1872 results about "Single strand" patented technology

The present disclosure provides new prime editor guide RNAs for prime editing, constructs for prime editing, and methods for using same. In addition, the present disclosure provides compositions and methods for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and / or targeted mutagenesis (e.g., insertion or deletion). The nucleotide change can include a single-nucleotide change (e.g., any transition or any transversion), an insertion of one or more nucleotides, or a deletion of one or more nucleotides. More in particular, the disclosure provides fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a prime editor RNA (PEgRNA). The prime editor guide RNA comprises an extension arm that provides a DNA synthesis template sequence which encodes a single strand DNA flap, which is homologous to an endogenous DNA sequence, but which contains the desired one or more nucleotide changes and which, following synthesis by the polymerase (e.g., reverse transcriptase), becomes incorporated into the target DNA molecule.

The invention discloses a kit for detecting tomato brown crinkled fruit virus based on enzyme-mediated dual-amplification nucleic acid amplification and a detection method. The detection system comprises a primer pair DNA (deoxyribonucleic acid) upstream primer (F5) and a DNA downstream primer (R6) for amplifying the tomato brown crinkled fruit virus movement protein gene, a probe (RNA primer (RNA1)) for detecting the MP gene, a nucleic acid amplification group enzyme system (recombinase, single-chain binding protein, polymerase and ATP (adenosine triphosphate) energy regeneration enzyme), a signal amplification group enzyme system (positioning enzyme and cutting enzyme), activating enzyme NTP, a buffer solution and RNase-free Water. The system composed of the freeze-drying protective agent is prepared into the pre-subpackaged freeze-dried balls. The kit disclosed by the invention can realize DNA detection of aM concentration, and besides rapid qualitative detection of the tomato brown crinkled fruit virus, early screening of the tomato brown crinkled fruit virus can also be realized due to high detection sensitivity.

Systems and methods for sequencing polynucleotides using nanopores are disclosed. In some embodiments, a polynucleotide including a single-stranded region and a double-stranded region, in which the single-stranded region is disposed through a nanopore. The polynucleotide can be moved relative to the nanopore by electric forces while one or more structural locks keep the polynucleotide close to the nanopore. A characteristic signal based on nanopore ionic current blockade and associated with the regions of the polynucleotide at or near the nanopore recognition zone is measured and used to infer the nucleobase sequence of the polynucleotide. In some examples, the double-stranded region is extended by a polymerase, and the polymerase is removed from the polynucleotide. In some examples, signals measured under different applied voltages provide nonredundant information regarding the polynucleotide sequence.

The invention discloses a preparation method of polydeoxyribonucleotide, and relates to the technical field of synthetic biology, and the preparation method comprises the following steps: S1, randomly selecting n ssDNAs from a salmon genome sequence; s2, carrying out cyclization on the ssDNA, so as to obtain cssDNA; s3, carrying out amplification on the cssDNA to obtain a repeated long single-stranded DNA (Deoxyribose Nucleic Acid); and S4, breaking the repeated long single-stranded DNA to obtain the PDRN. According to the method, a large amount of repeated long single-stranded DNA can be synthesized only by introducing a small amount of foreign proteins (T4DNA ligase and Phi29 DNA polymerase), the repeated long single-stranded DNA is broken by a physical breaking method, and a large amount of PDRN products with ideal molecular weight can be obtained in a short time.

The invention provides an intracellular mycobacterium tuberculosis in-situ nucleic acid detection method based on CRISPR / Cas and application, and the method comprises the following steps: S1, designing crRNA and a primer pair of a mycobacterium tuberculosis target gene sequence, and synthesizing a single-stranded DNA fluorescent probe; s2, preparing a standard solution containing a target gene sequence, and incubating with a CRISPR / Cas12a system; s3, extracting nucleic acids of different types of bacteria, uniformly mixing the nucleic acids of different types of bacteria with the CRISPR / Cas12a system, and incubating; s4, incubating an attenuated strain of mycobacterium tuberculosis and the DNA fluorescent probe, infecting macrophages, and incubating to obtain infected cells; s5, electrically transferring a CRISPR / Cas12a system into the infected cells in S4 by utilizing an electroporation technology, incubating, and detecting the intensity of a fluorescence signal; and S6, performing quantitative and positioning analysis on the detected fluorescence signal to complete detection, and directly introducing a CRISPR / Cas12a system into the macrophage by adopting an electroporation technology to realize intracellular in-situ detection, thereby avoiding the operation complexity of a nucleic acid extraction link and the loss of detection sensitivity in the prior art.

A nucleic acid molecule comprising: i) a first nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a nucleotide sequence comprising a cardiovascular disease associated gene to be silenced and wherein said cardiovascular gene is proprotein convertase subtilisin kexin 9 (PCSK9) there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand; and ii) a second nucleic acid comprising a double stranded inhibitory ribonucleic acid (RNA) molecule comprising a sense and an antisense strand designed with reference to a different cardiovascular disease associated gene to be silenced wherein said cardiovascular gene is lipoprotein (a) (Lp(a)) and wherein there is provided a single stranded deoxyribonucleic acid (DNA) molecule conjugated to the 5’ end of said sense strand wherein the single stranded deoxyribonucleic acid (DNA) molecule is substantially complementary to the single stranded deoxyribonucleic acid (DNA) molecule set forth in i) above and anneals by complementary base pairing to form a double stranded DNA linker that links the first and second double stranded inhibitory ribonucleic acid (RNA) molecules.

The invention discloses an SNP (Single Nucleotide Polymorphism) site and a KASP primer group for identifying or assisting in identifying clematis maple and application of the SNP site and the KASP primer group. The SNP site is located at the 2688th site of SEQ ID No.1 (sequence identifier number 1). The KASP primer group is composed of single-stranded DNA (deoxyribonucleic acid) as shown in SEQ ID No. 2, SEQ ID No. 3 and SEQ ID No. 4. The method comprises the following steps: carrying out next-generation sequencing and assembling on clematis maple for the first time, taking formed ultra-long scaffold as a reference genome ultra-long fragment, carrying out simplified genome sequencing on 15 clematis plants, and comparing the simplified genome sequencing with the reference genome ultra-long fragment to obtain difference sites, finally, the KASP molecular marker CA24547 is developed on the basis of the difference sites, and the KASP molecular marker CA24547 is verified. The KASP molecular marker developed by the invention can realize accurate identification of the clematis nepalensis in the seedling stage, and has important significance for protecting the clematis nepalensis.

The present invention relates to CRISPR / Cas based biosensing materials, assays, and methods. Specifically, the present technology relates to CRISPR / Cas-based ultra-sensitive detection methods for nucleic acid assays using specific molecular constructs, including constructs referred to as nucleic acid mediators, comprising single-stranded and double-stranded nucleic acid sequences of a cyclic conformation, and palindromic oligonucleotides. The materials and methods according to the invention can also be used to enhance the sensitivity of existing bioassays.

The invention relates to the technical field of micro-fluidic chips and biosensors, in particular to a paper folding type micro-fluidic paper chip analysis device for detecting ATP (adenosine triphosphate) based on a difunctional DNA tetrahedron and application of the paper folding type micro-fluidic paper chip analysis device. The analysis device comprises a detection layer and a washing layer, and is characterized in that a difunctional DNA tetrahedron system containing detection ATP is added into a reaction zone; wherein the bifunctional DNA tetrahedron comprises a capture DNA tetrahedron (B-TDN) and a detection DNA tetrahedron (D-TDN), and a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the B-TDN and a single-stranded DNA sequence extending from any vertex of the tetrahedron formed by the D-TDN can be complementary. The paper folding type micro-fluidic paper chip analysis device based on the difunctional DNA tetrahedron has the advantages of being small in size, low in scientific research cost, biodegradable, high in sensitivity, good in selectivity and the like, a new method is provided for rapid ATP detection in the medical field, and the paper folding type micro-fluidic paper chip analysis device is suitable for remote areas and economic outdated areas.

The invention discloses a colorimetric-fluorescent dual-signal salmonella detection method based on Ag-MoS2 nano-enzyme. The method is characterized by comprising the following steps: mixing a single strand S1, a single strand S2, a single strand S3, a single strand S4, a salmonella aptamer and cDNA (complementary deoxyribonucleic acid) to prepare a multivalent aptamer competitive probe solution; respectively dissolving a hairpin probe H1 and a hairpin probe H2 in PBS (Phosphate Buffer Solution); a step of preparing an Ag-MoS2 nano enzyme composite material; the method comprises the following steps: mixing and incubating a to-be-detected salmonella solution and a multivalent aptamer competitive probe, adding the Ag-MoS2 nano-enzyme composite material for performing CHA amplification reaction, then adding an H2O2 solution and a TMB solution for performing peroxidase catalytic reaction, respectively performing colorimetric method and fluorescence method detection, and calculating to obtain the salmonella concentration in the to-be-detected solution. The kit has the advantages of simplicity, rapidness, high sensitivity, strong specificity, high accuracy and real-time sterilization after detection.

The invention discloses a method and device for photo-thermal amplification and rapid detection of multiple genes and application. An amplification system containing a photo-thermal amplification material is irradiated by a coaxial reflection LED light source outside 405-500nm, and isothermal amplification of a target gene is realized. According to the invention, a large number of single-stranded DNA amplification products are obtained by using asymmetric RPA, and then qualitative and quantitative detection is carried out through photo-thermal test paper. A detection area adopts a sandwich capture amplification product and a photo-thermal signal probe to generate a temperature change signal related to the content of the amplification product, and the gene is quantified according to the temperature change signal. The device comprises an LED light source, a spectroscope, a plano-convex cylindrical lens, a photo-thermal amplification system bracket, a test paper detection bracket and a light-shielding heat-insulating shell. According to the method, the photo-thermal conversion effect of a nano material is utilized, so that portable, rapid and low-cost multi-sample amplification is realized, and the portability limitation of traditional equipment is overcome.

The invention relates to antisense oligonucleotides that are capable of bringing about specific editing of a target nucleotide (adenosine) in a target RNA sequence in a eukaryotic cell, wherein said oligonucleotide does not, in itself, form an intramolecular hairpin or stem-loop structure, and wherein said oligonucleotide comprises a non-complementary nucleotide in a position opposite to the nucleotide to be edited in the target RNA sequence.

The invention discloses a method for detecting a diallele editing cell based on a CRISPR / Cas12a technology. The invention provides crRNA, and the nucleotide sequence of a binding target of the crRNA is SEQ ID NO.6. The crRNA is obtained by in-vitro transcription of a transcription template; the transcription template is a product obtained by annealing a single-stranded DNA (Deoxyribose Nucleic Acid) molecule as shown in SEQ ID NO.10 and a single-stranded DNA molecule as shown in SEQ ID NO.13. The invention provides crRNA of the CD71 gene, a CRISPR / Cas12a system is constructed by using the crRNA, CRISPR / Cas12a detection is performed, screening of CRISPR / Cas9 induced CD71 gene diallele editing cells is realized, and the method is simple, convenient, quick and convenient for economically and effectively screening a large number of mutant cells.

A nanostructure comprising: a) a base plate sheet comprising a single-duplex layer comprised of DNA origami configured in a planar arrangement, b) a pore configured within the center of the base plate sheet, and c) a multi-duplex layer-thick wall comprising a single-duplex layer comprised of DNA origami that extends downward from the base plate sheet on the periphery of the pore, wherein the base plate sheet comprises a single-stranded DNA region on the bottom surface for lipid membrane anchor modification.

The invention relates to a metallized microbubble and a preparation method thereof, a construction method of a multifunctional composite probe, and a rapid bacterium capturing and sterilizing method, and belongs to the technical field of functional material preparation. The method aims at solving the technical problems that in the prior art, a metal coating on the surface of the hollow glass microbubble is not uniform, combination is not firm, and stability is poor. According to the technical scheme, the method is characterized by comprising the following steps: mixing surface-activated hollow glass microbubbles with a polyethyleneimine solution for reaction, and washing and drying to obtain polyethyleneimine-coated hollow glass microbubbles; then reacting with a single-stranded DNA buffer solution to obtain an intermediate product loaded with DNA; then introducing a silver nitrate solution to load silver ions on the surface of the compound; and finally, reducing through a sodium borohydride solution to prepare the metallized microbubbles. The metalized microbubbles prepared by the method can be used for constructing a multifunctional composite probe to realize efficient capture and synergistic antibiosis of targets such as bacteria.

The invention discloses a nucleic acid tetrahedron DNA-quercetin compound and application of the nucleic acid tetrahedron DNA-quercetin compound in treatment of retinal neovascular diseases. In the nucleic acid tetrahedron DNA-quercetin compound disclosed by the invention, a quercetin molecule is connected to one single-stranded DNA of four single-stranded DNAs of a nucleic acid tetrahedron through an azide coupling reaction. The compound is stable in physical property, quantitatively carried quercetin molecules guarantee the stability of the treatment effect of the compound, generation of new vessels can be effectively prevented, remodeling of retina non-perfusion area vessels can be promoted, retina vascular injury can be recovered, a new strategy is provided for treatment and prevention of retina neovascular diseases, and the compound is worthy of popularization and application. Meanwhile, compared with tFNAs-QUE obtained through an existing electrostatic adsorption method, the tFNAs-QUE synthesized through the azide coupling method has a better treatment effect, pathological neovascularization is greatly reduced, and meanwhile generation of physiological blood vessels in a non-vascular region is obviously promoted.

The present invention discloses a nucleic acid aptamer that specifically recognizes DEHP and its application, and belongs to the field of environmental safety biotechnology. The nucleic acid aptamer is any one of the following: (1) a single-stranded DNA sequence such as a nucleotide sequence shown in SEQ ID No.10; (2) a sequence that uses the nucleotide sequence shown in SEQ ID No.10 as a core sequence, is extended or shortened on both sides or on one side, and has the same function as the nucleotide sequence described in (1). The present invention is based on Capture‑SELEX technology, and after 10 rounds of repeated screening, a nucleic acid aptamer SEQ ID No.10 that can bind to DEHP with high affinity and high specificity is finally obtained. The nucleic acid aptamer is applied to the DEHP detection of actual samples, which has important application prospects for the analysis and detection of DEHP in blood samples, cell samples, food and drinking water.

The invention belongs to the technical field of biological medicines, and particularly relates to a DNA tetrahedron capable of carrying oligonucleotides and nucleic acid analogues as well as a preparation method and application of the DNA tetrahedron. The method comprises the following steps: cutting off one single-chain DNA in classical tetrahedral framework nucleic acid to form three short-chain DNAs, and self-assembling six single-chain DNAs again to form a DNA tetrahedron to obtain ORT; according to the invention, the mismatching and self-matching phenomena of tFNA are improved, the yield of tFNA is improved, and the tFNA has good structural stability, high bioavailability and excellent biocompatibility; the ORT prepared by the invention can be used as a drug carrier for loading nucleic acid and nucleic acid analogues, protein drugs or small molecule drugs, and the application of the ORT in the aspects of drug delivery, gene therapy, biosensing and the like is promoted.

Provided in the present invention are a method for preparing a strand-specific library for the rapid detection of various types of RNAs, and a high-throughput sequencing method. A polyadenylic acid (poly A) tail is artificially added at the 3' terminal of various RNAs by using a poly A polymerase, and a single-stranded cDNA is synthesized using a poly-deoxythymidine ribonucleotide primer with deoxyuridine under the action of a reverse transcriptase. The obtained single-stranded cDNA molecule is subjected to a series of reactions, and the reaction product is finally amplified by means of PCR to obtain a strand-specific library of various types of RNAs. The library sample can be subjected to on-machine sequencing.

The invention relates to the technical field of biomedical engineering, and discloses a preparation method of a nano-enzyme SERS (Surface Enhanced Raman Scattering) sensor for detecting diabetic retinopathy, which comprises the following steps: (1) synthesizing a bimetallic gold-palladium nanorod; (2) assembling a gold trioctahedron array; and (3) modifying the surface of the gold-palladium nanorod synthesized in the step (1) with single-stranded DNA1 to prepare a nano-enzyme probe, hybridizing an aptamer of a vascular endothelial growth factor (VEGF) with single-stranded DNA2, modifying the surface of the gold trioctahedron array assembled in the step (2) with the hybridized aptamer, preparing a capture substrate, and constructing the nano-enzyme SERS sensor by the nano-enzyme probe and the capture substrate. In the detection process, the VEGF triggers a competitive recognition mechanism to promote the nano-enzyme probe to be captured by the capture substrate and catalyze tetramethyl benzidine (TMB) to generate oxTMB with a strong SERS signal. The sensor has the advantages of high sensitivity, strong specificity and good accuracy, and can realize rapid quantitative detection of DR biomarkers in serum.

The present disclosure provides systems and methods for separating and characterizing oligonucleotides. Fixed dose combinations of small interfering RNAs can be subjected to denaturing ion pairing reverse phase liquid chromatography using optimal conditions to separate single RNA strands for purity analysis.

The invention relates to a T7RNA polymerase mutant for synthesizing single-stranded DNA as well as a screening method and application of the T7RNA polymerase mutant, and belongs to the technical field of enzyme engineering. The invention provides a method for directed evolution and screening of a T7RNA polymerase mutant, which comprises the following steps: knocking out exonuclease and mismatch repair protein from a chassis strain genome, knocking in single-stranded annealing protein, resistance gene containing deletion mutation and a single-stranded DNA synthesis template, and constructing to obtain a genetically engineered bacterium for screening; a T7RNA polymerase mutant library is constructed, recombinant plasmids with the mutant library are transferred into engineering bacteria, T7RNA polymerase mutants synthesize single-stranded DNA to repair deletion mutation of resistance genes, the transformed engineering bacteria can grow on a resistance plate, the T7RNA polymerase mutants obtained through multiple rounds of screening can efficiently synthesize the single-stranded DNA through in-vitro verification, and the T7RNA polymerase mutant can be used for preparing the single-stranded DNA. A brand new tool is provided for synthesis of single-stranded DNA, and the method has the potential of being applied to the fields of gene editing, evolutionary engineering and the like.

The invention discloses a preparation method of light-regulated single-stranded DNA and application of the light-regulated single-stranded DNA in CRISPR nucleic acid detection. According to the invention, Lambda excision enzyme and a primer modified with PC-Linker are introduced into a detection system based on RPA-CRISPR / Cas12a, and a one-tube nucleic acid detection system without PAM limitation is constructed. By introducing Lambda excision enzyme, the strict dependence of the traditional CRISPR / Cas12a detection technology on PAM sites is broken through, and the application range of the CRISPR / Cas12a detection technology is remarkably widened. And the system is integrated into one-tube detection, so that the risk of aerosol pollution caused by opening a cover for multiple times and transferring a sample is avoided while the experimental operation process is simplified. According to the research, CRISPR / Cas12a and CRISPR / Cas13a are further integrated into a single-tube reaction system, so that the probability of false negative results is reduced in a double-gene detection manner, and the detection accuracy is improved.

The application discloses a kind of carrier, kit and application for long-acting gene knockdown of animal parasitic nematode, belong to the field of animal parasitic disease prevention and control.The lentivirus vector in the application integrates specific coding sequence to the genome of the infected nematode, stably and continuously expresses short hairpin RNA (shRNA) in the nematode, efficiently produces primary single-stranded small RNA (siRNA), these siRNAs target homologous target gene mRNA to cause its degradation, realize stable and continuous gene silencing effect.The application breaks through the bottleneck of traditional animal parasitic nematode RNA interference (RNAi) technology with low efficiency and instability, significantly improves the timeliness of conventional RNAi, can be used to establish long-acting RNAi technology system of animal parasitic nematode, has application value in the research of host in vivo process such as nematode infection, parasitism and pathogenicity and animal parasitic nematode disease prevention and control.

The invention discloses a one-tube method visual nucleic acid detection kit based on a split G-quadruplex and a detection method. Common gene segments of RSV-A and RSV-B are amplified and transcribed to obtain a single-stranded RNA product, the single-stranded RNA product is specifically recognized by sgRNA *, the trans-cleavage activity of Cas13 is activated, and a Link-C * chain is cleaved; according to the method, Link-C * serves as a substrate, a split G-quadruplex serves as a visual signal output probe, degradation of Link-C * promotes formation of G4 / hemin DNAzyme, colorless ABTS is catalyzed to be oxidized into a green product, and visual detection of the respiratory syncytial virus is achieved; the detection kit comprises a split type G-quadruplex, an RPA (recombinase polymerase amplification) primer, Link-C *, sgRNA *, Cas13, hemin, ABTS and H2O2. The detection sensitivity is high, the specificity is strong, the whole detection process is carried out in one centrifugal tube, and the aerosol pollution is reduced.

The present invention discloses a recombinant glycosylase base editing system and its application. The recombinant glycosylase base editing system of the present invention is an APOBEC-nCas9-Ung1 or APOBEC(R33A)-nCas9-Rad51-Ung1 base editing system; the base editing system APOBEC-nCas9-Ung1 includes cytosine deaminase APOBEC, nuclease nCas9 and uracil DNA glycosylase Ung1; the base editing system APOBEC(R33A)-nCas9-Rad51-Ung1 includes cytosine deaminase APOBEC(R33A), nuclease nCas9, single-stranded DNA binding protein Rad51 and uracil DNA glycosylase Ung1. Experiments have shown that the recombinant glycosylase base editing system of the present invention can improve the editing efficiency of base C to base G in the genome sequence of an organism or a biological cell and the purity of the editing product.

The multi-channel electrochemical micro-fluidic chip detection platform provided by the invention realizes rapid, high-throughput, low-cost, high-sensitivity and specific detection on genital tract pathogens. Specifically, when a target exists, a target recognition probe Cas12a-crRNA compound specifically recognizes a target and forms a Cas12a-crRNA-Target triad, the trans-cleavage activity is activated, an activated Cas12a system cuts a G1 / G2 single-chain region in the signal amplification probe, then encapsulated methylene blue is released, the methylene blue further promotes a methyl bromide redox reaction, signal transduction is promoted, and the target recognition probe Cas12a-crRNA compound can be used for recognizing the target. Therefore, high-sensitivity and high-specificity quantitative detection of pathogens is realized. On the basis, through a multi-channel micro-fluidic chip, high-sensitivity detection of multiple targets can be realized at the same time by placing different crRNA sequences in reaction tanks respectively. In the embodiment of the invention, the multi-target ultra-sensitive detection of CT, CA, TV and NG is successfully realized, and the detection limit is as low as 0.8 fM.

This invention provides a process for sequencing single-stranded DNA by employing a nanopore and modified nucleotides.

The invention relates to adaptors for sequencing nucleic acids. The adaptors may be used to generate single stranded constructs of nucleic acid for sequencing purposes. Such constructs may contain both strands from a double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) template. The invention also relates to the constructs generated using the adaptors, methods of making the adaptors and constructs, as well as methods of sequencing double stranded nucleic acids.

The invention discloses a pyramid type thermal assembly efficacy skin care nanoparticle based on DNA nucleic acid and application thereof. According to the invention, skin-care active components are loaded into a nucleic acid framework in a thermal assembly innovation mode to obtain a'pyramid-shaped 'functional skin-care nano system, and the embedding rate, stability, infiltration capacity and bioavailability of the system are improved. According to the invention, four sections of nucleotide single chains with the length of 59 basic groups are designed as a tetrahedral framework sequence, so that the synthesis cost is reduced. According to the invention, a blending autonomous thermal assembly mode is developed to load skin-care active substances into a tetrahedral frame to prepare a'pyramid-shaped 'functional skin-care nano system, so that the embedding efficiency of the skin-care active substances is improved. Whitening active substances are wrapped, zebra fish experiments prove that the pyramid-shaped functional skin care (whitening) nanoparticles can more remarkably inhibit melanin synthesis and relieve and improve skin melanin pigmentation, and meanwhile, the system is proved to have a functional skin care effect.