23 results about "Base-Base Mismatch" patented technology

The invention discloses a reagent and a method for detecting low-frequency pancreatic cancer KRAS G12D gene mutation based on a Cas12a-Cas13a combined reaction system. The reagent comprises a single-stranded substrate DNA (Deoxyribonucleic Acid), a TRNA (Transcription Ribonucleic Acid), a 12CrRNA, a 13CrRNA, a 12Report and a 13Report; the single-stranded substrate DNA is derived from genome DNA of a to-be-detected sample, after the genome DNA of the to-be-detected sample is extracted, asymmetric PCR is carried out by utilizing a PCR primer pair, namely, the single-stranded substrate DNA is amplified and comprises wild type WT and mutant type MT; the mutant MT is completely matched with a recognition region of 12CrRNA, and a base mismatch exists between the mutant MT and a recognition region of 13CrRNA; a base mismatch exists between the wild type WT and a recognition region of 12CrRNA, and the wild type WT is completely matched with a recognition region of 13CrRNA; the TRNA is a pre-synthesized single-stranded RNA, has a base mismatch with a recognition region of the 12CrRNA, and is completely matched with a recognition region of the 13CrRNA; the 12Reporter is a DNA (Deoxyribose Nucleic Acid) probe of the FAM and BHQ double-standard genes, and the 13Reporter is an RNA (Ribonucleic Acid) probe of the FAM and BHQ double-standard genes. The nucleic acid cleavage activities of Cas12a and Cas13a are jointly utilized to realize multiple signal amplification, so that low-frequency detection of KRAS G12D mutation, which is simple to operate and low in cost, is realized.

The invention discloses a method and application for single nucleotide polymorphism identification based on crRNA spacer splitting, by splitting the crRNA spacer complementary to the target base to obtain a recognition probe, when the recognition probe is fully complementary to the target sequence, Cas12a protein is activated to produce a significant fluorescent signal; when there is a base mismatched with the split spacer in the target sequence, the crRNA spacer of the split is difficult to assemble with the incomplete matching sequence, and the Cas12 system is not activated and does not produce a fluorescent signal. The method of the present invention enhances the high specificity recognition ability of CRISPR / Cas12a to single base mismatch, so that the detection of SNP is not limited by the position of the mismatched base, and still shows high resolution in low mutation abundance, for preparing a detection kit for single nucleotide mismatch, to meet the actual detection requirements for clinical samples.

The invention relates to an intramolecular strand displacement reaction-based microRNA detection method and kit, and the kit is characterized in that the kit comprises four single-stranded sequences forming a DNA tetrahedron, a probe S and DNA polymerase; a probe T structure is designed at the top end of the DNA tetrahedron, and the probe T is used for capture of the probe S and intramolecular strand displacement reaction triggered by microRNA to be detected. The DNA tetrahedral structure is utilized to enhance the probe fixing efficiency and stability, and the detection sensitivity is improved; signal amplification is realized through intramolecular strand displacement reaction, and the detection limit is as low as 5 * 10 <-17 > mol / L; good selectivity is achieved, and single-base mismatch sequences can be distinguished; the kit has good performance in serum samples and has practical application potential.

The invention discloses a mutant gene enrichment method, a mutant gene detection method and a mutant gene detection kit based on Cas9 specific cleavage and isonucleotide magnetic beads. The method comprises the following steps: firstly, extracting free DNA; then identifying and cutting a completely matched wild type sequence by using Cas9 protein and specific sgRNA, and reserving the mutant DNA due to base mismatch; cas9 treated DNA and streptavidin magnetic beads containing a biotinylated oligonucleotide probe are incubated, guanine and cytosine in the probe are substituted by isoguanine and isocytosine respectively, and adenine and thymine are modified by locked nucleic acid, so that high-stability homodromous pairing is realized, and mutation DNA is selectively enriched; and finally, carrying out qPCR or sequencing detection on the enriched DNA. The kit has the characteristics of simplicity and convenience in operation, high specificity and high sensitivity, can be used for detecting single-base-level mutation, and is suitable for liquid biopsy, tumor early screening and genetic disease mutation detection.

The invention relates to a detection strategy of a programmable TMSD-CRISPR system for DNA / RNA (deoxyribonucleic acid / ribonucleic acid) single base mutation. Based on split crRNA and dynamic and thermodynamic regulation and control mechanisms of a dynamic nanotechnology, the purpose of identifying DNA / RNA single-base mutation at different sites is achieved by utilizing different kinetic rates of toehold-mediated strand displacement on strand displacement with a single-base mismatched target strand. According to the present invention, the identification factors for the ssDNA / dsDNA / RNA mutation are significantly enhanced (the ssDNA mutation is 169.4, the dsDNA mutation is 243.8, and the RNA mutation is 156.2), such that the DNA mutation detection with the abundance as low as 0.025% is achieved, and the single base mutation detection is allowed to be performed in the miR-21; in the research of a queue containing 75 clinical samples, the method successfully detects and accurately identifies all KRAS G12V variants at the accuracy rate of 100%, and shows the huge potential of the KRAS G12V variants in clinical molecular diagnosis. The programmable split TMSD-CRISPR has excellent specificity and adjustability, and can be used as a multifunctional platform for biomedical research, molecular diagnosis and precision medicine.

The application discloses a DecAgo mutant protein and application thereof, and belongs to the technical field of programmable nucleases, wherein the DecAgo mutant protein has an amino acid mutation at the 182th position, specifically T182D, relative to a wild-type DecAgo protein with a sequence as shown in SEQ ID NO. 1. Relative to the wild-type DecAgo protein, the DecAgo mutant protein is more accurate to a cleavage site of a target at normal temperature, and has lower tolerance to a 10-base single-base mismatch behind an 18 nt guide, thereby laying a foundation for development of a new nucleic acid manipulation tool. Meanwhile, the application provides a strategy for modifying Ago function, based on which similar nucleases can be further modified, and the development of a gene editing tool based on Ago nucleases is facilitated.

The invention discloses a composition for detecting type I and type II monkey pox virus typing and a typing detection kit and application thereof, the composition comprises an LNA-Taqman probe for detecting the monkey pox virus I, the nucleotide sequence of the LNA-Taqman probe is shown as SEQ ID NO. 1, the nucleotide sequence of the LNA-Taqman probe is shown as SEQ ID NO. 2, and the nucleotide sequence of the LNA-Taqman probe is shown as SEQ ID NO. The nucleotide sequence of the upstream primer for typing detection of the monkey pox virus is shown as SEQ ID NO. 2; and a monkey pox virus typing detection downstream primer, the nucleotide sequence of which is as shown in SEQ ID NO. 3. According to the invention, the characteristic that the hybridization stability is sharply reduced when a locked nucleic acid (LNA) probe is mismatched with a single base is utilized, and an LNA monomer is accurately designed at a monkey pox virus typing SNP key site of the probe. By means of the design, typing signal differences can be remarkably amplified, and highly homologous virus branches can be effectively distinguished. The problems of cross reaction and false positive caused by high mismatching tolerance of a traditional DNA probe are avoided, and the accuracy of typing identification is ensured.

The invention discloses a method for rapidly and accurately cutting RNA (Ribonucleic Acid) based on MfAgo nuclease, which belongs to the technical field of molecular biology and comprises the following steps: (1) designing and synthesizing gDNA complementary with a RNA target sequence cutting site, and phosphorylating the 5'end of the gDNA; (2) incubating MfAgo nuclease and the gDNA obtained in the step (1) in an enzyme digestion buffer solution to form an MfAgo-gDNA compound; (3) adding an RNA (Ribonucleic Acid) target into the MfAgo-gDNA compound obtained in the step (2); reacting at 55-75 DEG C for more than 15 minutes to cut the RNA target; the method has accurate single base recognition capability and is extremely sensitive to the mismatching of the bases of the guide and the Target, and the cutting efficiency can be obviously reduced or even completely disappear when the mismatching of the guide and the Target exists, so that the method has extremely low miss rate and is expected to be further used as a gene editing tool.

A production method for a cell in which only one allele is genome-edited includes: a step of introducing, into a cell, (A) at least one selected from the group consisting of (a1) a guide RNA in which one or more nucleotide residues are added to a 5′-end of a spacer sequence, (a2) a guide RNA containing a spacer sequence having single-base or multiple-base mismatches with respect to a target sequence, and (a3) an expression vector that can cause the guide RNA of (a1) or (a2) to be expressed, and (B) at least one selected from the group consisting of a Cas protein and an expression vector that can cause the Cas protein to be expressed.

The purpose of the present invention is to provide a technology for, by using a small number of gRNA or crRNA, simultaneously editing numerous genes that are functionally duplicated. It was found that a CasΦ protein derived from huge phages can cut a target sequence having a nucleotide length of 16 or more, and a mismatch sequence including a mismatch of 1 or 2 bases with respect to the target sequence. Accordingly, provided is a genome editing method that is for duplicated genes and that uses said CasΦ protein.

The present invention discloses a single-base mutation imaging method based on in situ ARMS-PCR and Cas labeling. This method belongs to the field of single-cell analysis. By adding base mismatch sites to PCR primers, effective amplification and specific identification of single nucleotide variation information within bacteria are achieved. At the same time, the amplified products are labeled by modified gRNA / Cas12a. This method can also accurately quantify Salmonella resistant bacteria in complex environments and obtain cellular heterogeneity and spatial location information.

The invention discloses a method for preparing a base mismatched specific binding protein as well as a related biological material and application thereof. The invention provides a method for preparing a base mismatched specific binding protein, which comprises the following step: expressing a coding gene of the base mismatched specific binding protein in organisms to obtain the base mismatched specific binding protein, the organisms are microorganisms, plants or non-human animals; the base mismatch specific binding protein comprises a base mismatch specific endonuclease and a structural domain which contributes to the affinity adsorption of the zymoprotein to the matrix carrier or promotes the expression of the zymoprotein. The base mismatch specific endonuclease is a CEL I zymoprotein or a CEL II zymoprotein of apios graveolens, and the structural domain is an MBP protein. The fusion protein disclosed by the invention is applied to methods for detecting DNA base errors or mutation, correcting base errors in a gene synthesis process and the like, and has the advantages of high efficiency, rapidness, convenience in automatic operation, low cost and the like.

The invention provides crRNA, a CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system and a kit for SMN1 gene mutation detection. The nucleotide sequence of the crRNA is as shown in SEQ ID NO: 31. The crRNA is very suitable for detecting SMN1 gene mutation through an RPA-Cas12a one-step method, the tolerance of the crRNA to single base mismatch in a target is low, and the SMN1 gene recognition specificity is obviously enhanced; moreover, 9-nt single-stranded DNA modification is added to the 3'terminal of the RPA-Cas12a, so that the cutting efficiency of Cas12a to a substrate in RPA-Cas12a one-step detection can be effectively reduced, amplification and accumulation of target DNA are promoted, the detection sensitivity is effectively improved (about 100 times), and the detection performance is improved. Furthermore, a lateral flow chromatography test strip is developed on the basis of the crRNA, non-SMA patients and SMA patients can be rapidly and visually distinguished, large expensive equipment and skilled operators are not needed for detection, and the lateral flow chromatography test strip has the advantages of high sensitivity, high specificity, low cost and the like, is suitable for large-scale screening, is high in universality and can be widely applied to the field of clinical detection. And a new means is provided for early screening and prevention of SMA.

This invention discloses a rapid plasmid assembly method suitable for high-throughput screening, comprising the following steps: Step 1, design and preparation of targeted primers; Step 2, high-fidelity PCR amplification of the target fragment; Step 3, double enzyme digestion of the vector and target fragment; Step 4, efficient homologous recombination ligation; Step 5, high-throughput transformation and screening; Step 6, verification of the recombinant plasmid. This invention significantly improves the specificity and efficiency of PCR amplification by optimizing primer design, employing a dual homologous arm structure, and strictly controlling the primer Tm value, thus avoiding the problem of target fragment amplification failure. It also optimizes the PCR amplification system and procedure, using a hot-start high-fidelity enzyme mixture to reduce non-specific amplification and base mismatches, ensuring the integrity and accuracy of the target fragment. Furthermore, it uses a buffer compatible with both enzymes, shortening the enzyme digestion reaction time and simultaneously treating the vector and target fragment, enabling direct recombination of the digestion products and simplifying the operation process.

The invention relates to a DNA (deoxyribonucleic acid) self-assembly technology, in particular to a method for programming a pH (potential of hydrogen) response range by reasonably designing a shadow chain. According to the invention, a shadow chain which has a nucleotide sequence similar to that of i-motif but lacks continuous C basic groups is designed, and a toehold-mediated chain displacement (TMSDR) reaction is utilized to assist space folding of the i-motif under an acidic condition. The hairpin shadow is formed by designing complementary effect of a base in the shadow and mismatching of the base and a C-A base, and alkaline pH sensitivity is achieved. The slightly acidic narrow pH sensing probe is designed by combining the two designs to balance the acid-base sensing force. According to the strategy provided by the invention, when the pH value is 6.5, the fluorescence signal is relatively highest, the integration of a pH sensing domain and a protein recognition domain is allowed, the cell surface can be positioned by connecting an aptamer, the extracellular pH value of a tumor can be detected, and non-invasive imaging of the solid tumor is realized.

This invention provides a kit for detecting the tumor driver gene TP53R248W. Using crRNA with the nucleotide sequence shown in SEQ ID NO: 5, the R248W mutant base is designed at the 5' end of the crRNA spacer sequence, and a new mismatched base C is introduced. When there are two base mismatches between the crRNA spacer sequence and the wild-type RNA, a complex will not form, and the Cas13a protein will not be activated. This kit can detect mixed plasmids with a mutation frequency as low as 0.01%, and can detect plasma concentrations as low as 10... 4 The kit contains copies / μL of the TP53R248W variant. This kit exhibits good sensitivity, high specificity, and is simple, rapid, and efficient to use.

The present invention belongs to the field of biochemistry, and specifically relates to a Taq enzyme mutant and its application in SNP-related research. The specific technical solution is as follows: a Taq enzyme mutant, which is mutated from wild-type C-Taq enzyme, and the amino acid sequence of wild-type C-Taq enzyme is shown in SEQ ID NO: 1. Based on the wild-type C-Taq enzyme, at least the following mutations are carried out: the threonine at position 506 of the wild-type C-Taq enzyme is mutated to lysine. The present invention analyzes the key active sites of wild-type C-Taq enzyme and performs site-directed mutagenesis, and uses multiple pairs of SNP genotyping primers for high and low GC target fragments to test the candidate mutant enzymes, and screens two enzymes with better discrimination ability for single-base mismatches. The single-base mismatch probe of the mutant enzyme provided by the present invention has a lower peak and higher genotyping accuracy. The mutant enzymes provided by the present invention can amplify well at high, medium and low GC sites, with wide universality; using these mutant enzymes can effectively save the cost of screening MGB probes.

The present invention is directed to provide novel RNA molecules, chimeric NA molecules, double-stranded RNA molecules, and double-stranded chimeric NA molecules. Specifically, an embodiment of the present invention is an RNA molecule for RNA interference to target a mutant allele with a point mutation, in which (1) the molecule has a nucleotide sequence complementary to a nucleotide sequence of a coding region of the mutant allele; and (2) when counted from the base at the 5′-end in a nucleotide sequence complementary to a nucleotide sequence of the mutant allele, (2-1) a base at position 5 or 6 is mismatched to a base in the mutant allele; (2-2) a position 10 or 11 corresponds to the position of the point mutation; and (2-3) a group at the 2′-position of a pentose at positions 6-8 or positions 7 and 8 is modified with, e.g., OCH3. In this RNA molecule, one or more ribonucleotides may be replaced by, e.g., a deoxyribonucleotide. The molecule may form a double-stranded RNA with a complementary strand.

This invention discloses a T4 RNA ligase 2 mutant, its preparation method, and its applications, relating to the fields of biology and enzyme engineering. The T4 ligase 2 mutant provided by this invention has mutations at at least one of the following positions in the wild-type T4Rnl2 sequence: positions 62, 103, 166, 168, 193, 217, 260, 297, 298, 303, 306, 311, 313, and 318. Compared to the wild type, the mutant exhibits significantly improved thermostability and maintains high ligase activity at higher temperatures, such as 45°C and 50°C. In addition to ligating native nucleic acid sequences, the mutant can also ligate modified non-native nucleic acids at high temperatures. The mutant provided by this invention enables high-temperature synthesis of ribonucleic acid, effectively reducing the generation of byproducts due to base mismatches, improving RNA synthesis efficiency, and facilitating large-scale, high-quality RNA production.

A production method for a cell in which only one allele is genome-edited includes: a step of introducing, into a cell, (A) at least one selected from the group consisting of (a1) a guide RNA in which one or more nucleotide residues are added to a 5′-end of a spacer sequence, (a2) a guide RNA containing a spacer sequence having single-base or multiple-base mismatches with respect to a target sequence, and (a3) an expression vector that can cause the guide RNA of (a1) or (a2) to be expressed, and (B) at least one selected from the group consisting of a Cas protein and an expression vector that can cause the Cas protein to be expressed.

The invention discloses a nucleotide analogue, a kit and application. The nucleotide analogue disclosed by the invention has a structure as shown in a formula I, wherein R1 is adenine, guanine, cytosine, thymine, uracil or analogues of the adenine or the pyrimidine; r2 / R3 is a fluorophore, a reactive active group, H or a molecule for increasing the binding capacity with a DNA chain; r4 is a monophosphate group or a polyphosphate group, and in the polyphosphate group, O between a first phosphate group and a second phosphate group at the 5'end is substituted by CH2; and L1 is a linker. The nucleotide analogue disclosed by the invention solves the problem that a chemical structure'scar 'is easy to remain in the elution process of a fluorescent dye connecting chain, and also solves the problems that the flexibility of a newly synthesized chain and the combination efficiency of polymerase are influenced, the base mismatch probability is increased, the sequencing read length and the data quality are limited and the like; longer read length sequencing can be realized, and the method has higher quality and lower error rate.

The invention discloses a T4RNA ligase 2 mutant and a preparation method and application thereof, and relates to the field of biology and enzyme engineering, the T4RNA ligase 2 mutant provided by the invention has mutation at at least one site of 62th, 103th, 166th, 193th, 260th, 297th, 298th, 303th, 306th, 311th, 313th and 318th sites of a wild type T4Rnl2 sequence, compared with the wild type, the thermal stability of the mutant is obviously improved, and the T4RNA ligase 2 mutant can be used for preparing a T4RNA ligase 2 mutant. High ligase activity can be maintained at high temperatures such as 45 DEG C and 50 DEG C; in addition to being connected with a natural nucleic acid sequence, the mutant can also be connected with a non-natural nucleic acid with modification at high temperature. According to the mutant provided by the invention, ribonucleic acid can be synthesized at high temperature, so that by-products caused by base mismatch are effectively reduced, the RNA synthesis efficiency is improved, and large-scale high-quality RNA production is facilitated.

The invention discloses a fusion protein with DNA (Deoxyribose Nucleic Acid) error correction enzyme activity as well as a preparation method and application thereof. The present invention provides a fusion protein comprising a base mismatch specific endonuclease and a domain that contributes to affinity adsorption of a zymoprotein to a matrix carrier or promotes expression of the zymoprotein. The base mismatched specific endonuclease is escherichia coli T7 bacteriophage endonuclease I, and the structural domain facilitating affinity adsorption of zymoprotein to a specific matrix carrier or promoting zymoprotein expression is a cellulose specific binding structural domain CBM3. The method can be used for DNA base error or mutation detection, base error correction in the gene synthesis process and the like.