10 results about "Nucleic acid cleavage" patented technology

This invention belongs to the field of programmable nuclease technology, and particularly relates to a TcAgo protein mutant and its encoding gene, a nucleic acid cleavage reagent containing the mutant, and its applications. The amino acid sequence of the TcAgo protein mutant is selected from one of SEQ ID NO. 2-4. The TcAgo protein mutant provided by this invention has advantages such as improved low-temperature and medium-temperature performance, high room-temperature catalytic activity, wide temperature adaptability, broadened substrate spectrum, and high stability. It is beneficial for overcoming high-temperature dependence, adapting to in vivo and room-temperature physiological environments, and promoting the development of gene editing, molecular diagnostic technology, biosensing, and other fields towards in vivo applications and mild reactions. It solves the problem of insufficient activity and limited application of thermophilic pAgo proteins, including TcAgo, under mild conditions in existing technologies.

The application discloses a nucleic acid cleavage amplification device. The nucleic acid cleavage amplification device comprises a pushing device, a liquid storage device and a chip. The liquid storage device is internally provided with multiple cavities, the pushing device extends into the interior of the liquid storage device, and the pushing device is slidably connected with the liquid storage device below. One end of the liquid storage device is snap-connected with the upper portion of the pushing device, and the other end of the liquid storage device is snap-connected with the chip. The liquid storage device is connected with the chip through a liquid inlet channel. The chip comprises a chip body, a quantitative groove, a waste liquid groove and a positioning support column. Multiple quantitative grooves and waste liquid grooves are arranged, the quantitative grooves and the waste liquid grooves are arranged on the chip body, the quantitative grooves and the waste liquid grooves are connected with the liquid storage device through quantitative channels and waste liquid channels respectively, and the positioning support column is arranged on one side of the bottom of the chip body. The chip further comprises a buffer channel, one end of the buffer channel is connected with the quantitative channel, the other end of the buffer channel is connected with the quantitative groove, and the cross-sectional area of the buffer channel is smaller than that of the quantitative channel, so that the accuracy of nucleic acid detection is improved.

The invention provides a PfAgo detection system for synchronously detecting staphylococcus aureus and escherichia coli, an RPA primer group, a kit and a method, and belongs to the technical field of pathogenic bacterium detection. The system comprises PfAgo protein, at least two fluorescent report probes respectively corresponding to staphylococcus aureus and escherichia coli, and at least two groups of specific guide DNA (deoxyribonucleic acid), namely guide gDNA. According to the invention, RPA isothermal amplification and PfAgo protein-mediated nucleic acid cleavage reaction are combined, so that high-specificity and high-sensitivity rapid detection of two pathogenic bacteria is realized. The method is short in detection time, easy and convenient to operate, free of complex instruments and suitable for on-site rapid screening and multiple detection of pathogenic bacteria in complex matrixes such as food and cosmetics.

The invention relates to a nested PCR (Polymerase Chain Reaction) method and application, and a targeted detection library building method improved based on the nested PCR library building method. According to the method, the nucleic acid cleavage activity of USER enzyme is utilized, and Taq DNA polymerase or an enzyme with both DNA polymerization activity and 5 '-3' excision enzyme activity is combined, so that a primer removal method is established and is used for replacing a purification step between a first round of PCR and a second round of PCR, and the problem of cross contamination between samples caused by purification operation of nested PCR is successfully solved. Meanwhile, the method can effectively remove non-specific amplification products and improve the specificity of targeted amplification, the experimental process is simpler and more convenient, and the nested PCR method can be applied to a targeted library building platform and a sequencing platform.

The present invention provides a genome modification method and a genome modification kit that can efficiently modify two or more alleles and modify relatively large regions. [Solution] A genome modification method for modifying two or more alleles of a chromosome genome, comprising: (a) introducing the following (i) and (ii) into a cell containing the chromosome; (i) a genome modification system comprising a sequence-specific nucleic acid cleavage molecule that targets a target region of the chromosome genome, or a polynucleotide encoding the sequence-specific nucleic acid cleavage molecule; (ii) two or more selection marker donor DNAs having mutually different selection marker genes (the number of types of selection marker donor DNAs is equal to or greater than the number of alleles to be modified); and (b) selecting the cell based on all the selection marker genes possessed by the two or more selection marker donor DNAs.

The invention discloses TtdAgo mutants with target nucleic acid cleavage activity at low temperatures and use thereof. Compared to wild-type TtdAgo, the TtdAgo mutant comprises mutations at amino acid position 527 and / or position 561 and / or position 593 and / or position 599. Relative to wild-type TtdAgo, these TtdAgo mutants not only exhibit significantly enhanced activity at moderate to low temperatures but also enable cleavage of target RNA, thereby effectively expanding the applicability range of pAgo.

The invention relates to the technical field of molecular biology and biological detection, in particular to a concentration dependent type dual-mode nucleic acid cleavage system and a concentration dependent type dual-mode nucleic acid cleavage method for mesophilic Clostridium perfringens Argonaute protein. The system is based on the cleavage behavior difference of CpAgo protein under different target single-stranded DNA (ssDNA) concentrations. When the concentration of the target ssDNA is micromole or above, the CpAgo shows non-specific ssDNA cleavage activity independent of a guide chain, and has wide compatibility to the length, sequence and various chemical modifications of a substrate; when the concentration of the target ssDNA is nanomole or below, the cleavage behavior of the target ssDNA is converted into a specific mode which strictly depends on a complementary guide chain. The dual-mode mechanism of the CpAgo and the efficient non-specific cutting capacity of the CpAgo at normal temperature are verified through electrophoresis, fluorescence kinetic analysis and comparison experiments with other prokaryotic Ago proteins. The system can be used for rapidly degrading exogenous nucleic acid or realizing high-specificity nucleic acid detection, and has the advantages of single-enzyme dual-mode, concentration gating, strong substrate compatibility and the like.

The application discloses a nucleic acid lysis buffer for RPA-CRISPR / RspCas13d detection and application thereof, and belongs to the technical field of biology. The nucleic acid lysis buffer is composed of 8-12 mM pH8 Tris-HCl, 0.8-1.2 mM EDTA, 0.05-0.15% Triton X-100, 0.8-1.2 mM DTT, 0.4-0.6% betaine, 0.8-1.2 U / μL mouse ribonuclease inhibitor and 0.08-0.12 mg / mL BSA, can quickly lyse and release nucleic acid in a sample to be detected under room temperature conditions, and does not need to be heated or a commercial nucleic acid extraction kit. The application further provides a buffer composition for RPA-CRISPR / RspCas13d detection. When the nucleic acid lysis buffer is combined with the buffer composition, RPA amplification, T7 transcription and RspCas13d detection can be simultaneously carried out in a single reaction tube at 37 DEG C, and one-step detection is realized. The application provides a fast and device-independent diagnosis method for pathogens such as CDV, and is especially suitable for on-site monitoring of endangered animals such as giant pandas and instant detection in an environment with limited resources.

The present invention relates to a method for generating ssDNA guides from dsDNA using an Argonaute protein. Said guides may be useful in applications, like selectively reducing repetitive sequences in sequencing libraries using Argonaute proteins. The invention further relates to a method for cleaving a nucleic acid molecule, a method for reducing the number of nucleic acid molecules comprising a target sequence and a method for selectively enriching and / or sequencing nucleic acid molecules in a sample, like a DNA sequencing library.

The invention provides a magnetic control ratio fluorescent biosensor based on RPA-CRISPR / Cas12a as well as a preparation method and application of the magnetic control ratio fluorescent biosensor, and belongs to the technical field of fluorescent biosensors and pathogenic bacterium detection. The magnetic control ratio fluorescence biosensor comprises a fluorescence reference signal element, a fluorescence response signal element and a specific recognition element, according to the magnetic control ratio fluorescent biosensor, high-sensitivity and specific detection of staphylococcus aureus and salmonella is realized through the magnetic and fluorescent characteristics of the fluorescent reference signal element, the fluorescent characteristic of the fluorescent response signal element and the nucleic acid cleavage activity of Cas12a; the magnetic control ratio fluorescent biosensor has a built-in calibration function, can reduce interference brought by the environment and instruments, has good selectivity to staphylococcus aureus and salmonella, has good anti-interference performance to other bacteria, and can be used for rapid detection in the field of food safety.