria and the richness of the bacterial genome information are improved. The invention further provides an intratumoral bacterium analysis method which can be used for screening generic cancer bacterial markers, and provides a cancer risk assessment machine learning model based on the intratumoral bacterium, so that the clinical application possibility of the intratumoral bacterium is improved.

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.

Provided herein are non-immunogenic, circular, non-integrating, non-viral DNA vectors, and methods of using such DNA vectors, which are capable of forming one or more specialized secondary structures, for example extended cruciform structures, and express one or more therapeutic genes. These DNA vectors capable of forming one or more specialized secondary structures, for example extended cruciform structures, provide for increased nucleus entry, episomal stability, and long-term transgene expression, and have unique and highly unexpectedly reduced immunogenicity. The one or more specialized secondary structures, for example extended cruciform structures, formed by the circular DNA vectors provided herein are stabilized through the interaction of two specifically oriented repeating sequences, for example AAV-derived inverted terminal repeat (ITR) sequences, separated by a small, non-repeating sequence, for example a non-repeating sequence comprising a small bacterial origin of replication (Ori).

The invention discloses a method for promoting endothelialization of a heart valve and an artificial heart valve.The method is characterized in that circular DNA is modified on the surface of a decellularized valve (DHV), DNA hydrogel of targeted thrombin is obtained on the surface of the DHV through rolling circle amplification, the DNA hydrogel plays a role of a heme catcher while playing a role of the heme catcher, and heme is recruited from blood; heme is further converted into biliverdin (BV) through heme oxygenase (HO1) loaded in DNA, so that continuous production of biliverdin is ensured, anti-inflammatory immune regulation and active oxygen removal are promoted, a regenerative immune microenvironment is created, and endothelialization of the valve is promoted.

Provided is a method with which it is possible to simply and exponentially amplify circular DNA, particularly long-chain circular DNA, in a cell-free system. Specifically, provided is a method for amplifying circular DNA, which comprises an isothermal reaction of a reaction mixture produced by mixing circular DNA having a replication initiation sequence (oriC) with a reaction solution containing the following enzyme groups: (1) a first enzyme group that catalyzes replication of the circular DNA, (2) a second enzyme group that catalyzes replication of the circular DNA, (3) a third enzyme group that catalyzes replication of the circular DNA, (4) a fourth enzyme group that catalyzes replication of the circular DNA, and (5) a third enzyme group that catalyzes replication of the circular DNA. (2) a second enzyme group which catalyzes a linking reaction of an okazaki fragment to synthesize two sister circular DNAs forming a catenary body; and (3) a third enzyme group that catalyzes a separation reaction of the two sister circular DNAs.

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 invention belongs to the technical field of immunodetection, and particularly relates to an unlabeled multiple immunodetection system based on core-shell photon bar code integrated rolling circle amplification and application. The non-labeled multiple immunodetection system comprises: 1) a probe-modified porous hydrogel-PhC shell microsphere, the probe is used for capturing a target nucleic acid molecule to be detected, the porous hydrogel-PhC shell microsphere is formed by coating a silica microsphere with a porous hydrogel, and the diameter of the porous hydrogel-PhC shell microsphere is 200-200 [mu] m; 2) an antibody modified by a DNA promoter; 3) a blocking / primer compound; and 4) a circular DNA. The detection system provided by the invention realizes high-sensitivity multiple quantitative detection, and the detection limit is within the range of 0.030 pg / mL. The detection system provided by the invention is verified through clinical sample analysis, has acceptable accuracy and high specificity, and proves that the provided detection system and method have good potential applicability in clinical screening and diagnosis.

Provided herein are improved methods for producing engineered bacterial cells of circular DNA vectors, circular DNA vectors produced by such methods, and pharmaceutical compositions containing such circular DNA vectors. The methods provided herein are suitable for large scale production of high purity compositions of circular DNA vectors.

Provided is a method capable of replicating or amplifying circular DNA, and particularly, long-chain circular DNA, in a cell-free system. Specifically, provided is a method for suppressing generation of a DNA multimer as a by-product, when circular DNA having a replication origin sequence (origin of chromosome (oriC)) is replicated or amplified by using the following enzyme groups: (1) a first enzyme group that catalyzes replication of circular DNA; (2) a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; and (3) a third enzyme group that catalyzes a separation of two sister circular DNAs. Moreover, also provided is a method comprising introducing oriC into circular DNA by using a transposon.

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 relates to a circular DNA probe for recognizing vibrio vulnificus and a preparation method of the circular DNA probe, specifically, a linear probe carrying DNAzyme is mixed with a looping primer, the mixture is connected under the action of T4DNA ligase to form the circular DNA probe, the circular DNA probe can be specifically recognized with a target and then is subjected to rolling circle amplification to form long-chain DNA, the long-chain DNA contains a large amount of DNAzyme formed by amplification and repeated GC-rich regions, and the long-chain DNA can be used for detecting the vibrio vulnificus. Intramolecular or intermolecular hydrogen bonds are spontaneously formed and are self-assembled to form the DNA hydrogel in a porous state, so that the structure promotes region limitation, and the detection sensitivity is greatly improved; target concentration gradient verification shows that the linear relation of the kit is good, R2 is equal to 0.9915, clinical sample verification shows that the specificity of the kit is high, and the kit can be used for visual and portable detection of vibrio vulnificus.

The invention discloses an ultrasensitive immunodetection method based on adjacent nucleic acid amplification signal amplification in the technical field of medical detection. The ultrasensitive immunodetection method comprises the following steps: step 1, immune binding; step 2, enzyme digestion release: releasing a hairpin loop DNA single strand; step 3, complementation and enzyme ligation: adding T4 ligase to cyclize the circular DNA single chain; step 4, rolling circle amplification; and step 6, detecting, and quantifying and positioning by qPCR (quantitative polymerase chain reaction) and an in-situ oligonucleotide fluorescence labeling detection method. According to the present invention, the hairpin ring structure on the oligonucleotide DNA chain coupled to the antibody protects the complementary sequence so as to avoid the generation of the non-specific circular DNA single chain, and the rolling circle amplification technology is combined to amplify the detection signal, such that the detection background signal intensity is inhibited while the specificity of the detection signal is enhanced so as to further improve the detection sensitivity.

The present disclosure relates to the technical field of sequencing, and in particular to a method and reagent for preparing a sequencing library. Provided in the present disclosure is a method for preparing a sequencing library. According to the method, by means of additionally adding an additive or an additive combination capable of reducing the DNA secondary structure and / or reducing the degradation of single-stranded DNA in the single-chain cyclization reaction and / or the rolling circle amplification reaction, and additionally adding an additive or an additive combination capable of reducing the DNA secondary structure and reducing the degradation of single-stranded DNA and / or reducing the stability of the double helix structure of double-stranded DNA in the rolling circle amplification reaction of the double-stranded circular library, the ability of the DNA ligase and / or the strand displacement DNA polymerase to bind to a target nucleic acid is enhanced, such that the non-uniform rolling circle amplification efficiency of different circular DNAs caused by the secondary structure is reduced, the probability that the loose single-stranded DNA is easily degraded is reduced, the amplification uniformity of regions with different GC contents and different sequence characteristics in the library is improved, and thus the sequencing quality and the yield of sequencing data are improved.

The invention provides a preparation method of a chromosome outer circular DNA library, a sequencing method, a kit and application thereof. The preparation method comprises the steps that total DNA in a sample is extracted, linear DNA in the total DNA is digested, a digestion product is obtained, and the digestion product comprises chromosome outer circular DNA; directly carrying out rolling circle amplification on the digestion product without purification to obtain an amplification product; an amplification product is directly subjected to library preparation without being purified, and the chromosome outer circular DNA library is obtained.

The present invention provides an efficient method for producing circular DNA. This method for producing circular DNA includes: (1) preparing circular DNA that will serve as a template, said circular DNA including (i) a replication initiation sequence capable of binding to an enzyme that has a DnaA activity, and (ii) a gyrase binding sequence, and said circular DNA not including a DnaA box cluster at a different position from the replication initiation sequence; (2) forming a reaction mixture of the circular DNA prepared in (1) and a reaction liquid including a first enzyme group that catalyzes replication of the circular DNA, a second enzyme group that synthesizes two sister circular DNAs forming a catenane, and a third enzyme group that catalyzes a separation reaction of the two sister circular DNAs; and (3) incubating the reaction mixture formed in (2) at a temperature of 80°C or lower, thereby obtaining a reaction product that includes the circular DNA, which has been amplified.

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.

The invention relates to novel ionizable amine lipids incorporating one or more sulphur atoms in the tail section. These lipids can be used in combination with other components to form lipid nanoparticles with oligonucleotides. 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, miRNA, pDNA, circular DNA, dsRNA, small biologically active molecules) into the cells.

The invention relates to a novel silicon-containing ionizable cation lipid family, which belongs to a first-generation lipid with the trademark of LipexSilTM, and at least one of two side chains comprises a silyl acetal joint. So far, lipids comprising a silyl acetal linker are unprecedented in the art and are effective as ionizable cationic lipids in formulations of empty or loaded lipid nanoparticles (LNPs). The novel linker according to the invention is designed by means of a borane catalyst [WO2022 / 129966]. The present invention describes the synthesis of lipids of formula (I), the formation and characterization of nanoparticles and biological experiments that lipid nanoparticles prepared with these novel lipids are effective in delivering their articles (e.g., RNA, DNA, mRNA, siRNA, pDNA, circular DNA, biologically active small molecules) into cells.

The invention discloses a preparation method of an RCA-Cas12a-DNA (deoxyribonucleic acid) tetrahedron sensor for detecting vancomycin in blood serum. The sensor comprises a target object vancomycin, a DNA chain TSP1, a DNA chain TSP2, a DNA chain TSP3 and a vancomycin aptamer Aptamer which are used for preparing a DNA tetrahedron, a DNA chain T, a DNA chain S, a T4 DNA ligase, exonuclease I and exonuclease III which are used for preparing circular DNA, a DNA chain P and phi29 DNA polymerase which are used for carrying out rolling circle amplification reaction, and LbCas12a nuclease, crRNA and ssDNA-FQ probes which are used for preparing a reporter. The aptamer is specifically combined with a target object, a non-nucleic acid target is converted into nucleic acid detection, and the application range of RCA and Cas12a is expanded; rCA is combined with Cas12a, so that a signal is amplified twice, the detection limit is lower, and the sensitivity is higher; compared with a traditional detection method, expensive instruments and equipment, complex and time-consuming sample pretreatment and skilled operators are not needed, and the method is more convenient and economical.

The present invention belongs to the field of gene detection. More specifically, the present invention provides a method for constructing a library and sequencing of extrachromosomal circular DNA, a double-stranded circular DNA for detecting extrachromosomal circular DNA, and a kit comprising the double-stranded circular DNA. By using the method for constructing a library and sequencing of extrachromosomal circular DNA of the present invention, extrachromosomal circular DNA with a content as low as 5 pg per milliliter of sample can be detected, and true negative results and false negative results can be accurately and effectively distinguished.

The present invention provides: a method for easily isolating a plurality of gene species from the gene species in a cell-free system; and a method for easily isolating and selecting a specific gene species in a cell-free system using indexes derived from individual gene species. The present invention is a DNA amplification method comprising: preparing a mixture of DNA that is circular DNA having a replication origin sequence capable of binding to an enzyme having a DnaA activity; preparing a reaction gel solution containing the mixture, a first enzyme group which catalyze the replication of the circular DNA, a second enzyme group which synthesize two sister circular DNA molecules that catalyze an Okazaki fragment linking reaction to form a catenane, a third enzyme group which catalyze the reaction for separating the two sister circular DNA molecules, and a gelatinizing agent; gelatinizing the reaction gel solution to produce an amplification gel plate containing the circular DNA; and subjecting the DNA to a circular DNA amplification reaction at a temperature of 80°C or lower using the gel plate to produce amplified 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.

The present invention provides a circular DNA template precursor for detecting Clostridium difficile, a preparation method thereof and an application thereof. The circular DNA template precursor includes a three-dimensional self-assembly reaction using a reaction system composed of H1 and H2 hairpin probes and a trigger strand Trigger. The three-dimensional self-assembly reaction includes Trigger triggering the H1 hairpin structure to form a T / H1 intermediate, the H1 hairpin structure being unfolded and binding to H2 to produce an H1 / H2 structure, and the DNA template of H2 being cyclized to obtain the circular DNA template precursor. When Clostridium difficile toxin is present, the trigger strand Trigger can be released again, and can continuously catalyze the hairpin structure of the H1 DNA hairpin probe without being consumed. By adopting the technical scheme of the present invention, signal cascade three-stage amplification is realized through three-dimensional self-assembly-amplification reaction and silver-enhanced gold nanoparticles, so as to realize highly sensitive and highly specific detection of Clostridium difficile infection.