# US 10,760,116 B2 · Analysis kit, analyzer, and methods for analyzing template nucleic acid or target substance > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,760,116 B2 - Document ID: US10760116B2 - Status: Granted - Kind: B2 - Application number: 15/755,898 - Filed: 2016-08-26 - Published: 2019-01-31 - Granted: 2020-09-01 - Earliest claimed: 2015-08-28 - Assignees: Kabushiki Kaisha Dnaform - Applicants: Kabushiki Kaisha Dnaform - Inventors: Yuji Tanaka; Yoshihide Hayashizaki; Koichiro Tsujimaru - Web page: https://patentgrep.com/patent/US10760116B2 ## CPC - C12Q 1/682: Signal amplification - C12M 1/00: Apparatus for enzymology or microbiology - C12Q 1/68: involving nucleic acids - C12Q 1/6818: involving interaction of two or more labels, e.g. resonant energy transfer - C12Q 1/6851: Quantitative amplification - C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes - G01N 21/64: Fluorescence; Phosphorescence - G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)} - C12N 15/09: Recombinant DNA-technology - C12Q 2600/16: Primer sets for multiplex assays - G01N 2021/6432: {Quenching} - 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 - C12M: APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; {APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention provides a method for analyzing a template nucleic acid, a method for analyzing a target substance, an analysis kit for a template nucleic acid or a target substance, and an analyzer for a template nucleic acid or a target substance, which are excellent in accuracy. The method for analyzing a template nucleic acid of the present invention includes the steps of: fractionating a sample containing a template nucleic acid into a plurality of template nucleic acid fractions; amplifying a target sequence and its complementary sequence in the template nucleic acid with respect to each of the plurality of template nucleic acid fractions in the presence of a nucleic acid amplification reagent; detecting generation or quenching of a signal that shows an amplification of the target sequence or the complementary sequence with respect to each of the plurality of template nucleic acid fractions after the amplification step; and discriminating a template nucleic acid fraction in which the generation or quenching of a signal that shows the amplification has been detected among the plurality of template nucleic acid fractions as an amplified fraction in which the target sequence or the complementary sequence has been amplified, wherein the nucleic acid amplification reagent contains a primer set that amplifies the target sequence and the complementary sequence and a signal generating substance that generates or quenches a signal in response to the amplification, and the signal generating substance generates a signal in a state where it is bound sequence-dependently and quenches a signal in a state where it is not bound or quenches a signal in a state where it is bound sequence-dependently and generates a signal in a state where it is not bound, and generation and quenching of a signal are reversible. ## Claims ### Claim 1 A method for analyzing a template nucleic acid, comprising: fractionating a sample comprising a template nucleic acid into a plurality of template nucleic acid fractions; amplifying a target sequence and its complementary sequence in the template nucleic acid with respect to each of the plurality of template nucleic acid fractions in the presence of a nucleic acid amplification reagent; detecting generation or quenching of a signal that shows an amplification of the target sequence or the complementary sequence with respect to each of the plurality of template nucleic acid fractions after the amplification; and discriminating a template nucleic acid fraction in which the generation or quenching of the signal that shows the amplification has been detected among the plurality of template nucleic acid fractions as an amplified fraction in which the target sequence or the complementary sequence has been amplified, wherein the nucleic acid amplification reagent comprises: a primer set that amplifies the target sequence and the complementary sequence; and a signal generating substance that generates or quenches the signal in response to the amplification, wherein the signal generating substance is selected from the group consisting of a substance that generates the signal in a state where it is bound sequence-dependently and quenches the signal in a state where it is not bound; and a substance that quenches the signal in the state where it is bound sequence-dependently and generates the signal in the state where it is not bound, generating and quenching of the signal by the signal generating substance are reversible; the signal generating substance includes a fluorogenic probe, which comprises at least two fluorescent atomic groups per molecule that exhibit an exciton effect, the fluorogenic probe is selected from the group consisting of a probe that generates the signal in a state where it is bound to the target and quenches the signal in a state where it is dissociated from the target and a probe that quenches the signal in the state where it is bound to the target and generates the signal in the state where it is dissociated from the target, the primer set optionally comprises a fluorogenic primer, which comprises at least two fluorescent atomic groups per molecule that exhibit an exciton effect, the fluorogenic primer is selected from the group consisting of a primer that generates the signal in the state where it is bound to the target and quenches the signal in the state where it is dissociated from the target and a primer that quenches the signal in the state where it is bound to the target and generates the signal in the state where it is dissociated from the target, the at least two fluorescent atomic groups that exhibit the exciton effect in the fluorogenic probe or the primer are included in a base, the base comprises a structure represented by the following formulae (16), (16b), (17), or (17b): wherein in the formulae (16), (16b), (17), and (17b), 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 an atomic group exhibiting fluorescence, and may be identical to or different from each other, L1, L2, and L3 are each a linker (a linking atom or an 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, where 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 a 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), and (17), E is an atomic group described in the item (i), and at least one 0 atom in a phosphoric acid linkage may be substituted with an S atom; in the formulae (16b), and (17b), 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 for analyzing the template nucleic acid according to claim 1, further comprising: recovering the amplified fraction from the plurality of template nucleic acid fractions after the discriminating. ### Claim 3 (depends on 2) The method for analyzing the template nucleic acid according to claim 2, further comprising: amplifying the target sequence and the complementary sequence in the template nucleic acid with respect to the amplified fraction after the discriminating, wherein the amplifying is a second amplifying. ### Claim 4 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, wherein the detecting is conducted by a melting curve analysis. ### Claim 5 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, further comprising: conducting an analysis by a melting curve analysis after the detecting. ### Claim 6 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, wherein the sample comprising the template nucleic acid comprises the nucleic acid amplification reagent, and in the fractionating, the sample comprising the template nucleic acid and the nucleic acid amplification reagent is fractionated into the plurality of template nucleic acid fractions. ### Claim 7 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, wherein the fractionating causes each of the plurality of template nucleic acid fractions to contain the nucleic acid amplification reagent. ### Claim 8 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, wherein the fractionating comprises forming an emulsion from the sample, the template nucleic acid fraction is a drop of the sample dispersed in the emulsion, and the detecting is detecting the generation or quenching of the signal with respect to the drop in the emulsion. ### Claim 9 (depends on 8) The method for analyzing the template nucleic acid according to claim 8, wherein in the detecting, the emulsion is caused to pass through a flow channel, and the generation or quenching of the signal is detected with respect to the drop at a predetermined site of the flow channel when the drop in the emulsion passes through the flow channel. ### Claim 10 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, wherein the fractionating is fractionating the sample into the plurality of template nucleic acid fractions by dispensing the sample to a chip provided with a plurality of template nucleic acid fraction formation portions on its surface. ### Claim 11 (depends on 10) The method for analyzing the template nucleic acid according to claim 10, wherein in the chip, a surface of the template nucleic acid fraction formation portion is hydrophilic and a surface of a region excluding the template nucleic acid fraction formation portion is hydrophobic, and the fractionating is fractionating the sample into the plurality of template nucleic acid fractions by applying the sample to the surface of the chip to separate the sample into the template nucleic acid fraction formation portions. ### Claim 12 (depends on 10) The method for analyzing the template nucleic acid according to claim 10, wherein in the chip, the template nucleic acid fraction formation portion is a dent of the surface of the chip, the region excluding the template nucleic acid fraction formation portion is a non-dent, and the fractionating is fractionating the sample by introducing the sample into the dents on the surface of the chip. ### Claim 13 (depends on 10) The method for analyzing the template nucleic acid according to claim 10, wherein in the chip, the template nucleic acid fraction formation portion is a dent of the surface of the chip, and an inner surface of the template nucleic acid fraction formation portion is hydrophilic, and the region excluding the template nucleic acid fraction formation portion is a non-dent, and a surface of the region excluding the template nucleic acid fraction formation portion is hydrophobic. ### Claim 14 (depends on 10) The method for analyzing the template nucleic acid according to claim 10, wherein the nucleic acid amplification reagent is arranged in the template nucleic acid fraction formation portion of the chip, and the fractionating causes the template nucleic acid fraction to contain the nucleic acid amplification reagent in the template nucleic acid fraction formation portion of the chip. ### Claim 15 (depends on 10) The method for analyzing the template nucleic acid according to claim 10, wherein the detecting is obtaining an image of the plurality of template nucleic acid fractions on at least one chip, and the discriminating is discriminating the template nucleic acid fraction on the chip in which the generation or quenching of a signal has been detected in the image as the amplified fraction. ### Claim 16 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, wherein the fractionating is fractionating the sample into the plurality of template nucleic acid fractions by dropping the sample. ### Claim 17 (depends on 1) The method for analyzing the template nucleic acid according to claim 1, wherein in the fractionating, an average volume of the plurality of template nucleic acid fractions is 0.0001 to 5000 nL.