Method for detecting polynucleotide sequence having gene mutation

A method for detecting a gene mutation(s), the method comprising the steps of:

• hybridizing a single-stranded circular DNA and a primer with a target polynucleotide containing:
• a first region;
• and a second region adjacent to the 3′-side of the first region and containing a mutation;
• performing a nucleic acid amplification reaction by rolling circle amplification based on formation of a complex of the target polynucleotide, the primer, and the single-stranded circular DNA;
• and
• detecting amplified nucleic acid with a detection reagent;
• wherein
• the single-stranded circular DNA contains:
• a sequence complementary to the first region of the target polynucleotide;
• and
• a primer-binding sequence adjacent to the 5′-side thereof;
• and
• the oligonucleotide primer contains:
• a region having a sequence complementary to the second region of the target polynucleotide;
• and
• a region adjacent to the 3′-side thereof and having a sequence complementary to the primer-binding sequence of the single-stranded circular DNA.

A method for detecting a gene mutation(s), the method comprising the steps of:

• hybridizing a first single-stranded circular DNA and a first primer with a target polynucleotide containing:
• a first region;
• and a second region adjacent to the 3′-side of the first region and containing a mutation;
• performing a nucleic acid amplification reaction by rolling circle amplification based on formation of a complex of the target polynucleotide, the first primer, and the first single-stranded circular DNA;
• hybridizing a second single-stranded circular DNA and a second oligonucleotide primer with an extended chain generated by the nucleic acid amplification reaction, and performing a nucleic acid amplification reaction based on formation of a complex of the extended chain, the second primer, and the second single-stranded circular DNA;
• and
• detecting amplified nucleic acid with a detection reagent;
• wherein
• the first single-stranded circular DNA contains:
• a sequence complementary to the first region of the target polynucleotide;
• a primer-binding sequence adjacent to the 5′-side thereof;
• and
• a sequence complementary to a second-single-stranded-circular-DNA-binding sequence;
• the first oligonucleotide primer contains:
• a region having a sequence complementary to the second region of the target polynucleotide;
• and
• a region adjacent to the 3′-side thereof and having a sequence complementary to the primer-binding sequence of the first single-stranded circular DNA;
• the second single-stranded circular DNA contains:
• the same sequence as the sequence, in the first single-stranded circular DNA, complementary to the second-single-stranded-circular-DNA-binding sequence;
• and
• a second primer-binding sequence adjacent to the 5′-side thereof;
• and
• the second oligonucleotide primer contains:
• the same sequence as the region adjacent to the 5′-side of the sequence, in the first single-stranded circular DNA, complementary to the second-single-stranded-circular-DNA-binding sequence;
• and
• a sequence adjacent to the 3′-side thereof and complementary to the second primer-binding sequence in the second single-stranded circular DNA.

A method for detecting a gene mutation(s), the method comprising the steps of:

• hybridizing a single-stranded circular DNA and a capture polynucleotide with a miRNA containing:
• a first region;
• and a second region located in the 3′-side thereof and containing a mutation;
• performing a nucleic acid amplification reaction by rolling circle amplification based on formation of a complex of the capture polynucleotide, the miRNA, and the single-stranded circular DNA;
• and
• detecting amplified nucleic acid with a detection reagent;
• wherein
• the single-stranded circular DNA contains:
• a miRNA-binding region complementary to the second region of the miRNA;
• and
• a second region in the 3′-side thereof;
• and
• the capture polynucleotide contains:
• a sequence complementary to the second region of the single-stranded circular DNA;
• and
• a miRNA-binding sequence complementary to the first region of the miRNA.

A method for detecting a gene mutation(s), the method comprising the steps of:

• hybridizing a first single-stranded circular DNA and a capture polynucleotide with a miRNA containing:
• a first region;
• and a second region adjacent to the 3′-side of the first region and containing a mutation;
• performing a nucleic acid amplification reaction by rolling circle amplification based on formation of a complex of the miRNA, the capture polynucleotide, and the first single-stranded circular DNA;
• hybridizing a second single-stranded circular DNA and a second oligonucleotide primer with an extended chain generated by the nucleic acid amplification reaction, and performing a nucleic acid amplification reaction based on formation of a complex of the extended chain, the second primer, and the second single-stranded circular DNA;
• and
• detecting amplified nucleic acid with a detection reagent;
• wherein
• the first single-stranded circular DNA contains:
• a miRNA-binding region complementary to the second region of the miRNA;
• a second region in the 3′-side thereof;
• and
• a sequence complementary to a second-single-stranded-circular-DNA-binding sequence;
• the capture polynucleotide contains:
• a sequence complementary to the second region of the single-stranded circular DNA;
• and
• a sequence complementary to the first region of the miRNA;
• the second single-stranded circular DNA contains:
• the same sequence as the sequence, in the first single-stranded circular DNA, complementary to the second-single-stranded-circular-DNA-binding sequence;
• and
• a second primer-binding sequence adjacent to the 5′ side thereof;
• and
• the second oligonucleotide primer contains:
• the same sequence as the region adjacent to the 5′-side of the sequence, in the first single-stranded circular DNA, complementary to the second-single-stranded-circular-DNA-binding sequence;
• and
• a sequence adjacent to the 3′-side thereof and complementary to the second primer-binding sequence in the second single-stranded circular DNA.