3 results about "Base-Base Mismatch" patented technology

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 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.