168 results about "Circular DNA" patented technology

Disclosed are compositions and methods for detecting small quantities of analytes such as proteins and peptides. The method involves associating a primer with an analyte and subsequently using the primer to mediate rolling circle replication of a circular DNA molecule. Amplification of the DNA circle is dependent on the presence of the primer. Thus, the disclosed method produces an amplified signal, via rolling circle amplification, from any analyte of interest. The amplified DNA remains associated with the analyte, via the primer, and so allows spatial detection of the analyte. The disclosed method can be used to detect and analyze proteins and peptides. Multiple proteins can be analyzed using microarrays to which the various proteins are immobilized. A rolling circle replication primer is then associated with the various proteins using a conjugate of the primer and a molecule that specifically binds the proteins to be detectable. Rolling circle replication from the primers results in production of a large amount of DNA at the sites in the array where the proteins are immobilized. The amplified DNA serves as a readily detectable signal for the proteins. The disclosed method can also be used to compare the proteins expressed in two or more different samples. The information generated is analogous to the type of information gathered in nucleic acid expression profiles. The disclosed method allows sensitive and accurate detection and quantitation of proteins expressed in any cell or tissue.

A helicase-mediated amplification method for circular DNA templates and target DNA sequences within the templates is provided. The method combines a DNA polymerase and a helicase preparation to amplify a target sequence as well as the entire circular DNA template.

Provided is kit for and a method of amplifying a nucleic acid using rolling cyclic amplification (RCA), including amplifying a nucleic acid together with formation of a single-strand circular DNA template using RCA by reacting a reaction solution including: (a) two hairpin oligos, (b) a target nucleic acid, (c) a DNA ligase,(d) an endonuclease, (e) a DNA polymerase, and (f) a primer.

The invention relates to a paired-end library construction method and a method for sequencing a genome by using the library. The paired-end library construction method comprises the following steps of: performing plasmid extraction on clone in a constructed genome DNA library, fragmenting the extracted plasmid DNA, performing methyl protection on the restriction endonuclease site on the fragmented DNA, adding a hairclip type joint, performing enzyme cutting and cyclization to obtain circular DNA, then amplifying the genome library paired-end fragment in the circular DNA through a pair of composite primers to obtain a paired-end sequence of ultra-long span, and finally performing high flux sequencing. The paired-end sequence obtained by using the method can be applied to splicing of a new species genome sequence so as to further improve the splicing quality.

The invention relates to a method for using a DNA tetrahedron as a scaffold on a nano-particle surface and initiating rolling circle amplification reaction; after the prepared DNA tetrahedron is assembled on the nano-particle surface, a section of single-stranded DNA extending on the DNA tetrahedron can be used as primer DNA; circular DNA complementary with a special aptamer sequence is used as an amplification template; the circular DNA is hybridized with the primer DNA while the rolling circle amplification reaction is initiated; and the prepared product is multiple long single-stranded DNA composed of multiple apatmer structures on the nano-particle surface. The method disclosed by the invention is capable of effectively regulating and controlling the DNA distribution density on the nano-particle surface, so that the hybridization efficiency of the primer DNA and the circular DNA is increased; by means of long single-stranded DNA composed of the multiple aptamer structures prepared through the rolling circle amplification technology, the CTC (Carbon Tetra Choride) capture efficiency is greatly increased; and thus, the method disclosed by the invention has potential application value in the research fields, such as cell detection, cell capture, cell imaging and the like.

Pharmaceutical compositions of the invention comprise covalently closed circular DNA formation inhibitors having a disease-modifying action in the treatment of diseases associated with the formation of covalently closed circular DNA that include hepatitis B infection, and any disease involving formation of covalently closed circular DNA.

The present invention provides a method for the rapid simultaneous production of a plurality of single-stranded DNA circles having a predetermined size and nucleotide sequence using pre-designed hairpin oligonucleotides containing complementary sequences for directing ligation to form dumbbell-shaped monomers followed by heat denaturation to yield single-stranded DNA circles.

The invention discloses an efficient whole-genome chromosome conformation capture technology (eHi-C). The invention provides an eHi-C sequencing technology for cells. The eHi-C sequencing technology comprises the following steps: 1) subjecting the cells to disintegration so as to obtain chromatin; 2) subjecting the chromatin to enzyme digestion, DNA end labeling, cyclization connection of a blunt end and DNA purification so as to obtain purified circular DNA; 3) introducing a circular co-precipitation DNA molecule used as internal reference; 4) carrying out ultrasonic breaking; 5) capturing all the labeled DNA fragments with immunomagnetic beads; and 6) preparing eHi-C sequencing library from the labeled DNA fragments. The technology provided by invention is low in the amount of needed cells, can easily acquire an experiment material, reduces time in acquisition of the material, greatly lowers cell culture cost and cost for reagent consumables and decreases test errors.

Disclosed are compositions and methods for detecting small quantities of analytes such as proteins and peptides. The method involves associating a DNA circle with the analyte and subsequent release and rolling circle replication of the circular DNA molecule. In the method, an amplification target circle is associated with analytes using a conjugate of the circle and a specific binding molecule that is specific for the analyte to be detected. Amplification target circles not associated with the proteins are removed, the amplification target circles that are associated with the proteins are decoupled from the specific binding molecule and amplified by rolling circle amplification. The amplification is isothermic and can result in the production of a large amount of nucleic acid from each primer. The amplified DNA serves as a readily detectable signal for the analytes.

The invention relates to a nested quantitative PCR detection method for virus covalently closed circular DNA (ccc DNA) of hepatitis b virus. The particular detection steps are: 1) extraction DNA of hepatitis b virus; 2) design and synthesis primers and probe: design an internal and an external primer at conservative region of both sides of negative chain gap of HVB DNA, with Taqman fluorescence probe arranged at the lower part of the negative chain gap; 3) Plasmid-SafeTM ATP-Dependent DNase restriction enzyme purification; 4) nested real time quantitative PCR amplification, comprising: common PCR-template with external primer amplification purification and real time quantitative PCR-fluorescence quantitative amplification to common PCR production with internal primer and fluorescence probe. The invention can improve the specificity and sensitivity of detection reactive to make the operation process and result analysis much more accurate, simple and time saving.

The invention relates to a reagent and method for preparation of an HBV (hepatitis B virus) persistently infected animal model. According to the invention, a construct able to conditionally induce HBV covalently closed circular DNA (cccDNA) molecules in the liver of mice is constructed, and the HBV persistently infected animal model is successfully established.

The invention provides a nucleic acid detection method based on a DNA (Deoxyribonucleic Acid) hairpin and RCA (Rolling Circle Amplification). The detection method comprises the steps of: modifying the DNA hairpin to a solid carrier, wherein the circular part of the DNA hairpin is complementary with a target DNA and the stem part of the DNA hairpin is complementary with an an annular DNA; adding the annular DNA, an enzyme, an amplification raw material and the like; complementing and pairing a target nucleic acid molecule and the loop of the DNA hairpin when the target nucleic acid molecule is added into a detection system; opening a hairpin-loop structure; hybridizing a base of the hairpin and the circular DNA; performing RCA; opening different hairpin-loop structures when the target nucleic acid molecular weights are different; and quantifying the target nucleic acid molecule by using an optical or electric method. According to the nucleic acid detection method, as the DNA hairpin is used as a detection probe of RCA, the step of detecting the molecule by RCA is simplified, the detection cost is reduced, the advantage of high sensitivity of RCA for nucleic acid detection is simultaneously kept, and the nucleic acid detection method is suitable for popularization and use.

The present invention relates to improved methods of introducing site-directed mutations into circular DNA without the need for subcloning. The invention comprises single-stage, polymerase-based, mutagenesis procedures which eliminate the need for the second-stage linear cyclic amplification reaction of the prior art. In accordance with the invention, production of complimentary mutagenized DNA strands is accomplished in separate, single-primer, linear amplification reactions carried out for each primer of a mutagenic primer pair. The mutagenized DNA strands produced in the separate primer reactions are combined and annealed and the resulting double-stranded mutagenized DNA intermediate may then be used directly to transform a host cell for further production of the desired mutant DNA. Major applications of this method include directed evolution and other areas that benefit from the development of diversity.

The invention relates to a method for preparing an antitumor drug carrier by using a rolling circle amplification technology. An amplification primer DNA is assembled on the surface of a nano-mateiral, a circular DNA amplification template and other amplification mixtures are added and then rolling circle amplification is performed, and the prepared product is a long single-stranded DNA antitumor drug carrier obtained by extension from the surface of nano-gold. The primer DNA is extended to a microsize scale by means of room temperature amplification and can be in comparison with a non-amplified gold-DNA control group by means of agarose electrophoresis; after amplification, the DNA becomes longer, resulting in delay in the electrophoresis experiment. Besides, in an atomic force microscopy image, the amplified long-chain DNA extending from the surface of the nano-material can be intuitively observed. The nano-scale biological composite structure constructed by the rolling circle amplification technology can be used for the researches on novel antitumor drug carriers.

The invention provides application of circular DNA (Deoxyribonucleic Acid) in detecting and imaging sense RNA (Ribonucleic Acid) and gene therapy. The circular DNA at least comprises one or more DNA probe fragments complementary to a detection target sense RNA sequence, and is formed by connecting and cyclizing 3' and 5' terminals of ssDNA (single-stranded Deoxyribonucleic Acid). The circular DNAand the detection target sense RNA form heterozygous double strands, and the heterozygous double strands are separated from a carrier, so that (1) the application in detecting and imaging through a corresponding fluorescence signal change is realized; (2) the sense RNA in the heterozygous double strands is enzymically decomposed, so that the aim of gene therapy is achieved.

The invention provides a method for detecting hepatitis B virus covalently-closed circular DNA (HBV ccc DNA) from the samples of peripheral blood, hepatic tissues and the like by utilizing two-color fluorescence polymerase chain reaction (PCR) technology, namely a PCR-two-color fluorescence probe method, and a kit therefor. The method comprises the following steps of: (1) collecting the samples; (2) extracting the DNA; (3) detecting the samples by a two-color fluorescence quantitative PCR in-vitro amplification method; and (4) analyzing the samples according to the fluorescence intensity of the amplification reaction of the corresponding samples after the amplification reaction is finished, thereby judging the existence of the HBV ccc DNA in the collected samples and accurately quantifying the HBV ccc DNA by using the standard curve of a positive quality control material to realize the real-time, fast and quantitative detection of the HBV ccc DNA.

The invention belongs to the technical field of nucleic acid and particularly relates to a preparation method of high-concentration circular DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). The method comprises the following steps of: mixing assistant template DNA (hereinafter referred to as a splint), ligase and a buffer solution of the ligase required by a cyclic reaction to prepare an initial system, preparing an addition solution from single-stranded DNA or RNA (hereinafter referred to as a cyclic strand) and the buffer solution of the ligase, adding a certain amount of the addition solution containing the cyclic strand into the initial system every other certain time, allowing a concentration of the cyclic strand in the system to increase 0.1-5 micrometers after adding the new cyclic strand each time, repeating the procedure for several times, after adding the addition solution containing the cyclic strand for the last time, allowing for reaction for 2-15h, and allowing a concentration mole ratio of the splint to the cyclic strand in the system to be (1-10):1. The method is simple and easy to operate, does not require extreme conditions, and can achieve high yield preparation of circular DNA or RNA at the ultrahigh concentration of the cyclic strand (greater than or equal to 10 micrometers).

The invention discloses an oligonucleotide composition and a method for detecting hepatitis B virus (HBV) rcDNA (Relaxed Circular deoxyribonucleic acid) and/ or cccDNA (Covalently Closed Circular DNA), a kit and application of the oligonucleotide composition. The oligonucleotide composition comprises first oligonucleotide, second oligonucleotide, or oligonucleotide complementary with the sequenceof the first oligonucleotide and oligonucleotide complementary with the sequence of the second oligonucleotide; the first oligonucleotide is specifically combined with at least one part of continuoussequence of the first sequence, and the second oligonucleotide is specifically combined with at least one part of continuous sequence of the second sequence; in addition, a distance between two continuous sequences is 30-350 nt; the first sequence is a sequence between DR (Direct Repeat) 2-DR1 of the HBV DR sequence of a cross-notch area which contains cccDNA or rcDNA, and the second sequence is asequence after the fifth basic group of the DR1. According to the oligonucleotide composition, the level of HBV rcDNA and/ or cccDNA can be effectively detected without being affected by integrated HBV DNA.

The invention discloses application of pomegranate in preparing a medicament for treating or preventing hepatitis B virus (HBV) infection. The application is implemented through the following steps: detecting the cytotoxicity of each pomegranate sourced medicament to HepG2.117, wherein the CC50 of punicalagin, gallic acid and punicalin is sequentially more than 500mu M, 334mu M and 218.86mu M; and then detecting the effects of the punicalagin, ellagic acid, gallic acid and punicalin to HBeAg and HBsAg, and the effect of punicalagin to DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) of virus. Results indicate that the four compounds can be used for inhibiting HBeAg and HBsAg, wherein the punicalagin can be used for remarkably reducing the cccDNA (covalently closed circular DNA). The punicalagin can be used for remarkably reducing the contents of HBeAg and cccDNA in an HepG2.2.15 cell, and does not have effect to the total HBV DNA in the cell, which indicates that the punicalagin takes the cccDNA as a target to directly clear the cccDNA in a liver cell. Currently, the anti-HBV infection medicament in clinical treatment cannot directly act on the cccDNA, and cannot completely remove the virus. Experiment results disclose that the medicament has the prospect of being developed into anti-HBV infection medicaments.

The invention relates to a precise quantification method of HBV (hepatitis B virus) cccDNA (covalent closed circular DNA). The precise quantification method of the HBV cccDNA comprises steps as follows: the copy number of a sample is quantitatively controlled within 10,000 copes through nucleic acid; primers and probes of cccDNA and rcDNA are designed and detected with a double-gap method; digital PCR (polymerase chain reaction) amplification is performed; analysis parameters are adjusted in digital PCR instrument analysis software, 1/2-1/3 of the average fluorescence intensity of positive particles in positive control holes is set as a positive threshold, the to-be-detected sample is analyzed and the copy number of cccDNA in the sample is calculated using software of the digital PCR instrument, and false positive caused by non-specific amplification is eliminated. The method solves the false positive interference problem of double-gap method and the PCR method for rcDNA; compared with other digital PCR detection methods for cccDNA, the method has the advantages that specific DNA enzyme is not required to treat the sample, closed pipe operation is realized, the cross-pollution risk is reduced, and the operation process is simplified.

The invention discloses a yellow crucian carp high-density SNP (single nucleotide polymorphism) marker screening method, which includes extracting genomic DNA, constructing a RAD library and high-throughput sequencing, filtering original sequence data, and detecting and screening SNP information sites; adding circular DNA and chitin in the process of extracting the genomic DNA to effectively improve the screening accuracy of SNP markers; and adding chitin fluid into a PCR (Polymerase Chain Reaction) amplification system to improve the reaction sensitivity of PCR amplification and help to obtain the complete high-density SNP sites. The high-density SNP sites obtained by the method can be applied to analyze the genetic diversity level, population genetic structure and the like of the yellowcrucian carp population. The method for obtaining the high-density SNP marker sites of the yellow crucian carp has the advantages of simplicity, high efficiency, high SNP marker screening accuracy, good repeatability, low cost and the like.

(PROBLEM) To establish a system enabling modification and proliferation of EB virus circular DNA in Escherichia coli and large-scale production of recombinant EB virus virions in the cells.

(Means for Resolution) The present invention enables modification and proliferation of a circular EB virus genome derived from Akata cells in Escherichia coli by inserting a DNA sequence of a bacterial artificial chromosome (BAC) into a circular EB virus DNA in Akata cells via homologous recombination. The recombinant EB virus can be produced in a large quantity by introducing the resulting genomic DNA into Akata cells.