# US 10,066,264 B2 · Method for analyzing target nucleic acid, kit, and analyzer

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- Number: US 10,066,264 B2
- Document ID: US10066264B2
- Status: Granted
- Kind: B2
- Application number: 14/414,324
- Filed: 2013-08-29
- Published: 2015-06-04
- Granted: 2018-09-04
- Earliest claimed: 2012-08-30
- Assignees: Kabushiki Kaisha Dnaform
- Applicants: Kabushiki Kaisha Dnaform
- Inventors: Yoshihide Hayashizaki; Masayoshi Itoh; Takahiro Arakawa; Kengo Usui; Sotaro Uemura; Yasumasa Mitani
- Web page: https://patentgrep.com/patent/US10066264B2

## CPC

- C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C09B 23/04: one >CH- group, e.g. cyanines, isocyanines, pseudocyanines
- C09B 23/06: three >CH- groups, e.g. carbocyanines
- C12Q 1/682: Signal amplification
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q 1/6853: using modified primers or templates
- C12Q 2600/158: Expression markers
- 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
- C09B: ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES {, e.g. PIGMENTS}; MORDANTS; LAKES (fermentation or enzyme using processes to synthesise a desired chemical compound C12P)

## Abstract

The present invention is to provide a method for analyzing a target nucleic acid, by which the target nucleic acid can be analyzed rapidly and easily. In order to achieve the above object, the present invention provides a method for analyzing a target nucleic acid in a sample, including the step of: analyzing the target nucleic acid in the sample by bringing the sample into contact with a label and with a primer or probe that can hybridize to the target nucleic acid. The primer or probe is immobilized on a solid phase. The label does not emit light when the primer or probe does not hybridize to the target nucleic acid, whereas the label emits light when the primer or probe has hybridized to the target nucleic acid. The analysis is carried out by detecting the light emitted from the label.

## Claims

### Claim 1

A method for analyzing a target nucleic acid in a sample, the method comprising the step of:
analyzing the target nucleic acid in the sample by bringing the sample into contact with a label and with a primer that can hybridize to the target nucleic acid,
wherein the primer is immobilized on a solid phase, and the primer is a nucleic acid molecule comprising at least one of structures represented by the following formulae (16), (16b), (17), (17b), (18), and (18b),
the label is covalently bound to the primer so as to be part of the primer,
the label is a fluorescent dye moiety that exhibits an exciton effect, and the label does not emit light when the primer does not hybridize to the target nucleic acid, whereas the label emits light when the primer has hybridized to the target nucleic acid,
the analysis is carried out by detecting light emission from the label caused by hybridization of the primer with the target nucleic acid,
the target nucleic acid that has hybridized with the primer is removed by washing, the primer is hybridized again with another target nucleic acid, and the analysis is carried out again by detecting light emission from the label,
the primer is hybridized to the target nucleic acid by bringing the primer into contact with the sample, thereby causing an amplification reaction of the target nucleic acid, and
the analysis of the target nucleic acid is carried out by further measuring the degree of amplification of the target nucleic acid in the amplification reaction over time:
where in the formulae (16), (16b), (17), (17b), (18), and (18b),
B is an atomic group having a natural nucleobase (adenine, guanine, cytosine, thymine, or uracil) skeleton or an artificial nucleobase skeleton,
E is:
(i) an atomic group having a deoxyribose skeleton, a ribose skeleton, or a structure derived from either one of them; or
(ii) an atomic group having a peptide structure or a peptoid structure,
Z11 and Z12 are each the fluorescent dye moiety that exhibits an exciton effect, and may be identical to or different from each other,
L1, L2, and L3 are each a linker (a linking atom or a linking atomic group), the main chain length (the number of main chain atoms) thereof is arbitrary, L1, L2, and L3 each may or may not contain each of C, N, O, S, P, and Si in the main chain, L1, L2, and L3 each may or may not contain each of a single bond, a double bond, a triple bond, an amide bond, an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond, and a thioester bond in the main chain, and L1, L2, and L3 may be identical to or different from each other,
D is CR, N, P, P═O, B, or SiR, and R is a hydrogen atom, an alkyl group, or an arbitrary substituent, and
b is a single bond, a double bond, or a triple bond,
or alternatively,
in the formulae (16) and (16b), L1 and L2 are each the linker, L3, D, and b may not be present, and L1 and L2 may be bonded directly to B, provided that:
in the formulae (16), (17), and (18), E is an atomic group described in the item (i), and at least one O atom in a phosphoric acid linkage may be substituted with an S atom;
in the formulae (16b), (17b), and (18b), E is an atomic group described in the item (ii); and
in the formulae (17) and (17b), the respective Bs may be identical to or different from each other, and the respective Es may be identical to or different from each other.

### Claim 2 (depends on 1)

The method according to claim 1, wherein
there are two or more kinds of the target nucleic acids, and the respective target nucleic acids are detected separately.

### Claim 3 (depends on 1)

The method according to claim 1, wherein
two or more kinds of the primers are used.

### Claim 4 (depends on 1)

The method according to claim 1, wherein
a surface of the solid phase on which the primer is immobilized is a flat surface, a flat chip surface, a spherical surface, or a three-dimensional surface.

### Claim 5 (depends on 1)

The method according to claim 1, wherein
a surface of the solid phase is coated with a coating for reducing a background.

### Claim 6 (depends on 5)

The method according to claim 5, wherein
the coating for reducing a background is provided by graft polymerization.

### Claim 7 (depends on 1)

The method according to claim 1, wherein
in the formulae (16), (17), (16b), (17b), (18), and (18b), the main chain length (the number of main chain atoms) of each of L1, L2, and L3 is an integer of 2 or more.

### Claim 8 (depends on 1)

The method according to claim 1, wherein
in the formulae (16), (17), (16b), (17b), (18), and (18b), Z11 and Z12 are each independently a group derived from any one of thiazole orange, oxazole yellow, cyanine, hemicyanine, other cyanine dyes, methyl red, azo dyes, biotin, and derivatives thereof.

### Claim 9 (depends on 1)

The method according to claim 1, wherein
Z11 and Z12 are each independently an atomic group represented by any one of the following formulae (7) to (9):
where in the formulae (7) to (9),
X1 and X2 are S, O, or Se,
n″ is 0 or a positive integer,
R1 to R10 and R13 to R21 are each independently a hydrogen atom, a halogen atom, a lower alkyl group, a lower alkoxy group, a nitro group, or an amino group,
one of R11 and R12 is a linking group that is bound to L1 or L2 in the formulae (16), (17), (16b), and (17b), and the other is a hydrogen atom or a lower alkyl group,
when a plurality of R15s are present in the formula (7), (8), or (9), they may be identical to or different from each other,
when a plurality of R16s are present in the formula (7), (8), or (9), they may be identical to or different from each other, and
X1, X2, and R1 to R21 in Z11 and X1, X2, and R1 to R21 in Z12 may be identical to or different from each other, respectively.

### Claim 10 (depends on 9)

The method according to claim 9, wherein
in the formulae (7) to (9),
in R1 to R21, the lower alkyl group is a linear or branched alkyl group with a carbon number of 1 to 6, and the lower alkoxy group is a linear or branched alkoxy group with a carbon number of 1 to 6.

### Claim 11 (depends on 9)

The method according to claim 9, wherein
in the formulae (7) to (9),
in R11 and R12, the linking group is a polymethylene carbonyl group with a carbon number of 2 or more and is bound to L1 or L2 in the formulae (16), (16b), (17), and (17b) in a carbonyl group moiety.

### Claim 12 (depends on 9)

The method according to claim 9, wherein
Z11 and Z12 are each independently an atomic group represented by the formula (7) or (8), and
Z11 and Z12 represented by the formula (7) or (8) is a group represented by the following formula (19) or (20):
where in the formulae (19) and (20),
X1, R1 to R10, R13 and R14, and R11 and R12 are identical to those in the formulae (7) to (9).

### Claim 13 (depends on 12)

The method according to claim 12, wherein
Z11 and Z12 are each independently an atomic group represented by the above formula (19),
where in the formula (19),
X1 is S,
R1 to R10 are hydrogen atoms, and
one of R11 and R12 is a linking group that is bound to L1 or L2 in the formulae (16), (17), (16b), and (17b), and the other is a methyl group.

### Claim 14 (depends on 12)

The method according to claim 12, wherein
Z11 and Z12 are each independently an atomic group represented by the above formula (19),
where in the formula (19),
X1 is S,
R1, R4, R5, R6, R7, R9, and R10 are hydrogen atoms,
R2, R3, and R12 are methyl groups,
R8 is a halogen atom, and
R11 is a linking group that is bound to L1 or L2 in the formulae (16), (17), (16b), (17b), (18), and (18b).

### Claim 15 (depends on 9)

The method according to claim 9, wherein
Z11 and Z12 are each independently an atomic group represented by the above formula (7),
where in the formula (7),
X1 is S,
n″ is 1,
R1 to R10, R15, R16, and R17 are hydrogen atoms,
R11 is a linking group that is bound to L1 or L2 in the formulae (16), (17), (16b), (17b), (18), and (18b), and
R12 is a methyl group.

### Claim 16 (depends on 9)

The method according to claim 9, wherein
Z11 and Z12 are each independently an atomic group represented by any one of the following formulae:
where in each of the above chemical formulae,
n is a positive integer.

### Claim 17 (depends on 1)

The method according to claim 1, wherein
in the formulae (16), (17), (16b), (17b), (18), and (18b),
B is an atomic group having a natural nucleobase (adenine, guanine, cytosine, thymine, or uracil) skeleton.

### Claim 18 (depends on 1)

The method according to claim 1, wherein
in the formulae (16), (17), (16b), (17b), (18), and (18b),
B is an atomic group having an artificial nucleobase skeleton, and the artificial nucleobase is 2-amino-6-(N,N-dimethylamino)purine pyridin-2-one, 5-methylpyridin-2-one, 2-amino-6-(2-thienyl)purine, pyrrole-2-carbaldehyde, 9-methylimidazo[(4,5)-b]pyridine, 5-iodo-2-oxo(1H)pyridine 2-oxo-(1H)pyridine, 2-amino-6-(2-thiazolyl)purine, 7-(2-thienyl)-imidazo[4,5-b]pyridine, bromothymine, azaadenine, or azaguanine.

### Claim 19 (depends on 1)

The method according to claim 1, wherein
in the formulae (16), (17), (16b), (17b), (18), and (18b),
B is an atomic group having an artificial nucleobase skeleton, and the artificial nucleobase is Py, Py der., Pu, or Pu der.,
the Py is an atomic group having a covalent bond to E in the 1-position and a covalent bond to a linker moiety in the 5-position in a six-membered ring represented by the following formula (11):
the Py der. is an atomic group in which at least one of all the atoms of the six-membered ring of the Py has been substituted with an N, C, S, or O atom, and the N, C, S, or O atom optionally may have an electric charge, a hydrogen atom, or a substituent,
the Pu is an atomic group having a covalent bond to E in the 9-position and a covalent bond to a linker moiety in the 8-position in a condensed ring represented by the following formula (12):
and the Pu der. is an atomic group in which at least one of all the atoms of a five-membered ring of the Pu has been substituted with an N, C, S, or O atom, and the N, C, S, or O atom optionally may have an electric charge, a hydrogen atom, or a substituent.

### Claim 20 (depends on 1)

The method according to claim 1, wherein
the structure represented by the formula (16) is a structure represented by the following formula (16-1) or (16-2),
the structure represented by the formula (16b) is a structure represented by the following formula (16b-1) or (16b-2),
the structure represented by the formula (17) is a structure represented by the following formula (17-1),
the structure represented by the formula (17b) is a structure represented by the following formula (17b-1)
the structure represented by the formula (18) is a structure represented by the following formula (18-1), and
the structure represented by the formula (18b) is a structure represented by the following formula (18b-1):
where in the formulae (16-1), (16-2), (16b-1), (16b-2), (17-1), (17b-1), (18-1) and (18b-1),
l, m and n′ are arbitrary, l, m and n′ may be identical to or different from each other, l, m and n′ each may or may not contain each of C, N, O, S, P, and Si in a main chain thereof, and l, m and n′ each may or may not contain each of a single bond, a double bond, a triple bond, an amide bond, an ester bond, a disulfide bond, an imino group, an ether bond, a thioether bond, and a thioester bond in the main chain,
B, E, Z11, Z12, and b are identical to those in the formulae (16), (16b), (17), (17b), (18), and (18b), and
in the formulae (16-1), (16-2), (17-1), and (18-1), at least one 0 atom in a phosphoric acid linkage may be substituted with an S atom.

### Claim 21 (depends on 20)

The method according to claim 20, wherein
in the formulae (16-1), (16-2), (16b-1), (16b-2), (17-1), (17b-1), (18-1) and (18b-1),
l, m, and n are each an integer of 2 or more.

### Claim 22 (depends on 1)

The method according to claim 1, wherein
the nucleic acid molecule comprises at least one of nucleotide structures represented by the following chemical formulae 106, 110, 113, 117, 120, 122, 123, 124, and 114-2, geometric isomers and stereoisomers thereof, and salts thereof:
where in the chemical formulae 106, 110, 113, 117, 120, 122, 123, 124, and 114-2,
n is a positive integer.

### Claim 23 (depends on 16)

The method according to claim 16, wherein
the linker length n is in the range from 2 to 6.

### Claim 24 (depends on 1)

The method according to claim 1, wherein
the amplification reaction of the target nucleic acid is caused by a bridge PCR method.

### Claim 25 (depends on 24)

The method according to claim 24, wherein
a primer pair is used as the primer,
each of primers in the primer pair comprises the label covalently bound to the primer and thus comprises the label as part thereof,
the labels covalently bound to the respective primers are each a fluorescent dye moiety that exhibits an exciton effect,
the labels are different from each other, and
in the bridge PCR method, the presence or absence of a mutation in a plurality of loci in the target nucleic acid is detected simultaneously or the expression levels of the plurality of loci are analyzed simultaneously by: adapting the labels to cause no fluorescence or fluorescence of one to three colors and carrying out fluorescent color analysis; or adapting the respective labels to exhibit fluorescence intensities different from each other and measuring the difference in fluorescence intensity.

### Claim 26 (depends on 24)

The method according to claim 24, wherein
a primer pair is used as the primer,
each of primers in the primer pair comprises the label covalently bound to the primer and thus comprises the label as part thereof,
the labels covalently bound to the respective primers are each a fluorescent dye moiety that exhibits an exciton effect,
the labels are different from each other, and
in the bridge PCR method, the proportion of mutations in the entire sample containing the target nucleic acid is determined by: adapting the labels to cause no fluorescence or fluorescence of one to three colors and carrying out fluorescent color analysis; or adapting the respective labels to exhibit fluorescence intensities different from each other and measuring the difference in fluorescence intensity.

### Claim 27 (depends on 24)

The method according to claim 24, wherein
a primer pair is used as the primer,
each of primers in the primer pair comprises the label covalently bound to the primer and thus comprises the label as part thereof,
the labels covalently bound to the respective primers are each a fluorescent dye moiety that exhibits an exciton effect,
the labels are different from each other, and
in the bridge PCR method, the quality of the sample containing the target nucleic acid is checked by: adapting the labels to cause no fluorescence or fluorescence of one to three colors and carrying out fluorescent color analysis; or adapting the respective labels to exhibit fluorescence intensities different from each other and measuring the difference in fluorescence intensity.

### Claim 28 (depends on 1)

The method according to claim 1, wherein
the amplification reaction of the target nucleic acid is caused by an isothermal amplification method.

### Claim 29 (depends on 1)

The method according to claim 1, wherein
two or more spots on the primer are immobilized on the solid phase in an arbitrary positional relationship.

### Claim 30 (depends on 1)

The method according to claim 1, wherein
the target nucleic acid is RNA,
the method further comprises the step of causing a reverse transcription reaction of the RNA, and
the reverse transcription reaction is caused prior to the amplification reaction or at the same time with the amplification reaction on the solid phase having the primer immobilized thereon.

### Claim 31 (depends on 1)

The method according to claim 1, wherein
the amplification reaction is caused using DNA polymerase, RNA polymerase, reverse transcriptase (reverse transcription polymerase), or RNA-dependent RNA polymerase.

### Claim 32 (depends on 1)

The method according to claim 1, wherein
the presence or absence of a mutation in the target nucleic acid is detected by carrying out melting curve analysis after the amplification reaction.

### Claim 33 (depends on 1)

The method according to claim 1, wherein
the melting curve analysis is carried out using a probe, and
the probe comprises a fluorescent dye moiety that exhibits an exciton effect.

### Claim 34 (depends on 33)

The method according to claim 33, wherein
two or more kinds of the probes each comprising a fluorescent dye moiety that exhibits an exciton effect are used.