11 results about "Base-Base Mismatch" patented technology

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 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.

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.

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.

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 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.

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.