11 results about "Rolling circle replication" patented technology

The invention provides a method for sorting cell units and application thereof. The method for sorting the cell units comprises the following steps: specifically binding a first probe and a second probe with a target nucleic acid sequence, wherein the interval between binding sites of the first probe and binding sites of the second probe does not exceed a preset length; providing an insertion fragment, a skeleton fragment and a ligase, and setting the nucleic acid sequences of the insertion fragment and the skeleton fragment to be that when the first probe and the second probe are specifically combined with a target nucleic acid sequence, the insertion fragment and the skeleton fragment can be connected into a single-chain loop; performing rolling circle replication on the single-chain loop to obtain a long single-chain molecule, the long single-chain molecule comprising a plurality of repetitive units; combining a third probe with the repeating unit, the third probe carrying a detectable signal; and sorting the cellular units based on the detectable signal. According to the method, the target cell group can be efficiently selected, so that high-quality single cell transcriptome sequencing data can be obtained.

The invention belongs to the technical field of gene engineering, and relates to a plasmid of bacillus, an artificial recombinant vector as well as a construction method and application thereof, the plasmid is called pBso for short, and the nucleotide sequence of the plasmid is shown as SEQ ID NO.1. In a plasmid sequence, a protein coded by an open reading frame is a replication element Rep protein of a gram-positive bacterium rolling circle replication type plasmid, and the nucleotide sequence of the protein is shown as SEQ ID NO.2. The artificial recombinant vector is constructed by carrying out gene recombination on plasmids of the bacillus. The construction method of the artificial recombinant vector comprises the following steps: cloning a replication element Rep protein by using a molecular cloning means to construct the artificial recombinant vector with neomycin resistance. The replication element Rep protein of the plasmid pBso of the bacillus can be used for constructing an artificial recombinant vector, stably exists in the bacillus subtilis and the bacillus amyloliquefaciens and is subjected to replication and passage, and the steps of obtaining the engineering bacillus are simplified.

Provided herein are methods and compositions for the detection of target nucleic acids using target reporter constructs (TRCs) which comprise target sequences complementary to the target nucleic acid. Further provided are methods of replicating the TRCs using rolling circle replication and / or rolling circle amplification to produce replicated TRCs which can be detected using probe sequences within the replicated TRCs.

The invention discloses a method for detecting in-situ release of RNA by exosome based on rolling circle replication amplification, and belongs to the technical field of exosome detection. The invention designs and develops a detection probe taking a sulfydryl hairpin probe and a Padlock probe as core components, the sulfydryl hairpin probe can target an exosome membrane protein CD63, and after substances such as the Padlock probe and Phi 29 DNA polymerase are added, an RCA amplification reaction is initiated. Triton X-100 can punch an exosome membrane so that the exosome membrane can release miRNA located in the exosome membrane, and the miRNA is combined with an amplification chain to release a hidden G4 chain body sequence. The added Hemin is combined with the G4 to form a G4 conjoined compound, and the compound can display peroxidase catalytic activity and can catalyze H2O2 to be decomposed to generate oxygen free radicals, so that a detectable signal is generated. The detection method provided by the invention can be used for carrying out high-sensitivity detection on the low-concentration exosome miRNA.

The invention relates to the technical field of phytopathogen molecule detection, and discloses a rolling circle replication-based high-sensitivity fusarium pseudograminearum detection method which comprises the following steps: extracting a to-be-detected genome of a sample; preparing closed-loop DNA (deoxyribonucleic acid); carrying out mixed reaction on the closed-loop DNA and exonuclease to obtain a purified circular DNA template; mixing the purified annular template, a rolling circle amplification primer, Phi29 DNA polymerase, dNTPs and a buffer solution, and carrying out rolling circle replication to obtain super-long single-stranded DNA; and mixing the rolling circle replication product, Cas12b protein, sgRNA, an ssDNA fluorescence report probe and a buffer solution, and carrying out trans-cleavage to release a fluorescence signal. According to the invention, the necessary in-vitro transcription step in the traditional isothermal amplification-CRISPR coupling technology is eliminated, the detection time is shortened, and meanwhile, the reagent cost and the operation error risk are reduced.

The present invention discloses a method for preparing a hydrogel based on the activity of phi29 DNA polymerase rolling circle replication of long-chain base-modified nucleic acids, belonging to the technical field of biomaterials. The method of the present invention is operated as follows: In a rolling circle amplification reaction system containing phi29 DNA polymerase, a pairable artificial nucleotide with an alkynylamino modification at the 5th position of the base part is incorporated to prepare a nucleic acid with an amino linker on some bases; the obtained nucleic acid is concentrated and annealed, and the annealed product is mixed with a chemical crosslinking agent in a phosphate buffer solution and incubated to obtain the hydrogel. The present invention synthesizes nucleic acids with modified bases by an enzymatic method, provides covalent binding sites, and solves the problem of low stability of DNA hydrogels based on hydrogen bond binding by introducing covalent bonds. A large amount of nucleic acids can be quickly obtained through rolling circle amplification, providing a simple and highly universal production method for the formation of novel nucleic acid hydrogels.

The present invention discloses a DNA hydrogel with cascade reaction function, its preparation method and application. The carrier is a pure DNA system, which is formed by means of rolling circle replication. During the rolling circle replication process, an AS1411 nucleic acid aptamer sequence with targeting function and an antisense DNA sequence of hypoxia-inducible factor HIF-1α are generated. A nucleic acid aptamer capable of targeting tumor cells is designed on the linear template, and cholesterol is designed on the nucleic acid aptamer sequence. After high-speed centrifugation and standing, self-assembly forms a DNA hydrogel. This DNA hydrogel has been successfully applied to the drug sustained-release system, has good biocompatibility, and simultaneously loads chlorin e6 (Ce6), heme and glucose oxidase (GOx), showing broad prospects in the fields of inhibiting the growth of malignant tumor cells and biomedicine, etc.

The invention relates to the technical field of hydrogel preparation, and discloses a preparation method of DNA hydrogel, which comprises the following steps: a, using plasmid as a template, and melting double strands of the plasmid template through alkaline denaturation; b, adding a random primer, enabling the plasmid template to be combined with the random primer in a renaturation process, and producing a large amount of double-stranded DNA with an ultra-long chain length and a high-branch topological structure as a DNA hydrogel precursor through hyper-branched rolling circle replication amplification; c, adding a dehydrating agent into the DNA hydrogel precursor solution, and realizing self-assembly by utilizing dense physical entanglement among double-stranded DNA molecules under the induction of the dehydrating agent to form a DNA aggregate; and d, collecting the DNA aggregate, drying the DNA aggregate to remove the residual dehydrating agent, resuspending the DNA aggregate in a buffer solution to enable the DNA aggregate to absorb water and swell, and finally forming the DNA hydrogel with a stable structure. According to the invention, the design and preparation process of the DNA hydrogel can be greatly simplified, the availability of the DNA hydrogel is improved, and the transformation of the DNA hydrogel from basic research to practical application is promoted.

Provided herein are methods and compositions for the detection of target nucleic acids using target reporter constructs (TRCs) which comprise target sequences complementary to the target nucleic acid. Further provided are methods of replicating the TRCs using rolling circle replication and / or rolling circle amplification to produce replicated TRCs which can be detected using probe sequences within the replicated TRCs.

The invention discloses a high-fidelity single cell whole genome amplified polymerase mutant and a kit and application thereof. The polymerase mutant is a Phi29 DNA polymerase mutant which is any one of M1, M2, M3 and M4, and the amino acid sequences of the polymerase mutant are shown as SEQ ID NO.2-5 respectively. The Phi29 DNA polymerase mutant disclosed by the invention contains four mutation sites and can be stored for a long time under a room temperature condition. Compared with a wild type, the Phi29 DNA polymerase mutant M4 disclosed by the invention has excellent thermal stability. Under the reaction condition of 60 DEG C, 90% or above of enzyme activity can still be kept after 6 hours. The mutant shows higher amplification uniformity, reaction rate and fidelity in DNA amplification reaction, can be used for catalyzing multiple replacement amplification and rolling circle replication reaction, and can be used for preparing a PGT detection kit.

The invention discloses a method for determining the stability of polymerase. The invention provides a method for detecting the stability of DNA (deoxyribonucleic acid) polymerase. The stability of the DNA polymerase is detected according to the principle that the DNA polymerase is reversibly inactivated and then activated. According to the scheme, operation is easy and convenient, the requirement for raw materials needed for amplification is low, radioactive isotopes, fluorescent markers and other fluorescent components do not need to be added in the amplification process, and pollution and interference caused by fluorescence are eliminated. The method is more suitable for reaction conditions of reagent development of high-throughput sequencing, whole genome amplification, isothermal amplification, rolling circle replication and the like, is more suitable for judgment of enzyme stability, provides reference for selection of modified enzyme or judgment of use conditions of specific enzyme, is convenient and time-saving, and reduces expenditure.