JP5707719B2 - Target base sequence identification method - Google Patents

Description

  The present invention relates to a method for discriminating whether or not a double-stranded nucleic acid contained in a sample has a target base sequence using a strand displacement reaction by spontaneous interaction of a partial double-stranded nucleic acid probe.

  Information on the human genome continues to increase due to the decoding of the human genome, especially the international hapmap project that creates SNP (Single Nucleotide Polymorphism) maps. Furthermore, the relationship between the obtained genome information and the individual's constitution is found, the difference in the individual constitution is grasped at the genetic level, and the diagnosis, treatment, prevention and drug administration according to the individual characteristics are enabled. Research aimed at the realization of "medical care according to individual genetic information" (custom-made medical care) is being developed on a large scale all over the world. The difference in gene here means a difference in the base sequence of the genome of an individual, and the main difference is a single base difference (SNP). Recently, it has been found that the difference in the number of times a short base sequence is repeated (copy number) (Copy Number variation: CNV) has spread throughout the genome, and the relationship between this CNV difference and disease has also been pointed out. ing.

  In recent years, genetic tests have been performed to examine the genotype of each individual. Genetic testing includes a method of detecting the presence or absence of nucleic acid amplification or hybridization using a primer or probe having a base sequence identical or complementary to the base sequence of each genotype. In general, a nucleic acid to be subjected to genetic testing is a double-stranded nucleic acid. Therefore, in order to efficiently and advantageously promote a hybridization reaction between a primer or probe and a sample nucleic acid, in advance, A denaturation treatment is performed to make the double-stranded nucleic acid strand into a single strand. The denaturation treatment mainly includes a thermal denaturation treatment and a chemical denaturation treatment. In general, a thermal technique is often used, and a temperature control operation near 100 ° C. is usually performed. For example, in PCR widely used as a nucleic acid amplification reaction, each cycle has a heat denaturation step. In the LAMP method and SMAP method in which a nucleic acid amplification reaction is performed in an isothermal environment, the sample nucleic acid must be heat-treated (95 ° C., several minutes) in the initial reaction stage. Thus, denaturation treatment such as heat denaturation is essential in the reaction process of nucleic acid reagents used in many genetic tests.

  However, such heat treatment not only complicates the reaction process, but also has a great influence on the stability and reproducibility of the reaction. Furthermore, since a thermal load is applied to the apparatus and reaction vessel for performing the heat treatment, it is necessary to add a heat resistance function to them, but in order to ensure the heat resistance function, many development costs and time are required. Will also spend money.

  On the other hand, there is a concept of branch migration as a reaction form in which a wide range of chain-length interactions are induced based on local interactions between nucleic acid chain molecules. Branch migration is a phenomenon observed in gene recombination, and analysis of branch migration is also underway to elucidate the homologous recombination mechanism of genes (see, for example, Non-Patent Documents 1 to 4). In these studies, a technique for analyzing a strand displacement reaction caused by the action of a single-stranded nucleic acid on a double-stranded nucleic acid has been adopted, and the nucleic acid has a complementary double-stranded structure. It does not include a discussion of the reaction mechanism that goes from state to branch migration.

  Several methods for identifying a target base sequence using branch migration have been disclosed. A double-stranded nucleic acid probe having a protruding end can spontaneously interact with a single-stranded nucleic acid in solution. Therefore, the target base is determined by examining the extent to which a double-stranded nucleic acid is newly formed by one nucleic acid strand constituting a double-stranded nucleic acid probe and another single-stranded nucleic acid (the degree of strand displacement reaction). Methods for identifying sequences are disclosed (for example, see Patent Documents 1 to 5 and Non-Patent Documents 5 to 9). In these methods, first, a fluorescent substance or the like is used so that the detected signal is different between a double-stranded state and a single-stranded state or a state where a new double-stranded nucleic acid is newly formed with another single-stranded nucleic acid. The double-stranded nucleic acid probe is labeled in advance using Then, PCR is performed using this labeled double-stranded nucleic acid probe as a primer, the signal of the obtained PCR product is detected, and compared with the signal of the double-stranded formation state, two new strands are obtained by the strand displacement reaction. It is detected whether or not a strand-forming nucleic acid has been formed. By detecting the signal after mixing the labeled double-stranded nucleic acid probe with the sample nucleic acid that has been previously single-stranded, and comparing it with the signal in the double-stranded formation state, two new double-stranded nucleic acid probes can be obtained by the strand displacement reaction. It can be detected whether or not a strand-forming nucleic acid has been formed.

  These methods are nucleic acid identification systems using branch migration, but all reports have a form in which a single-stranded nucleic acid chain and a double-stranded nucleic acid chain are reacted, and a nucleic acid probe and a sample nucleic acid. Among these, at least one of the nucleic acid strands is a system that provides an environment in which the nucleic acid strand can exist as a single strand to execute the reaction. For this reason, when a strand displacement reaction is caused by branch migration between the double-stranded nucleic acid probe and the double-stranded nucleic acid, PCR is performed which requires a heat denaturation step, or the LAMP method, etc. Similarly to the above, a denaturation step such as heat treatment is provided before the chain substitution reaction.

International Publication No. 02/30946 Pamphlet US Pat. No. 5,804,375 US Pat. No. 6,593,091 International Publication No. 05/012571 Pamphlet US Patent Application Publication No. 2005/0227257 US Pat. No. 5,928,862

Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 1991, Vol. 26 (3/4), pages 227-259. Biswas, two others, Journal of Molecular Biology, 1998, Vol. 279, No. 4, pages 795-806. Reynaldo, 3 others, Journal of Molecular Biology, 2000, Vol. 297, No. 2, pages 511-520. Neschastnova, 6 others, Biochemistry, 2002, 41, 7795-7801. Li, 3 others, Nucleic Acids Research, 2002, volume 30, number 2, page e5. Cheng, 2 others, Nucleic Acids Research, 2004, Vol. 32, No. 7, page e61. Hirata, 4 others, Nucleic Acids Symposium Series, 2004, Vol. 48, No. 7, pp. 267-268. Ishii, 4 others, Nucleic Acids Symposium Series, 2006, Vol. 50, No. 7, pp. 325-326. Huang, 9 others, Nucleic Acids Research, 2007, Vol. 35, No. 16, p. E101.

  Under such circumstances, the present invention discriminates a target base sequence by effectively reacting a sample nucleic acid forming a double strand with a double-stranded nucleic acid probe without requiring a heat denaturation step. It aims to provide a method.

  As a result of intensive research to solve the above problems, the present inventor has adjusted the ratio of the length of the protruding end region to the total length of the double-stranded nucleic acid probe in the double-stranded nucleic acid probe having a protruding end. The present inventors have found that the target base sequence can be identified by strand displacement reaction without subjecting both the sample nucleic acid and the double-stranded nucleic acid probe to heat denaturation in advance.

That is, the present invention
(1) A method for identifying a target base sequence,
In the reaction solution, the sample nucleic acid in the state of forming a double strand and the partially double-stranded nucleic acid probe are allowed to coexist, and the degree of the strand displacement reaction is measured to determine the base in the sample nucleic acid. An identification step for identifying the identity between the sequence and the target base sequence,
The double-stranded nucleic acid probe is a first probe that is a single-stranded nucleic acid comprising a base sequence complementary to a target base sequence, and a single-stranded nucleic acid comprising a base sequence complementary to the first probe. Consisting of two probes,
The ratio of the base length of the second probe to the base length of the first probe ([base length of the second probe] / [base length of the first probe]) is 0.5 or more and less than 1.
The method for identifying a target base sequence, wherein the temperature of the reaction solution is 40 ° C. or higher and lower than 65 ° C.,
(2) The method for identifying a target base sequence according to (1) above, wherein the temperature of the reaction solution is within a range of 40 to 60 ° C.,
(3) The method for identifying a target base sequence according to (1) or (2), wherein the GC content in the protruding end region of the first probe is 50% or more,
(4) The target according to any one of (1) to (3), wherein the 3 ′ ends of the first probe and the second probe are modified with an inhibitor of elongation reaction by polymerase. Identification method of base sequence,
(5) The target base sequence identification method according to any one of (1) to (4), wherein at least one of the first probe and the second probe includes a nucleic acid analog,
(6) The identification of the target base sequence according to any one of (1) to (5), wherein the first probe and the second probe are labeled with different types of labeling substances, respectively. Method,
(7) The target base according to (6), wherein the first probe or the second probe is labeled with a labeling substance in a double-stranded forming region in the partial double-stranded nucleic acid probe. How to identify the sequence,
(8) The target base according to (6) or (7), wherein energy transfer is possible between a labeling substance that labels the first probe and a labeling substance that labels the second probe. How to identify the sequence,
(9) The target base sequence according to (6) or (7), wherein either one of the first probe and the second probe is labeled with a labeling substance that can bind to a solid phase carrier. Identification method,
(10) The method for identifying a target base sequence according to any one of (1) to (9), wherein the sample nucleic acid is a double-stranded nucleic acid amplified by a nucleic acid amplification reaction,
Is to provide.

  The target base sequence identification method of the present invention can identify a target base sequence in a sample nucleic acid, which is a double-stranded nucleic acid, with high accuracy and high accuracy without performing a heat denaturation treatment that places a heavy load.

In one aspect of the method for identifying a target base sequence of the present invention, a nucleic acid presumed to be present in a reaction solution is schematically shown in comparison with a conventional method that requires a heat denaturation step. . In Example 1, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a blunt end probe. In Example 1, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a protrusion terminal probe. In Example 2, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid. In Example 3, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid. In Example 3, it is the figure which showed the relationship between the fluorescence intensity ratio and the variation | mutation gene containing density | concentration at the time of 80-minute progress after reaction start. In Example 4, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid. In Example 5, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to the reaction liquid containing a Type1 probe. In Example 5, it is the figure which showed the result of having performed fluorescence time-measurement with respect to each reaction liquid containing a Type2 probe. In Example 5, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a Type3 probe. In Example 5, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a Type4 probe. In Example 6, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a Type1 probe. In Example 6, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a Type2 probe. In Example 6, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a Type3 probe. In Example 6, it is the figure which showed the result of having performed fluorescence time-measurement with respect to each reaction liquid containing a Type4 probe. In Example 6, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a Type5 probe. In Example 6, it is the figure which showed the result of having performed fluorescence time-lapse measurement with respect to each reaction liquid containing a Type6 probe.

  In the present invention and the present specification, “identifying a target base sequence” means identifying whether a sample nucleic acid to be identified is a nucleic acid having a target base sequence.

  The method for identifying a target base sequence of the present invention relates to a method for identifying a target base sequence using as an index the degree of occurrence of a strand displacement reaction that occurs between a partially double-stranded nucleic acid probe and a sample nucleic acid. In the present invention, a double-stranded nucleic acid having a protruding end formed by annealing two nucleic acid strands having a base length ratio within a specific range is used as a probe to form a double strand with the probe. A strand displacement reaction can be caused between the sample nucleic acid in a closed state. That is, without subjecting the sample nucleic acid to a denaturation treatment such as heat denaturation in advance, it uses the probe's spontaneous interaction to react with the target base sequence in the sample nucleic acid to perform strand displacement. By causing a reaction, a specific base sequence can be identified.

  The partial double-stranded nucleic acid probe used in the present invention includes a first probe that is a single-stranded nucleic acid that is composed of a base sequence complementary to a target base sequence, and a single probe that is composed of a base sequence complementary to the first probe. A second probe which is a strand nucleic acid. The first probe and the second probe are single-stranded nucleic acids having different lengths (base lengths). In the partial double-stranded nucleic acid probe, the 3 ′ end side of the first probe may be a protruding end, the 5 ′ end side of the first probe may be a protruding end, and both ends of the first probe protrude. The terminal may be sufficient. As the partial double-stranded nucleic acid probe used in the present invention, it is preferable that the 3 ′ end side or both ends of the first probe are protruding ends.

  In the partial double-stranded nucleic acid probe used in the present invention, the ratio of the base length of the second probe to the base length of the first probe ([base length of the second probe] / [base length of the first probe]) is 0.5 or more and less than 1. The greater the ratio between the base length of the second probe and the base length of the first probe, the higher the discrimination ability of the target base sequence, but the tendency for spontaneous strand displacement efficiency to decrease. Conversely, the smaller the ratio of the base length of the second probe to the base length of the first probe, the lower the ability to discriminate the target base sequence, but the higher the efficiency of spontaneous strand displacement. By making the ratio of the base length of the second probe and the base length of the first probe within the above range, the sample nucleic acid in a state of forming a double strand can be efficiently displayed while exhibiting sufficient discrimination ability. It can cause a strand displacement reaction.

  As long as the ratio of the base length of the second probe to the base length of the first probe is within the above range, the length of the first probe and the second probe is not particularly limited, but the length of the second probe In other words, the longer the (the region in which the first probe and the second probe form a base pair in the partial double-stranded nucleic acid probe), the higher the temperature of the reaction solution or the longer the reaction time is. is there.

  In the double-stranded nucleic acid probe, since the spontaneous interaction ability is affected by the sequence species of the protruding end region, the base sequence of the protruding end region is not included in the sample nucleic acid in order to increase the efficiency of spontaneous strand displacement. It is preferable that the sequence be capable of stably binding to the target base sequence. In the partially double-stranded nucleic acid probe used in the present invention, the GC content in the protruding end region on the 3 ′ end side of the first probe is preferably 50% or more. The higher the GC content in the protruding end region, the higher the spontaneous strand displacement efficiency and the more efficient strand displacement reaction with the sample nucleic acid.

  Here, the “protruding end region in the partial double-stranded nucleic acid probe” refers to a region in the first probe that does not form a base pair with the second probe. In the case of a partially double-stranded nucleic acid probe in which both ends are protruding ends, it is more preferable that the total GC content is 50% or more for each protruding end (single-stranded) region at each end.

  In the present invention, the first probe and the second probe may be DNA strands, RNA strands, or DNA and RNA chimeric strands. Further, it may contain a nucleic acid analog. A nucleic acid chain containing a nucleic acid analog means a nucleic acid analog that is used in place of a natural nucleic acid such as DNA as part or all of the nucleotides constituting the nucleic acid chain.

  In the present invention and the specification of the present application, a nucleic acid analog is a non-natural nucleic acid and has the same function as DNA or RNA, which are natural nucleic acids. That is, a nucleic acid analog can form a chain by a phosphodiester bond in the same manner as DNA and the like, and a primer or probe formed using a nucleic acid analog is formed using only a natural nucleic acid. Similar to primers and probes, they can be used for PCR and hybridization. Examples of such nucleic acid analogs include PNA (polyamide nucleotide derivatives), LNA (BNA), ENA (2'-O, 4'-C-Ethylene-bridged nucleic acids), and complexes thereof. Here, PNA is obtained by substituting a main chain composed of phosphoric acid and pentose of DNA or RNA with a polyamide chain. LNA (BNA) is a compound having two cyclic structures in which an oxygen atom at the 2 'site of a ribonucleoside and a carbon atom at the 4' site are bonded via methylene.

  The first probe and the second probe may be nucleic acid strands that can be annealed with each other, and the nucleic acid species may be different from each other. For example, both the first probe and the second probe may be DNA strands, one of them may be a DNA strand, and the other may be an RNA strand. Furthermore, only one of the first probe and the second probe may contain a nucleic acid analogue, or both may contain a nucleic acid analogue.

  Furthermore, the nucleic acid or nucleic acid analog constituting the first probe and the second probe may be modified. The substance used for the modification of the nucleic acid or the nucleic acid analog is not particularly limited as long as the effect of the present invention is not impaired, and a substance usually used for the modification of the nucleic acid or the like can be used. Modified nucleic acids and modified nucleic acid analogs include, for example, nucleic acids modified with functional groups such as amino groups, carboxyvinyl groups, phosphate groups, methyl groups, nucleic acids modified with 2-O-methylation with methyl groups, phosphorothioated Examples thereof include nucleic acids modified with a labeling substance to be described later, such as modified nucleic acids and fluorescent substances.

  In addition, it is desirable that the 3 ′ ends of the first probe and the second probe have an elongation-inhibiting structure, and each may be modified with an inhibitor of an elongation reaction by polymerase. When the 3 'end of each probe is modified with an inhibitor, the first probe and the second probe are prevented from being consumed as primers for the extension reaction even when polymerase is mixed in the reaction solution. can do. For this reason, when the PCR product is used as a sample nucleic acid, the reaction solution after PCR can be directly added to the reaction solution as a sample nucleic acid without purification.

  Examples of such an inhibitor include an amino group, a phosphate group, a methyl group, and a thiol group. Since the nucleotide at the 3 'end of each probe is aminated, phosphorylated, methylated, thiolated or the like, the extension reaction by the polymerase starting from the probe can be inhibited.

  The first probe and the second probe used in the present invention can be prepared using a nucleic acid amplification reaction, but are preferably prepared by a known chemical synthesis. This is because the chemical synthesis method has a higher degree of freedom in the introduction position of the labeling substance. Examples of the chemical synthesis method include triester method and phosphorous acid method. For example, a large amount of single-stranded DNA is prepared using an ordinary automatic synthesizer (APPLIED BIOSSYSTEMS 392, etc.) using a liquid phase method or a solid phase synthesis method using an insoluble carrier, and then annealed. By doing so, double-stranded DNA can be prepared.

  Specifically, in the method for identifying a target base sequence of the present invention, a strand displacement reaction is carried out by allowing a sample nucleic acid in the form of a double strand and a partially double-stranded nucleic acid probe to coexist in a reaction solution. And a step of identifying the identity between the base sequence in the sample nucleic acid and the target base sequence by measuring the degree of occurrence of.

  In the present invention, a sample nucleic acid in a state where a double strand is formed is reacted with a partially double-stranded nucleic acid probe in a state where a double strand is formed. For example, after adding a sample nucleic acid and a partially double-stranded nucleic acid probe in a double-stranded state to the reaction solution, the temperature of the reaction solution is adjusted without performing heat denaturation treatment or chemical denaturation treatment. By setting the desired temperature below 65 ° C., the sample nucleic acid and the partial double-stranded nucleic acid probe can be subjected to a strand displacement reaction while forming a double strand. When using a sample nucleic acid that has been subjected to heat denaturation treatment, after adding a sufficient amount of time at a temperature lower than the Tm value of the sample nucleic acid (for example, room temperature) before adding it to the reaction solution, Subject to strand displacement reaction. In addition, when using a PCR product as a sample nucleic acid, a partially double-stranded nucleic acid probe is added to the PCR reaction solution in advance, and the temperature of the reaction solution is less than 65 ° C. without performing heat denaturation after the PCR reaction is completed. The chain substitution reaction may be performed at a desired temperature. By shifting from the annealing step, which is the final step of PCR, to the direct strand displacement reaction, the PCR product can be subjected to the strand displacement reaction while forming a double strand.

  The sample nucleic acid used in the present invention is not particularly limited as long as it is a double-stranded nucleic acid, and may be a nucleic acid extracted from a living organism or a nucleic acid amplified by a nucleic acid amplification reaction. . When the amplification product is a single-stranded nucleic acid, it can be used after being double-stranded by a primer extension reaction or the like. Examples of such a nucleic acid amplification reaction include PCR method, LAMP method, SMAP method, NASBA method, RCA method and the like.

  In the present invention, the temperature of the reaction solution in which the sample nucleic acid and the partially double-stranded nucleic acid probe coexist is set to less than 65 ° C. This is because if the temperature of the reaction solution is too high, the partially double-stranded nucleic acid probe and the sample nucleic acid are dissociated and become single-stranded. In particular, the temperature of the reaction solution is preferably set to a temperature equal to or lower than the dissociation temperature of the partially double-stranded nucleic acid probe.

  The temperature of the reaction solution for performing the strand displacement reaction may be any temperature at which both the sample nucleic acid and the partial double-stranded nucleic acid probe are in a state of forming a double strand. It is preferable to adjust appropriately according to the chain length of the first probe. For example, when using a partially double-stranded nucleic acid probe with a relatively long chain length of the first probe, by using a higher reaction temperature than when using a relatively short chain length of the first probe, The strand displacement reaction can proceed efficiently. Specifically, in the present invention, the temperature of the reaction solution is preferably 40 to 60 ° C.

  You may add the substance which has the effect | action which promotes the spontaneous interaction of a partial double strand formation nucleic acid probe more effectively to a reaction liquid. Such substances include polycations. Since polycations function as a lubricant, it is expected to accelerate the spontaneous interaction of the partially double-stranded nucleic acid probe by acting with a sample nucleic acid in the presence of the polycations. Among these, it is preferable to add a polycation having a hydrophilic side chain. Examples of the polycation having a hydrophilic side chain include polymerizing one or more of amino acids such as lysine, arginine, histidine, ornithine, sugars such as glucosamine, and cationic monomers such as allylamine and ethyleneimine. And polymers having these derivatives as main chains, polysaccharides such as dextran, amylose and cellulose, polyethers such as polyethylene glycol, and substances having these derivatives as side chains. Among them, dextran side chain modified poly (L-lysine) (PLL-g-Dex) and dextran side chain modified guanidized poly (L-lysine) (GPLL-g-Dex) (for example, Nucleic Acids Symposium Series (2006) vol. .50, p27-28, or Nucleic Acids Symposium Series (2007) vol.51, p339-340) is preferably added to the reaction solution.

  Furthermore, other substances can be added to the reaction solution as long as the interaction between the sample nucleic acid and the partially double-stranded nucleic acid probe is not inhibited. For example, a buffer that adjusts the pH of the reaction solution, a monovalent or divalent cation necessary for double-stranded formation, an organic solvent that affects the stability of the double-stranded nucleic acid, and the like can be added. . Examples of the buffer include Tris hydrochloric acid. Examples of the cation include sodium ion and magnesium ion. Examples of the organic solvent include dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and the like.

  The reaction time of the strand displacement reaction may be sufficient to reach the equilibrium state, taking into consideration the target base sequence sequence and base length, the second probe sequence and base length, reaction temperature, reaction solution composition, etc. Thus, it can be determined as appropriate.

  If a sufficient amount of new double-stranded nucleic acid formed by the strand displacement reaction is present in the reaction solution, it is determined that the sample nucleic acid has the target base sequence. On the other hand, when a sufficient amount of new double-stranded nucleic acid does not exist, no strand displacement reaction occurs between the sample nucleic acid and the partial double-stranded nucleic acid probe, and the sample nucleic acid has no target base sequence. It can be determined that it does not have. In the present invention, “measuring the degree of occurrence of strand displacement reaction” means the ratio of the sample nucleic acid and the probe causing the strand displacement reaction to the entire partial double-stranded nucleic acid probe added to the reaction solution (sample The ratio of the first probe annealed with the nucleic acid).

  The degree to which the strand displacement reaction has occurred may be measured only after the strand displacement reaction (end point), or may be performed over time (in real time) during the strand displacement reaction. Moreover, it may be performed at the start and end of the strand displacement reaction, and by comparing the measured values of both, it may be discriminated whether or not the sample nucleic acid has the target base sequence.

  By first labeling the first and second probes of the partially double-stranded nucleic acid probe with a labeling substance, the degree of strand displacement reaction is measured quantitatively or semi-quantitatively using the labeling substance as an index. can do. In particular, the first probe and the second probe are preferably labeled with different types of labeling substances. In such a case, the double-stranded nucleic acid formed by the strand displacement reaction is labeled only with one kind of labeling substance, and the partial double-strand formation is labeled with two kinds of labeling substances. It can be detected separately from the nucleic acid probe.

  In addition, when one of the two types of labeling substances is a substance that can bind to a solid phase alone, a sample nucleic acid and a partial double strand are obtained by performing a widely used solid-liquid separation operation. The degree to which a strand displacement reaction has occurred with the formed nucleic acid probe can be measured. For example, either the first probe or the second probe is labeled with the labeling substance A, the other probe is labeled with the labeling substance B that can bind to the solid phase alone, and the reaction solution after the strand displacement reaction is The solid phase is allowed to contact the labeling substance B. Thereafter, the labeling substance A in the double-stranded nucleic acid bound to the solid phase carrier is detected. When the strand displacement reaction occurs, the ratio of the double-stranded nucleic acid labeled with the labeling substance A in the double-stranded nucleic acid bound to the solid phase carrier decreases.

  As the labeling substance, either a non-radioactive substance or a radioactive substance may be used, but a non-radioactive substance is preferably used. Non-radioactive labeling substances include fluorescent substances [for example, fluorescein derivatives (fluorescein isothiocyanate, etc.), rhodamine and derivatives thereof (tetramethylrhodamine isothiocyanate, etc.)], chemiluminescent substances (for example, Acridine, etc.). Further, the labeling substance can be indirectly detected by using a substance that specifically binds to the labeling substance. Examples of such a labeling substance include biotin, a ligand, a specific nucleic acid, or a protein hapten. In the case of biotin, the avidin or streptavidin that specifically binds to this substance is specifically bound to the labeling substance, and in the case of a hapten, the antibody that specifically binds to this is the ligand. When the receptor is a specific nucleic acid or protein, a nucleic acid that specifically binds to the receptor, a nucleic acid binding protein, a protein having an affinity for the specific protein, or the like can be used. As the hapten, a compound having 2,4-dinitrophenyl group or digoxigenin can be used, and biotin or a fluorescent substance can also be used as a hapten. Any of these labeling substances may be used alone or in combination of a plurality of kinds if necessary (see JP-A-59-93099, JP-A-59-148798, JP-A-59-204200). )).

  In particular, in the present invention, two kinds of labeling substances capable of transferring energy to each other (for example, a donor labeling substance that generates fluorescence by excitation and an acceptor labeling substance that absorbs the fluorescence) are used, and between these labeling substances. It is preferable to measure the degree of chain substitution reaction using the degree of energy change due to energy transfer as an index.

  Energy transfer between the labeling substances means that at least two kinds of labeling substances, ie, a donor labeling substance that generates energy and an acceptor labeling substance that absorbs energy generated from the donor labeling substance are in close proximity to each other. This refers to the transfer of energy from a donor labeling substance to an acceptor labeling substance. For example, when the two kinds of labeling substances are fluorescent substances, the acceptor labeling substance absorbs the fluorescence generated by exciting the donor labeling substance and measures the fluorescence emitted by the acceptor labeling substance, or the donor labeling substance is excited. The quenching of the donor labeling substance caused by the absorption of the fluorescence generated by the acceptor labeling substance can be measured (PCR Methods and applications 4,357-362 (1995), Nature Biotechnology 16, 49-53 (1998)). Note that energy transfer may occur even if there is no overlap between the fluorescence wavelength of the donor labeling substance and the absorption wavelength of the acceptor labeling substance. Such energy transfer is also included in the present invention. Further, the acceptor labeling substance may be a quenching substance. As such, the quencher includes, for example, DABCYL and black hole.

The two kinds of labeling substances capable of transferring energy to each other are not particularly limited as long as they are capable of transferring energy in the state of being close to each other. Among them, fluorescent substances and delayed fluorescent substances are preferable, and in some cases, chemiluminescent substances and bioluminescent substances Substances can also be used. As a combination of such labeling substances, fluorescein and its derivatives (for example, fluorescein isothiocyanate) and rhodamine and its derivatives (for example, tetramethylrhodamine isothiocyanate, tetramethylrhodamine-5- (and-6) hexanoic acid) Etc.), a combination of fluorescein and DABCYL, etc., and any combination can be selected from these (Nisotopic DNA Probe Technologies. Academic Press (1992)). In addition, it may be a combination of molecules in which thermal energy is released when close to each other. Examples of combinations of such labeling substances include Alexa Fluor (registered trademark) 488 (manufactured by Invitrogen), ATTO 488 (manufactured by ATTO-TEC GmbH), Alexa Fluor (registered trademark) 594 (manufactured by Invitrogen), and ROX ( Examples thereof include a combination of 1 selected from the group consisting of Carboxy-X-rhodamine) and BHQ (registered trademark, Black hole quencher) -1 or BHQ (registered trademark) -2.
In addition, since guanine has the ability to quench when FAM adjoins (Nucleic acids Research 2002, vol.30.no.9 2089-2195), you may utilize this. For example, in a partially double-stranded nucleic acid probe in which the 5 ′ end of the first probe is a blunt end and the 3 ′ end is a protruding end, the second probe is labeled when the 5 ′ end of the first probe is labeled with FAM. When the 3 ′ terminal base of the probe is guanine, the second probe may not be labeled with a labeling substance.

  As a method for introducing a labeling substance into the first probe or the second probe, a general method for introducing a label into a nucleic acid can be employed. For example, a method of directly introducing a labeling substance into a nucleic acid (Biotechniques 24, 484-489 (1998)), a method of introducing a labeling substance-bound mononucleotide by a DNA polymerase reaction or an RNA polymerase reaction (Science 238, 336-3341). (1987)), a method of introducing a PCR reaction using a primer into which a labeling substance has been introduced (PCR Methods and Applications 2, 34-40 (1992)) and the like.

  When a labeling substance is introduced into the first probe or the second probe, each probe and the labeling substance may be bound via a linker. The linker can be appropriately selected from linkers generally used for labeling nucleic acids. Examples of such a linker include 1-5 base oligonucleotides. When an oligonucleotide is used as the linker, the linker of the first probe and the linker of the second probe may be the same base sequence, may be complementary to each other, or may be a random base sequence. Good.

  When the protruding end region in the partial double-stranded nucleic acid probe is modified with a labeling substance or the like, the ability of the partial double-stranded nucleic acid probe to spontaneously interact with the sample nucleic acid tends to decrease. Therefore, the position where the labeling substance is introduced into the first probe or the second probe is preferably a region where both probes form a base pair, that is, a double-stranded forming region in the partial double-stranded nucleic acid probe. In particular, when the partially double-stranded nucleic acid probe has a blunt end, it is preferable to introduce a labeling substance into the blunt end sites of the first probe and the second probe. For example, when the first probe and the second probe are labeled with a labeling substance capable of transferring energy to each other, energy transfer occurs when both are annealed (in the case of a partially double-stranded nucleic acid probe). It is necessary to label the position. By introducing a labeling substance into the blunt end site of both probes, energy transfer can be caused by the partially double-stranded nucleic acid probe.

  In the method for identifying a target base sequence of the present invention, a high-temperature treatment process in which a sample nucleic acid is denatured in advance to become a single strand is not performed. Therefore, by performing gene detection by the target base sequence identification method of the present invention, it is possible to simplify and simplify the detection reaction and its detection device system. In addition, it is possible to reduce the cost and time spent for the development of the detection device system and the like, and further improvement in the stability and reproducibility of the detection reaction is also expected.

  In particular, as will be shown in Example 2 below, the method for identifying a target base sequence of the present invention is such that a sample nucleic acid is previously made into a single strand, and then a strand displacement reaction is performed by spontaneous interaction of a partially double-stranded nucleic acid probe. It is superior in base sequence discrimination performance than the conventional method. That is, the method for identifying a target base sequence of the present invention can detect a target base sequence in a sample nucleic acid in a state of forming a double strand at a low noise level without complicated operations. When a target base sequence is identified using a partial double-stranded nucleic acid probe, the performance of base sequence discrimination can be improved compared to a nucleic acid discrimination method using an existing heat denaturation system by not performing a heat denaturation step. This is a finding first discovered by the present inventors.

  The reason why the method for identifying a target base sequence of the present invention has an excellent identification performance is not clear, but it is presumed that it is due to the reaction mechanism shown below. FIG. 1 schematically shows a nucleic acid presumed to be present in a reaction solution in one embodiment of the method for identifying a target base sequence of the present invention, as compared with a conventional method requiring a heat denaturation step. It is a figure. The upper row (“variable temperature reaction DSP”) shows the conventional method of performing hybridization by lowering the temperature of the reaction solution after the thermal denaturation step, and the lower row (“isothermal reaction DSP”) is less than 65 ° C. without performing the thermal denaturation step. The method of this invention which performs chain | strand displacement reaction at the temperature of this is shown. In the figure, “T1” and “T2” indicate each nucleic acid strand constituting the sample nucleic acid, “P1” indicates the first probe, and “P2” indicates the second probe. “P1” is composed of a base sequence complementary to the partial region of “T1”.

  In the conventional method subjected to heat denaturation treatment, the nucleic acid in the reaction solution includes the original sample nucleic acid (A), the partially double-stranded nucleic acid probe (B) in which the first probe and the second probe are annealed, It can be divided into three types of hybridization groups of two types of double-stranded nucleic acids (C) newly formed by recombination of the strands between the sample nucleic acid and the partial double-stranded nucleic acid probe. In the reaction solution, non-competitive hybridization proceeds between the single-stranded sample nucleic acid and the single-stranded partial double-stranded nucleic acid probe, and the base sequence-specific nucleic acid. Hybridization is expected to be difficult.

  On the other hand, in the method of the present invention in which heat denaturation treatment is not performed, there is no non-competitive hybridization environment, and the original sample nucleic acid (A), partial double-stranded nucleic acid probe (B), spontaneous Due to the interaction, the first probe anneals with “T1” of the original sample nucleic acid (C). Thus, not a non-competitive hybridization, but a sequence-specific reaction proceeds due to a spontaneous interaction between the partially double-stranded nucleic acid probe and the sample nucleic acid. It is thought that identification will be performed.

  Since the identification method of the present invention is excellent in the identification performance of the target base sequence, it can be suitably used for identification of gene mutations. Specifically, the base sequence of the region containing the mutation site of the gene mutation is used as the target base sequence. At this time, the target base sequence may be designed so that the site that recognizes the mutation site in the first probe is provided in a region that forms a base pair when annealed with the second probe. You may design so that it may provide in the area | region (protruding terminal area | region) which becomes a single strand when it anneals.

  In the present invention, the gene mutation means a difference in the base sequence of a gene existing between individuals of the same organism species, and the mutation site means a different site in the base sequence. Specifically, a difference in the base sequence is caused by substitution, deletion, or insertion of one or more bases in the base sequence. Examples of such gene mutations include SNP and CNV polymorphism. In addition, in the present invention, gene mutation refers to a somatic mutation that is a difference in the base sequence of a gene existing between cells in the same individual, in addition to a congenital mutation such as a gene polymorphism such as SNP. Also includes acquired mutations.

  In the identification method of the present invention, target gene mutations are called cancer-related genes, genes related to genetic diseases, genes related to drug metabolism and efficacy, viral genes, and mutations in bacterial genes. Examples of cancer-related genes include k-ras gene, BRAF gene, PTEN gene, ALK gene, EGFR gene, N-ras gene, p53 gene, BRCA1 gene, BRCA2 gene, or APC gene. Examples of genes related to genetic diseases include genes reported to be associated with various inborn errors of metabolism. Regarding drug metabolism, genes such as cytochrome P450 and transporter, which are enzymes involved in drug metabolism, can be mentioned. Examples of viral genes and bacterial genes include genes such as hepatitis C virus and hepatitis B virus. In addition, HLA, which is a human leukocyte antigen gene that is not necessarily directly related to the cause of illness, is also important in relation to transplantation compatibility and side effects of drugs. Furthermore, gene mutations encoded in mitochondria have been suggested to be associated with diseases, and mutations in these genes can also be targeted.

  The identification method of the present invention is useful in clinical examinations and the like because of its high identification accuracy and simplicity. When considering the practicality of genetic testing in the medical field, simplification of the measurement process is very important. By the identification method of the present invention, not only germ cell mutations such as SNP but also somatic mutations can be identified with high accuracy and simplicity.

  For example, K-ras is a signal transduction system protein and a proto-oncogene. It has been reported that mutations have occurred in the K-ras gene in many cancer cells. In particular, mutations involving amino acid substitutions are remarkably observed in codons 12 and 13 of the K-ras gene, and it is known that 13 types of mutation patterns exist. Recently, it has been clarified one after another that EGFR antibody drugs (cetuximab, panitumumab), etc., which are anticancer agents, cannot exert their efficacy in patients with mutations in the K-ras gene. Such anticancer drug treatment requires not only side effects but also high costs. Therefore, it has been proposed as a part of custom-made medicine to test K-ras mutation before treatment and to select and treat only effective patients.

  Also, cetuximab, which is an EGFR antibody drug, is used as a colorectal cancer therapeutic drug. The annual incidence of colorectal cancer was just under 100,000, and the number of deaths in 2005 was 40,800. There is a tendency for the EGFR antibody drug manufacturer to estimate that 40,000 colorectal cancer patients will be the target of EGFR antibody drug treatment in 4 years, due to the increasing trend of eating habits in the West. If the estimate is correct, the K-ras inspection market is expected to exceed 400 million yen in four years in Japan alone.

  However, the conventional identification method has a problem that it is difficult to quickly identify somatic mutations with sufficient accuracy, and there are many false positives. Since the identification method of the present invention can rapidly identify somatic mutations with high accuracy, it can be expected not only to improve accuracy in clinical examinations but also to reduce medical costs.

  The target base sequence identification method of the present invention can also identify a target base sequence in a sample nucleic acid in a living body by directly introducing a partially double-stranded nucleic acid probe into a cell or a living body. . For example, by modifying the first probe and the second probe in advance with a labeling substance that can be detected from outside the organism, the degree of occurrence of the strand displacement reaction in vivo can be measured in vivo.

  In addition, by introducing a partial double-stranded nucleic acid probe used in the method for identifying a target base sequence of the present invention directly into an organism and spontaneously interacting with a double-stranded nucleic acid having a target base sequence, It is also possible to arbitrarily inhibit the function of the double-stranded nucleic acid.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated concretely, this invention is not limited to the following Example. In the following Examples, a double-stranded probe (including a partial double-stranded nucleic acid probe used in the present invention) having at least one end as a protruding end is a protruding end probe, and both ends are blunt ends 2 This strand-forming probe is called a blunt end probe. Further, each probe and nucleic acid chain used in the examples were prepared by a conventional chemical synthesis method unless otherwise specified.

[Example 1]
Spontaneous interaction between a protruding end probe and a double-stranded nucleic acid having a target base sequence was examined.
Specifically, a region containing a mutation site of a gene mutation is used as a target base sequence, and a double-stranded formation comprising a protruding end probe for detecting a normal type and a base sequence of a normal allele or a mutant allele in a reaction solution Nucleic acid (oligo DNA: 49 bp) was allowed to coexist and the degree of strand displacement reaction was measured. As a control, the same measurement was performed using a blunt end probe.
The protruding end probe has a base sequence complementary to a normal type allele, the 5 ′ end is modified with a fluorescent substance Alexa Fluor 488, and the 3 ′ end is modified with an amino group. DPS-W25-1 A probe (corresponding to the first probe of the present invention) and a DPS-W21-2 probe comprising a base sequence complementary to the DPS-W25-1 probe and modified at the 3 ′ end with a quencher Black hole quencher 1 It was formed with a protruding end probe (corresponding to the second probe of the present invention). This protruding end probe is a single strand of 4 bases on the 3 ′ end side of the DPS-W25-1 probe, and the GC content in this protruding end region is 50%. The blunt end probe has a base sequence complementary to the DPS-W25-1 probe, a DPS-W25-2 probe whose 3 ′ end is modified with a quencher Black hole quencher 1, and a DPS-W25-1 Formed with a probe.
Table 1 shows the base sequence of each nucleic acid chain constituting the double-stranded nucleic acid of each probe and normal allele and mutant allele. In Table 1, “Ale488” indicates Alexa Fluor 488 labeling, “BHQ1” indicates Black hole quencher 1 labeling, and “wT49” and “rc-wT49” constitute double-stranded nucleic acids of normal alleles. “MT49” and “rc-mT49” indicate the nucleic acid strands constituting the double-stranded nucleic acid of the mutant allele, and the numbers in the right column indicate the corresponding sequence numbers in the sequence listing. In each base sequence, the underlined base is the mutation site.

  First, according to the composition described in Table 2, Sol. A1 (blunt end probe solution) and Sol. A2 (protruding end probe solution) was prepared at room temperature and allowed to stand for 15 minutes. The buffer solution and dNTP mix attached to Takara Taq (registered trademark) Hot Start Version (manufactured by TAKARA BIO) were used for the 10 × PCR buffer and 2.5 mM each dNTP described in Tables 2 and 3, respectively. In addition, “Ale594 se dye” described in Table 2 means the fluorescent substance Alexa fluor 594 succinimidyl ester (manufactured by Invitrogen). This fluorescent substance was added to correct measurement errors between wells in the measurement of fluorescence intensity.

  Sol. Apart from A1 etc., various solutions (Sol. B1-8) were prepared according to the composition described in Table 3. In Table 3, “NTC” is a negative control that does not contain a double-stranded nucleic acid. In addition, “Wt” is a normal genotype, “Ht” is a mutant hetero genotype, and “Mt” is a homozygous genotype. It is.

  Next, Sol. Equal amount (20 μL) of A1 in Sol. It added to B1-4 and the reaction solution of a total amount of 40 microliters was prepared. Similarly, Sol. Equal amount (20 μL) of A2 in Sol. It added to B5-8 and the reaction solution of a total amount of 40 microliters was prepared. These reaction solutions were set in a fluorescence detection device LightCycler-330 (manufactured by Roche) immediately after preparation, and first kept at 50 ° C./40° C. for 50 minutes (temperature control region A), The temperature is raised to 95 ° C. at a rate of temperature increase of 20 ° C./second and held for 30 seconds (temperature adjustment region B), and then lowered to 40 ° C. at a temperature decrease rate of 0.1 ° C./second and held for 30 seconds (temperature adjustment region C). Under the temperature control conditions, fluorescence time-lapse measurement was performed. The measurement with the fluorescence detection apparatus was performed with an excitation wavelength of 470 nm, a fluorescence photometry wavelength of Alexa fluor 488 of 520 nm (gain 1), and a fluorescence photometry wavelength of Alexa fluor 594 succinimidyl ester of 640 nm (gain 1).

  FIG. 2 is a diagram showing the results of performing fluorescence time-lapse measurement on each reaction solution. FIG. 2A shows the result of the reaction solution containing the blunt end probe, and FIG. 2B shows the result of the reaction solution containing the protruding end probe. In the figure, “NTC”, “Wt”, “Ht”, and “Mt” indicate the type of the double-stranded nucleic acid solution (Sol. B) added to the reaction solution, as in Table 3. . In the figure, the Y-axis represents the intensity ratio (F1 / F2) between the fluorescence (F1) of Alexa fluor 488 modified on one side of the probe and the fluorescence (F2) of Alexa fluor 594 added for the purpose of correcting the difference between wells. ). It shows that the quenching effect by Black hole quencher 1 with respect to Alexa fluor 488 is decreasing, so that F1 / F2 becomes high. That is, by monitoring this intensity ratio, it is possible to suggest the state of interaction between the double-stranded nucleic acid probe and the double-stranded nucleic acid.

  In the blunt-ended probe, no change in the fluorescence intensity ratio was observed for any genotype double-stranded nucleic acid in the temperature control region A (40 ° C. isothermal environment). On the other hand, in the protruding end probe, the mutant homozygous type (Mt) showed no change in the fluorescence intensity ratio as in the negative control (NTC) not containing the double-stranded nucleic acid, whereas the normal type ( Significant intensity ratio changes appeared in Wt) and mutant heterozygous (Ht). In particular, in the normal type (Wt), an increase in the intensity ratio approximately twice that of the mutant heterotype (Ht) was observed. From this, it can be seen that the protruding end probe interacts with the double-stranded nucleic acid even in an isothermal environment, and it is clear that the reaction is promoted in a base sequence-specific manner and has genotyping ability. became.

  By performing rehybridization of the temperature control region C after the heat denaturation treatment of the temperature control region B, it is possible to hybridize a nucleic acid having a target base sequence specifically with the probe sequence and the probe. In the temperature control region C, the fluorescence intensity ratio (genotyping ability) corresponding to the genotype of the double-stranded nucleic acid could be confirmed even with the blunt end probe. However, in view of the maximum value of the fluorescence intensity ratio in the temperature control region B, it can be seen that the reaction efficiency after the heat denaturation treatment is by no means high. This is because, in competitive hybridization of a nucleic acid chain obtained by single-stranded a relatively long double-stranded nucleic acid and a relatively short probe, long nucleic acid chains having a high Tm This is considered to be a result of hybridization with a dominant advantage.

  In contrast, with the protruding end probe, genotyping ability with extremely high reaction efficiency could be recognized in the temperature control region C. What is surprising here is that the spontaneous genotyping ability in the temperature control region A responded with high efficiency comparable to the genotyping ability in the temperature control region C.

[Example 2]
The nucleic acid sequence detection accuracy by the spontaneous interaction of the protruding end probe was investigated.
The protruding end probe is a DPS-W21 + 5A probe in which the DPS-W25-1 probe is modified with the fluorescent substance Alexa Fluor 488 via a 5-base poly A sequence, and the amino group at the 3 ′ end is removed from the modification. And the DPS-W21-2 probe was formed with a DPS-W21 + 5A probe adapted to be modified by the quencher Black hole quencher 1 via a 5-base poly A sequence. Table 4 shows the base sequences of the DPS-W21 + 5A probe and the DPS-W21 + 5A probe. In Table 4, “Ale488”, “BHQ1”, and the numbers in the right column are the same as those in Table 1.

  Specifically, first, various solutions (Sol. A1 to A3) were prepared at room temperature according to the composition described in Table 5 and allowed to stand for 15 minutes. Sol. Separately from A, various solutions (Sol. B1 to B1) were prepared according to the compositions shown in Table 6. Sol. B1 is a negative control containing no double-stranded nucleic acid. B2 represents a normal genotype that matches the protruding end probe, Sol. B3 is a double-stranded nucleic acid solution prepared by assuming a genotype of a mutant homozygous mismatch with a protruding terminal probe. In Tables 5 and 6, “10 × PCR buffer”, “Ale594 se dye”, “wT49”, “rc-wT49”, “mT49”, and “rc-mT49” are the same as those in Tables 2 and 3.

  Next, Sol. A1-3 are Sol. Each was added to B1 to B3 to prepare a total 20 μL reaction solution. These reaction solutions were set in a fluorescence detection device LightCycler-330 (manufactured by Roche) immediately after preparation, and first kept at 20 ° C./second for 40 minutes (temperature control region A). The temperature is raised to 55 ° C. at a rate of temperature increase of 20 ° C./s and held for 20 minutes (temperature control region B), further increased to 95 ° C. at a rate of temperature increase of 20 ° C./s and maintained for 30 seconds (temperature control region C). The fluorescence time-lapse measurement was performed under the temperature control condition of decreasing to 40 ° C. at a temperature decrease rate of 20 ° C./second and holding for 20 minutes (temperature control region D). The measurement using the fluorescence detection apparatus was performed with the excitation wavelength of 470 nm, the fluorescence photometry wavelength of Alexa fluor 488 of 520 nm (gain 1), and the fluorescence photometry wavelength of Alexa fluor 594 succinimidyl ester of 640 nm (gain 15).

  FIG. 3 is a diagram showing the results of performing fluorescence time-lapse measurement on each reaction solution. As a result, the S / N ratio in the temperature control region A was 12, the S / N ratio in the temperature control region B was 11, and the S / N ratio in the temperature control region D was 3. From these results, the partial double-stranded nucleic acid probe is not subjected to denaturation treatment, compared to the case where the sample nucleic acid is subjected to heat denaturation treatment at 95 ° C. or the like to be single-stranded and then interacted (temperature control region D). It was clarified that the target base sequence can be identified with a higher S / N ratio by interacting with the sample nucleic acid in the state of forming a double strand (temperature control regions A and B).

[Example 3]
We investigated quantitative detection of target nucleic acids by spontaneous interaction of protruding end probes.
Specifically, genomic DNA is extracted from cell line whose genomic DNA is normal type and cell line whose genomic DNA is mutant type, respectively, and PCR is performed using this as a template. A plurality of sample nucleic acid solutions having different contents from the mutant-type double-stranded nucleic acid are prepared, and the extent of the strand displacement reaction is determined by using these sample nucleic acid solutions and a protruding end probe for detecting a normal type. It was measured. NA17247 cell line was used as a normal type, and NA17222 cell line was used as a mutant type. Each cell line was obtained from the Coriell cell bank.

  First, each PCR solution was prepared so that it might become the composition of Table 7. In Table 7, “NA17247 (W) 100%” means that the genomic DNA contained in the PCR solution is all genomic DNA derived from NA17247, and “NA17222 (M) 100%” It means that all genomic DNA contained in the DNA is genomic DNA derived from NA17247, and “NA17247 (W) / 222 (M) X%” is the genomic DNA contained in the PCR solution, This means that X% of genomic DNA derived from NA17247 is contained. In Table 7, “10 × PCR buffer” and “2.5 mM each dNTP” are the same as those in Table 2. Furthermore, “Hs Taq TAKARA” indicates Taq polymerase attached to Takara Taq (registered trademark) Hot Start Version (manufactured by TAKARA BIO).

  The prepared PCR solution was set in LightCycler-330 (manufactured by Roche), and PCR was performed. PCR reaction conditions were 95 ° C. for 3 minutes, followed by 45 cycles of 95 ° C. (30 seconds) → 68 ° C. (30 seconds) as one cycle. In addition, the temperature increase rate and temperature decrease rate during PCR were both 20 ° C./second. Table 8 shows the base sequences of the F33 primer and R30 primer. In Table 8, the numbers in the right column are the same as those in Table 1. By PCR using these primers, double-stranded nucleic acid solutions containing PCR products (197 bp) of both the normal gene sequence region and the mutant gene sequence region in different quantitative ratios are obtained.

  Apart from PCR, Sol. A (protruding end probe solution) was prepared at room temperature and allowed to stand for 15 minutes. “DPS-W25-1” and “DPS-W21-2” in Table 9 are the same as those in Table 2.

  Subsequently, Sol. A reaction solution was prepared by dispensing 1.5 μL each of A. Immediately after preparation, these reaction solutions are set in a fluorescence detector LightCycler-330 (manufactured by Roche), first kept at 40 ° C. at a temperature rising rate of 20 ° C./second for 40 minutes (temperature control region A), and then The temperature is raised to 50 ° C. at a temperature rising rate of 20 ° C./second and held for 40 minutes (temperature control region B), and further heated to 60 ° C. at a temperature rising rate of 20 ° C./second and held for 40 minutes (temperature control region C). The temperature is increased to 95 ° C. at a rate of 20 ° C./second and held for 30 seconds (temperature control region D), and then lowered to 40 ° C. at a temperature decrease rate of 0.1 ° C./second and held for 30 seconds (temperature control region E). The fluorescence time-lapse measurement was performed under the adjusting conditions. In addition, the measurement by a fluorescence detection apparatus was performed by setting the excitation wavelength to 470 nm, the fluorescence photometry wavelength of Alexa fluor 488 to 520 nm (gain 100), and the fluorescence photometry wavelength of Alexa fluor 594 succinidine ester to 640 nm (gain 100).

  FIG. 4 is a diagram showing the results of performing fluorescence time-lapse measurement on each reaction solution. As a result, both a decrease in the fluorescence intensity ratio and an increase in the fluorescence intensity ratio were observed from the temperature adjustment region A to B and in the aging process of the temperature adjustment region B. However, in the temperature control regions A to C, when the equilibrium was reached, the fluorescence intensity ratio correlated with the normal gene content ranking was shown (the lower the normal sequence base content, the lower the fluorescence intensity ratio). ) In addition, genotyping by rehybridization is shown from the temperature control region D to E, and the fluorescence intensity ratio depending on the content of the normal sequence base could be confirmed even in this temperature control region. The result of the temperature control region E was similar to the fluorescence intensity ratio pattern of the previous temperature control region B. From these results, it was clarified that even when a PCR product was used as a sample nucleic acid, a target base sequence could be identified using a strand displacement reaction based on a spontaneous interaction of a double-stranded nucleic acid probe.

  FIG. 5 shows the relationship between the fluorescence intensity ratio and the mutant gene-containing concentration at one time point in the temperature control region B (sampling point: 80 minutes after the start of the reaction). As a result, a high correlation with an R-square value of 0.9993 is obtained, and the interaction between the double-stranded nucleic acid probe and the sample nucleic acid in a double-stranded state exhibits high quantitativeness. Became clear.

[Example 4]
Genotyping of genomic DNA using a protruding end probe was examined. Specifically, a protruding end probe was added in advance to the reaction solution of the genomic DNA amplification reaction, and genomic DNA genotyping was attempted in a homogeneous system.
In addition to the NA17247 cell line (normal type) and NA17222 cell line (mutant type) used in Example 3, the genomic DNA was NA17204 cell line (normal type), NA17252 cell line (mutant heterotype), and NA17266 cell. DNA extracted from line (mutant heterotype) was used. Each cell line was obtained from the Coriell cell bank.

  First, each PCR solution was prepared so that it might become the composition of Table 10. “DPS-W25-1” and “DPS-W21-2” in Table 10 are the same as in Table 2, and “F33” and “R30” are the same as in Table 9. The prepared PCR solution was set in LightCycler-330 (manufactured by Roche), PCR was performed under the same conditions as in Example 3 (temperature control region A), and subsequently lowered to 40 ° C. at a temperature decrease rate of 4 ° C./second. Hold for 10 minutes (temperature control region B), then hold at 10 ° C. with a temperature increase rate of 4 ° C./s and hold for 10 minutes (temperature control region C), and further increase to 50 ° C. with a temperature increase rate of 4 ° C./s and hold for 10 minutes (Temperature control region D), and finally, the temperature was raised to 55 ° C. at a temperature increase rate of 4 ° C./second and held for 10 minutes (temperature control region E). In all steps of the temperature control region A to E, fluorescence time measurement was performed. The measurement with the fluorescence detection apparatus was performed with the excitation wavelength of 470 nm, the fluorescence photometry wavelength of Alexa fluor 488 of 520 nm (gain 15), and the fluorescence photometry wavelength of Alexa fluor 594 succinimidyl ester of 640 nm (gain 15).

FIG. 6 is a diagram showing the results of performing fluorescence time-lapse measurement on each reaction solution. Immediately after the end of PCR (immediately after the temperature control region A), the PCR product (corresponding to the sample nucleic acid of the present invention) is in a double-stranded state, and then maintained in an isothermal environment (temperature control region B). Genotyping was attempted using the spontaneous interaction of the protruding end probe. As a result, in the process of transition from the temperature control region B to E after PCR, the protruding end probe shows a high fluorescence intensity ratio in the normal type gene (NA17204, NA17247), and in the mutant heterogeneous gene (NA17252, NA17266). An intermediate value of the fluorescence intensity ratio between the normal type and the mutant homotype was shown. In particular, in the temperature control region D (50 ° C.), the fluorescence intensity ratio tends to converge for each genotype, and the high genotyping ability of the protruding end probe could be confirmed.
In the temperature control region A, a remarkable difference in fluorescence intensity ratio was observed between NTC1 (negative control 1) not containing Taq polymerase and NTC2 (negative control 2) not containing genomic DNA. It has been found that this is due to the presence or absence of the polymerase enzyme.

[Example 5]
The GC content and spontaneous interaction in the protruding end region of the protruding end probe were examined.
Specifically, a region containing a mutation site of a genetic mutation different from the genetic mutation detected in Example 1 having a normal type (G12G) and two types of mutant types (G12D and G12V) is used as a target base sequence. In the reaction solution, a protruding end probe for detecting a normal type and a double-stranded nucleic acid (oligo DNA: 54 bp) consisting of the base sequence of each genotype allele are coexisted to measure the degree of strand displacement reaction. did.

As the protruding terminal probes, four types of types 1 to 4 were used.
The Type 1 probe is formed by a G12G / LP25 probe and a G12G / SP21 probe, and the 4 bases on the 3 ′ end side of the G12G / LP25 probe are single-stranded, and the GC content in this protruding end region is 50%. is there.
Type 2 probes are G12G / LP25 probe and G12G / SP17_V. 3 probes, 6 bases on the 3 ′ end side and 2 bases on the 5 ′ end side of the G12G / LP25 probe are single-stranded, and the GC content in the protruding end region on the 3 ′ end side is 50 %.
The Type 3 probe is G12G / LP21_V. 3 probes and G12G / SP17_V. 3 probes, and G12G / LP21_V. The 4 bases on the 3 ′ end side of 3 probes are single-stranded, and the GC content in this protruding end region is 75%.
The Type 4 probe is G12G / LP25_V. 2 probes and G12G / SP21_V. 2 probes, G12G / LP25_V. The 4 bases on the 3 ′ end side of 2 probes are single-stranded, and the GC content in this protruding end region is 75%.
G12G / LP25 probe, G12G / LP21_V. 3 probes, and G12G / LP25_V. In each of the two probes, the 5 ′ end is modified with a fluorescent substance Alexa Fluor 488, and the 3 ′ end is modified with an amino group. On the other hand, G12G / SP21 probe, G12G / SP17_V. 3 probes, and G12G / SP21_V. In both probes, the 3 ′ end is modified with a quencher Black hole quencher 1.
Table 11 shows the base sequence of each nucleic acid chain constituting the double-stranded nucleic acid of each probe and each genotype allele. In Table 11, “Ale488”, “BHQ1”, and the numbers in the right column are the same as those in Table 1. “T54-G12G” and “rcT54-G12G” indicate nucleic acid chains constituting the double-stranded nucleic acid of the normal type (G12G) allele, and “T54-G12D” and “rcT54-G12D” are mutant types (G12D). ) Nucleic acid strands constituting the double-stranded nucleic acid of the allele, and “T54-G12V” and “rcT54-G12V” represent the nucleic acid strands constituting the double-stranded nucleic acid of the mutant (G12V) allele. In each base sequence, the underlined base is the mutation site.

  First, according to the composition described in Table 12, various solutions (Sol. A1 to 16) were prepared at room temperature and left for 15 minutes. Sol. Separately from A1 etc., various solutions (Sol. B1-16) were prepared according to the composition described in Table 13. In Tables 12 and 13, “10 × PCR buffer”, “2.5 mM each dNTP”, and “Ale594 se dye” are the same as in Table 2.

  Next, Sol. A1-16 is Sol. B1 to 16 were added to prepare a reaction solution having a total amount of 20 μL. These reaction solutions were set on a fluorescence detection device LightCycler-330 (manufactured by Roche) immediately after preparation, and first maintained at 40 ° C. at a temperature increase rate of 20 ° C./second for 30 minutes (temperature control region A). Hold at 30 ° C. for 30 minutes at a temperature increase rate of 20 ° C./second (temperature control region B), then maintain at 30 ° C. for 30 minutes at a temperature increase rate of 20 ° C./second (temperature control region C), then increase the temperature The temperature is maintained at 55 ° C. for 30 minutes at a rate of 20 ° C./second (temperature control region D), and then maintained at 60 ° C. for 30 minutes at a temperature increase rate of 20 ° C./second (temperature control region E). The temperature was raised to 95 ° C. at a rate of 95 ° C./second and held for 1 minute (temperature control region F), then lowered to 40 ° C. at a temperature drop rate of 0.1 ° C./second and held for 1 minute (temperature control region G). After increasing to 95 ° C. at a temperature rate of 20 ° C./second and holding for 1 minute (temperature control region H), Down to the 40 ° C. at a rate of temperature 20 ° C. / sec to 1 minute hold at (temperature control region I) of temperature control conditions, it was subjected to fluorescence measured over time. The measurement using the fluorescence detection apparatus was performed with the excitation wavelength of 470 nm, the fluorescence photometry wavelength of Alexa fluor 488 of 520 nm (gain 1), and the fluorescence photometry wavelength of Alexa fluor 594 succinimidyl ester of 640 nm (gain 15).

  FIG. 7 is a diagram showing the results of performing fluorescence time-lapse measurement on each reaction solution. FIGS. 7A to 7D show the results using Type 1-4 probes, respectively. In each figure, the base sequence of the protruding end probe used is shown. In the base sequence, bold letters are mutation sites, and bases surrounded by squares are protruding end regions in the probe.

  As a result, the S / N ratio was higher in the case of using the Type 4 probe (FIG. 7D) than in the Type 1 probe (FIG. 7A), and there was a significant difference in the spontaneous strand displacement efficiency. Here, in the Type 1 probe, the GC content of the double-stranded forming region of the protruding end probe is 57%, the GC content of the protruding end region is 50%, and the G12G / LP25 probe having a longer base length (the first of the present invention) The GC content (corresponding to the probe) is 56%. On the other hand, in the Type 4 probe, the GC content of the double-stranded forming region of the protruding end probe is 52%, the GC content of the protruding end region is 75%, and G12G / LP25_V. The GC content of 2 probes (corresponding to the first probe of the present invention) is 56%. That is, the Type 1 probe and the Type 4 probe have the same GC content in the nucleic acid chain corresponding to the first probe of the present invention. Nevertheless, since the type 4 probe has higher base sequence discrimination performance, the GC content ratio in the protruding end region contributes to the spontaneous strand displacement efficiency of the partially double-stranded nucleic acid probe. It was speculated that the higher the GC content ratio, the higher the spontaneous strand displacement efficiency.

  Further, in the Type 4 probe (FIG. 7D), the mutation discrimination accuracy (S / N ratio) using the spontaneous strand displacement reaction at 40 ° C. (temperature control region A) before heat denaturation treatment was 7-12. On the other hand, the mutation discrimination accuracy (S / N ratio) by rehybridization at 40 ° C. (temperature control region G and temperature control region I) after heat denaturation treatment was 2 or less, both of which were low. From these comparisons, it was found that the base mutation can be identified with higher accuracy by using the spontaneous strand displacement reaction without performing the heat denaturation treatment.

[Example 6]
The chain length ratio of two nucleic acid strands constituting the protruding end probe and the spontaneous interaction were examined.
Specifically, the region containing the mutation site of the same gene mutation as that detected in Example 1 is used as the target base sequence, and the protruding end probe for detecting the normal type and the base of the allele of each genotype in the reaction solution The degree of occurrence of strand displacement reaction was measured by coexisting with a double-stranded nucleic acid (oligo DNA: 150 bp) comprising a sequence.

As the protruding terminal probes, six types of Type 1 to 6 were used.
The Type 1 probe is formed by the DSP-W25-1 probe and the DSP-W21-2 probe used in Example 1, and 4 bases on the 3 ′ end side of the DSP-W25-1 probe are single-stranded, The GC content in this protruding end region is 50%.
The Type 2 probe is formed by the DSP-W25-1 probe and the DSP-W17-2 probe used in Example 1, and includes 4 bases on the 3 ′ end side and 4 on the 5 ′ end side of the DSP-W25-1 probe. The base content is single-stranded, the GC content in the protruding end region on the 3 ′ end side is 50%, and the GC content in the protruding end region on the 5 ′ end side is 75%.
The Type 3 probe is formed by the DSP-W25-1 probe and the DSP-W16-2 probe used in Example 1, and the 9 bases on the 3 ′ end side of the DSP-W25-1 probe are single-stranded, The GC content in this protruding end region is 44%.
The Type 4 probe is formed of a DSP-W25-LNA probe and a DSP-W21-2 probe, and the 4 bases on the 3 ′ end side substituted with LNA are single-stranded, and the GC in this protruding terminal region The content is 50%. The DSP-W25-LNA probe is obtained by replacing the 4 bases on the 3 ′ end side of the DSP-W25-1 probe used in Example 1 with LNA.
The Type 5 probe is formed by a DSP-W40-1 probe and a DSP-W36-2 probe, and the 4 bases on the 3 ′ end side of the DSP-W40-1 probe are single strands. The content is 50%.
The Type 6 probe is formed by a DSP-W16-1 probe and a DSP-W12-2 probe, and the 4 bases on the 3 ′ end side of the DSP-W16-1 probe are single strands. The content is 75%.

The DSP-W25-1 probe, DSP-W25-LNA probe, DSP-W40-1 probe, and DSP-W16-1 probe are all modified at the 5 ′ end with the fluorescent substance Alexa Fluor 488, and 3 ′. The terminal is modified with an amino group. On the other hand, the DSP-W21-2 probe, the DSP-W17-2 probe, the DSP-W16-2 probe, the DSP-W36-2 probe, and the DSP-W12-2 probe all have a quenching substance Black hole at the 3 ′ end. It has been modified by quencher 1.
Table 14 shows the base sequences of the nucleic acid strands constituting the double-stranded nucleic acid of each probe (excluding those listed in Table 1) and alleles of each genotype. In Table 14, “Ale488”, “BHQ1”, and the numbers in the right column are the same as those in Table 1. In addition, “wT150” and “rc-wT150” indicate nucleic acid strands constituting the double-stranded nucleic acid of the normal allele, and “mT150” and “rc-mT150” indicate the double-stranded nucleic acid of the mutant allele. The nucleic acid strand which comprises is shown. In each base sequence, the underlined base is the mutation site.

  First, according to the composition described in Tables 15 and 16, various solutions (Sol. A1 to 18) were prepared at room temperature and left for 15 minutes. Sol. Apart from A1 etc., various solutions (Sol. B1-18) were prepared according to the compositions described in Tables 17 and 18, and allowed to stand for 15 minutes. In Tables 15 to 18, “10 × PCR buffer” and “Ale594 se dye” are the same as those in Table 2.

  Next, Sol. A1-18 is Sol. B1 to 18 were added to prepare a reaction solution having a total volume of 20 μL. These reaction solutions were set in a fluorescence detection device LightCycler-330 (Roche) immediately after preparation, and first maintained at 40 ° C. at a temperature increase rate of 20 ° C./second for 15 minutes (temperature control region A). Maintained for 15 minutes at a temperature increase rate of 20 ° C./s and maintained at 45 ° C. for 15 minutes (temperature control region B), then maintained at a temperature increase rate of 20 ° C./s for 50 minutes and maintained for 15 minutes (temperature control region C). The temperature is maintained at 55 ° C. for 15 minutes at a rate of 20 ° C./second (temperature control region D), then is maintained at 60 ° C. for 15 minutes at a temperature increase rate of 20 ° C./second (temperature control region E), and then the temperature increase rate is 20 The temperature is raised to 65 ° C. at 65 ° C./second and held for 15 minutes (temperature adjustment region F), then raised to 95 ° C. at a temperature increase rate of 20 ° C./second and held for 1 minute (temperature adjustment region G). The temperature is adjusted to 40 ° C./second and held for 15 minutes (temperature adjustment region H). In the matter, it was subjected to fluorescence measured over time. The measurement using the fluorescence detection apparatus was performed with the excitation wavelength of 470 nm, the fluorescence photometry wavelength of Alexa fluor 488 of 520 nm (gain 1), and the fluorescence photometry wavelength of Alexa fluor 594 succinimidyl ester of 640 nm (gain 15).

  FIG. 8 is a diagram showing the results of performing fluorescence time-lapse measurement on each reaction solution. 8A to F show the results using Type 1 to 6 probes, respectively. In each figure, the base sequence of the protruding end probe used is shown. In the base sequence, the bold letters are mutation sites, the bases enclosed in squares are the protruding end regions in the probe, and the bases marked with “L” in the upper left are LNAs.

  The type 1 probe and the type 2 probe have the same base length of the long chain probe, but are different in the base length of the short chain probe. Comparing these results, the reaction efficiency of the strand displacement reaction was higher when the Type 2 probe (FIG. 8B) was used than the Type 1 probe (FIG. 8A), but the S / N ratio was higher than that of the Type 2 probe. It was higher when the probe was used. From these results, the ratio of the base lengths of the first probe (long chain) and the second probe (short chain) ([base length of the second probe] / [base length of the first probe]) is determined as the reaction efficiency and base. This is considered to be one of the factors governing the discriminating ability of the sequence. The larger the base length ratio, the higher the discriminating ability of the base sequence, but the lower the spontaneous strand displacement efficiency, and conversely the smaller the base length ratio (0 Less than .5), the discriminating ability was lowered, but the spontaneous strand displacement efficiency tended to be increased.

  In addition, almost the same results were obtained with the Type 1 probe (FIG. 8A) and the Type 4 probe (FIG. 8D). Therefore, even when the nucleic acid in the protruding end region of the partial double-stranded nucleic acid probe is replaced with a nucleic acid analog such as LNA, the reaction efficiency and base sequence of the spontaneous strand displacement reaction is almost the same as that of the probe before substitution. It was found to show discriminative ability.

  The method for identifying a target base sequence according to the present invention identifies a target base sequence with high accuracy in a simple reaction system with a simple reagent configuration and without using a device having high heat resistance (eg, 90 ° C. or higher). Because it can be used, it can be used in fields such as clinical gene diagnosis business.

Claims (10)

A method for identifying a target base sequence, comprising:
In the reaction solution, the sample nucleic acid in the state of forming a double strand and the partially double-stranded nucleic acid probe are allowed to coexist, and the degree of the strand displacement reaction is measured to determine the base in the sample nucleic acid. An identification step for identifying the identity between the sequence and the target base sequence,
The double-stranded nucleic acid probe is a first probe that is a single-stranded nucleic acid comprising a base sequence complementary to a target base sequence, and a single-stranded nucleic acid comprising a base sequence complementary to the first probe. Consisting of two probes,
The ratio of the base length of the second probe to the base length of the first probe ([base length of the second probe] / [base length of the first probe]) is 0.5 or more and less than 1.
The method for identifying a target base sequence, wherein the temperature of the reaction solution is 40 ° C or higher and lower than 65 ° C.   The method for identifying a target base sequence according to claim 1, wherein the temperature of the reaction solution is in the range of 40 to 60 ° C.   The method for identifying a target base sequence according to claim 1 or 2, wherein the GC content in the protruding end region of the first probe is 50% or more.   The method for identifying a target base sequence according to any one of claims 1 to 3, wherein the 3 'ends of the first probe and the second probe are modified with an inhibitor of elongation reaction by polymerase.   The method for identifying a target base sequence according to claim 1, wherein at least one of the first probe and the second probe includes a nucleic acid analog.   The method for identifying a target base sequence according to any one of claims 1 to 5, wherein the first probe and the second probe are labeled with different types of labeling substances, respectively.   The method for identifying a target base sequence according to claim 6, wherein the first probe or the second probe is labeled with a labeling substance in a double-stranded forming region in the partial double-stranded nucleic acid probe. .   8. The method for identifying a target base sequence according to claim 6, wherein energy transfer is possible between a labeling substance that labels the first probe and a labeling substance that labels the second probe.   The target base sequence identification method according to claim 6 or 7, wherein either one of the first probe and the second probe is labeled with a labeling substance that can bind to a solid phase carrier.   The method for identifying a target base sequence according to claim 1, wherein the sample nucleic acid is a nucleic acid amplified by a nucleic acid amplification reaction.

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