# US 8,206,902 B2 · Method of amplifying nucleic acid and method of detecting mutated nucleic acid using the same

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- Number: US 8,206,902 B2
- Document ID: US8206902B2
- Status: Granted
- Kind: B2
- Application number: 10/583,706
- Filed: 2004-12-24
- Published: 2007-08-16
- Granted: 2012-06-26
- Earliest claimed: 2003-12-25
- Assignees: Riken; Kabushiki Kaisha Dnaform
- Inventors: Yasumasa Mitani; Takanori Oka; Yoshihide Hayashizaki; Toshizo Hayashi
- Web page: https://patentgrep.com/patent/US8206902B2

## CPC

- C12Q 1/6853: using modified primers or templates
- C12Q 1/6816: characterised by the detection means (C12Q1/6804 takes precedence)
- C12Q 1/6827: for detection of mutation or polymorphism
- C12Q 1/6858: Allele-specific amplification
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

A primer set that allows a target nucleic acid to be amplified specifically and efficiently. The primer set of the present invention includes at least two primers that allow a target nucleic acid sequence to be amplified. A first primer included in the primer set contains, in its 3′ end portion, a sequence (Ac′) that hybridizes to a sequence (A) located in the 3′ end portion of the target nucleic acid sequence. The first primer also contains, on the 5′ side of the sequence (Ac′), a sequence (B′) that hybridizes to a complementary sequence (Bc) to a sequence (B) that is present on the 5′ side with respect to the sequence (A) in the target nucleic acid sequence. A second primer included in the primer set contains, in its 3′ end portion, a sequence (Cc′) that hybridizes to a sequence (C) located in the 3′ end portion of a complementary sequence to the target nucleic acid sequence. The second primer also contains, on the 5′ side of the sequence (Cc′), a folded sequence (D-Dc′) that contains, on the same strand, two nucleic acid sequences that hybridize to each other.

## Claims

### Claim 1

A primer set comprising at least two primers that allows a target nucleic acid sequence to be amplified,
wherein a first primer included in the primer set contains, in its 3′ end portion, a sequence (Ac′) that hybridizes to a sequence (A) located in the 3′ end portion of the target nucleic acid sequence, and also contains, on the 5′ side of the sequence (Ac′), a sequence (B′) that hybridizes to a complementary sequence (Bc) to a sequence (B) that is present on the 5′ side with respect to the sequence (A) in the target nucleic acid sequence, and
a second primer included in the primer set contains, in its 3′ end portion, a sequence (Cc′) that hybridizes to a sequence (C) located in the 3′ end portion of a complementary sequence to the target nucleic acid sequence, and also contains, on the 5′ side of the sequence (Cc′), a folded sequence (D-Dc′) that contains, on the same strand, two nucleic acid sequences that hybridize to each other.

### Claim 2 (depends on 1)

The primer set according to claim 1, further comprising a third primer that hybridizes to the target nucleic acid sequence or the complementary sequence thereto,
wherein the third primer does not compete with other primers for hybridization to the target nucleic acid sequence or the complementary sequence thereto.

### Claim 3 (depends on 1)

The primer set according to claim 1, wherein in the first primer, when no intervening sequence is present between the sequence (Ac′) and the sequence (B′), a ratio (X−Y)/X is in a range of −1.00 to 1.00, where X denotes the number of bases contained in the sequence (Ac′) while Y indicates the number of bases contained in a region flanked by the sequence (A) and the sequence (B) in the target nucleic acid sequence, and when an intervening sequence is present between the sequence (Ac′) and the sequence (B′) in the primer, a ratio {X−(Y−Y′)}/X is in a range of −1.00 to 1.00, where X and Y denote the same as described above, and Y′ indicates the number of bases contained in the intervening sequence.

### Claim 4 (depends on 1)

The primer set according to claim 1, wherein in the second primer, the folded sequence (D-Dc′) has a length of 2 to 1000 nucleotides.

### Claim 5 (depends on 1)

The primer set according to claim 1, wherein at least one primer included in the primer set has a solid-phase support or a site that can bind to a solid-phase support.

### Claim 6 (depends on 5)

The primer set according to claim 5, wherein the solid-phase support is one selected from the group consisting of a water-insoluble organic polymer support, a water-insoluble inorganic polymer support, a synthetic polymer support, a phase transition support, a metal colloid, and a magnetic particle.

### Claim 7 (depends on 5)

The primer set according to claim 5, wherein the site that can bind to a solid-phase support is selected from the group consisting of biotin, avidin, streptoavidin, an antigen, an antibody, a ligand, a receptor, a nucleic acid, and a protein.