JP2009232778A - Method for detecting variation in target nucleic acid, and array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for quickly detecting variations in a target nucleic acid. <P>SOLUTION: The method uses (a) a query probe having a capture sequence hybridizing with a capture probe and a query sequence complementary to a base sequence adjacent to a specific site in the target nucleic acid in a manner that a nucleotide species complementary to a nucleotide species adjacent to a specific site of the target nucleic acid is present at the 3' terminal and (b) a labeled nucleotide having a query nucleotide complementing a nucleotide species of a specific site in the target nucleic acid and having a labeled element to each mutation constituting base relating to the variation in the target nucleic acid and possibly detectable in a specific site. Variations can be detected quickly by using these components. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a technique for detecting a mutation on a gene containing single nucleotide polymorphisms in a target nucleic acid.

  It has been found that genetic mutations including SNPs in humans and the like are related not only to constitution and genetic diseases, but also to the prevalence of specific diseases, disease diagnosis, prognosis of treatment, selection of drugs and treatments, and the like. A plurality of SNPs and gene mutations are usually related, and in order to promote personalized medicine, it is required to comprehensively detect or quantify as many SNPs and mutations as possible.

  Therefore, a method using DNA computer technology has been proposed as a method for efficiently detecting or quantifying SNP or the like (Non-patent Document 1). In this method, as shown in FIG. 6, a solid phase carrier holding a number of capture probes having different sequences is prepared in advance. Two kinds of 5 ′ query probes having a tag sequence for labeling and a query sequence complementary to a specific partial sequence containing a specific base of SNP, and a query sequence complementary to a common partial sequence adjacent to the specific partial sequence And a 3 ′ query probe having an identification tag sequence complementary to the capture. The tag sequences 1 and 2 for labeling of the two kinds of 5 'query probes are formed so that the respective query sequences can be distinguished, for example, capable of labeling with different labeling substances such as Cy3 and Cy5. Further, the identification sequence is complementary to the capture probe.

  In this method, as shown in FIG. 6, first, a target nucleic acid in a nucleic acid in a test sample is amplified by multiplex PCR or the like (amplification step). Next, the 5 'query probe and the 3' query probe are ligated by a hybridization reaction and a ligation reaction of the PCR amplification product, the 5 'query probe, and the 3' query probe (ligation / coding step). Next, labeling is performed by PCR using the tag sequence for labeling of the obtained linking molecule (labeling step). By such ligation and labeling, a linking molecule having a labeling substance associated with a specific base of SNP and having one kind of identification tag sequence is obtained. Further, the labeled linking molecule is detected by an array in which capture probes complementary to the identification tag sequence are immobilized (detection step).

  In this method, when a linking molecule having two different labeling substances is hybridized to one capture probe via an identification tag sequence, the SNP type (based on a plot of signal intensity based on each labeling substance ( 2 types of homo and 1 type of hetero) are detected.

Analytical Biochemistry 364 (2007) 78-85

  The method described in Non-Patent Document 1 includes a base sequence set independently of a partial sequence containing a specific base without preparing a solid phase carrier on which a capture probe corresponding to the specific partial sequence in a target nucleic acid is immobilized. There is an advantage that a universal solid-phase carrier on which a capture probe having s is immobilized can be used. In addition, since two types of base substitutions constituting the SNP are detected with one capture probe using two types of labeling substances, there is also an advantage that a large number of SNPs can be comprehensively detected.

  However, in the above method, many steps are required to prepare a sample used in the detection process, and at least a dozen hours are required as a whole. Moreover, in order to suppress the influence which the enzyme used at a connection / encoding process and a labeling process has on a detection result, it was necessary to carry out from the connection / encoding process to the detection process (about 7 hours) on the same day. For this reason, in consideration of daily work time, the amplification process and the concatenation / coding process must be performed on different days.

  Furthermore, in the above method, SNP type discrimination must be based on signal intensity plots, and if there is a difference in fading between labeled substances, the accuracy of SNP type discrimination may not be sufficiently secured. It can be.

  Accordingly, an object of the present invention is to provide a method for rapidly detecting or quantifying a mutation in a target nucleic acid, a probe set for the method, and the like. Another object of the present invention is to provide a method for detecting or quantifying mutations with higher accuracy and a probe set for the method.

  The present inventors considered that it is important to simultaneously realize a ligation / coding step and a labeling step in order to rapidly detect a mutation such as a single nucleotide polymorphism in a target nucleic acid, and conducted various studies. As a result, a nucleotide sequence that is complementary to a capture sequence previously hybridized with a capture probe provided on a solid phase carrier and a base sequence adjacent to the mutation site and complementary to the nucleotide species adjacent to the mutation site is located at the 3 ′ end. Encoding and labeling at once by using a query probe comprising a query sequence having a query nucleotide having a nucleotide species complementary to the nucleotide species of the mutation site in the target nucleic acid at the 5 ′ end and a labeling element The knowledge that can be realized. The present invention provides the following means based on these findings.

[1] A method for detecting a mutation in a target nucleic acid using a plurality of types of capture probes immobilized on a solid support,
The following (a) and (b) for each mutated base that may be detected at a specific site for the mutation in the target nucleic acid:
(A) a capture sequence that hybridizes with the capture probe and a complementary query sequence to a base sequence adjacent to a specific site in the target nucleic acid, and a nucleotide species adjacent to the specific site in the target nucleic acid A query probe comprising at the 3 ′ end a nucleotide species to be
(B) a labeled nucleotide having a query nucleotide that complements the nucleotide species at the specific site in the target nucleic acid and comprising a labeling element;
The following steps (1) to (3):
(1) A linking step for hybridizing the target nucleic acid and the query probe for each of the mutated bases, and linking a labeled nucleotide to the 3 ′ end of the query probe to obtain a linking molecule;
(2) a contact step in which the linking molecule obtained for each of the mutation-constituting bases and the capture probe are brought into contact with each other so as to be hybridizable on the solid phase carrier;
(3) a signal information acquisition step of acquiring signal information based on the labeling element for each capture probe on the solid phase carrier;
A detection method is carried out.
[2] The detection method according to [1], wherein the labeled nucleotide has only the query nucleotide as a nucleotide species, and is formed such that a ligation reaction is terminated by the query nucleotide.
[3]
The query probe comprises a capture sequence that hybridizes with a different capture probe for each of the mutated bases,
The labeled nucleotides comprise mutually identical labeling elements,
The detection method according to [1] or [2].
[4] The presence or absence of the mutation or the ratio of the constituent mutant bases is expressed by the following formula:
[Ratio of signal intensity obtained for a capture probe hybridized with a linking molecule obtained for one of the mutation-constituting bases] / [Signal obtained for a capture probe hybridized with all ligation molecules obtained for the mutation Sum of strength]
The detection method according to [3], wherein detection is performed based on.
[5] Regarding the mutation,
The query probe comprises a capture sequence that hybridizes with the same capture probe between the mutated bases,
The labeled nucleotide comprises a different labeling element for each of the mutated bases.
The detection method according to [1] or [2].
[6] Any one of [1] to [5], wherein the contacting step includes supplying a mixture of each linking molecule in the linking step performed for each of the mutated bases onto one solid phase carrier. The detection method according to.
[7] In detecting a plurality of mutations for one or more types of target nucleic acids, the steps (1) to (3) are collectively performed for each of the plurality of mutations. [1] to The detection method according to any one of [6].
[8] The detection method according to [7], wherein the plurality of target nucleic acids are prepared by multiplex PCR for nucleic acids in a specimen.
[9] A kit for detecting a mutation in a target nucleic acid using a plurality of types of capture probes immobilized on a solid phase carrier,
The following (a) and (b) for each mutated base that may be detected at a specific site for the mutation in the target nucleic acid:
(A) a capture sequence that hybridizes with the capture probe and a complementary query sequence to a base sequence adjacent to a specific site in the target nucleic acid, and a nucleotide species adjacent to the specific site in the target nucleic acid A query probe comprising at the 3 ′ end a nucleotide species to be
(B) a labeled nucleotide having a query nucleotide that complements the nucleotide species at the specific site in the target nucleic acid and comprising a labeling element;
A kit comprising:
[10] A kit for detecting a mutation in a target nucleic acid,
A solid support comprising a plurality of capture probes;
The following (a) and (b) for each mutated base that may be detected at a specific site for the mutation in the target nucleic acid:
(A) a capture sequence that hybridizes with the capture probe and a complementary query sequence to a base sequence adjacent to a specific site in the target nucleic acid, and a nucleotide species adjacent to the specific site in the target nucleic acid A query probe comprising at the 3 ′ end a nucleotide species to be
(B) a labeled nucleotide having a query nucleotide that complements the nucleotide species at the specific site in the target nucleic acid and comprising a labeling element;
A kit comprising:

  Since the present invention uses the query probe and the labeled nucleotide as described above, the query probe and the target nucleic acid are hybridized, and the labeled nucleotide is linked to the query lobe, thereby detecting a mutated base in the target nucleic acid. Thus, it is possible to obtain a linking molecule that can be hybridized with a capture probe having a predetermined sequence on a solid phase carrier and that has a labeling element at once. By using this linking molecule as a hybridized sample to be supplied onto the solid phase carrier and acquiring signal intensity information based on the labeling element provided in the linking molecule, the presence or absence and the ratio of the mutated base can be detected. Therefore, the conventional connection / coding process and labeling process can be omitted / simplified, and the detection process can be reached quickly.

  Furthermore, by using a query probe having a capture sequence that hybridizes with a different capture probe for each mutated base and a labeled nucleotide that uses the same identification element between the mutated bases, the resulting linking molecule is mutated base Capture sequences that can be identified by different capture probes on the solid phase carrier according to the type of the linking molecule and the same identification element as the linking molecule according to the other mutated base. For this reason, it is possible to suppress the variation between the label elements and improve the qualitative detection accuracy and the quantification degree.

  In the present specification, “nucleic acid” refers to all DNA and RNA including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA and synthetic RNA, peptide nucleic acid, morpholino nucleic acid, methylphosphonate nucleic acid, and Includes artificially synthesized nucleic acids such as S-oligonucleic acid. Moreover, it may be single-stranded or double-stranded. Further, in this specification, the “target nucleic acid” refers to an arbitrary nucleic acid having an arbitrary sequence and a single-stranded state having a mutation to be actually detected. Usually, in many organisms, chromosomal DNA is a double-stranded DNA consisting of complementary strands. In the present invention, either the sense strand (coding strand) or the antisense strand (template strand) can be detected. Good. The target nucleic acid is typically a genetic index in humans and non-human animals such as constitution, genetic disease, onset of specific diseases such as cancer, disease diagnosis, treatment prognosis, drug and treatment selection, etc. Examples include nucleic acids that may have a base sequence. Examples of the index include polymorphisms such as SNP and congenital or acquired mutations. Further, nucleic acids derived from microorganisms such as pathogenic bacteria and viruses are also included in the target nucleic acid.

  As the target nucleic acid, the sample described later or its nucleic acid fraction can be used as it is, but preferably all of the plurality of target nucleic acids were amplified by PCR amplification reaction, more preferably multiplex PCR amplification reaction. It is preferable to use an amplification product.

  As used herein, “sample” refers to a sample that may contain a target nucleic acid. Samples include cells, tissues, blood, urine, saliva and the like, and any sample containing nucleic acid can be used. Fractions containing nucleic acids from these various samples can be obtained by those skilled in the art with reference to conventional techniques as appropriate.

  As used herein, “mutation” refers to a change including any one or more of substitution, deletion, insertion and addition of one or more bases on a gene. “Mutation” is used in a broad sense including single nucleotide polymorphisms and other polymorphisms. “Polymorphism” typically includes a single nucleotide polymorphism. As used herein, “mutant constituent base” refers to a base constituting a normal type and a mutant type in one mutation. For example, in the single nucleotide polymorphism in which A is substituted with G, there are two types of “mutant constituent bases”.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate.

[Method for detecting mutation or polymorphism in target nucleic acid]
The detection method of the present invention comprises a linking step for linking a query probe and a labeled nucleotide, a contacting step for bringing a linking molecule and a capture probe into contact, and a signal intensity information acquisition step. The detection method of the present invention applies to one or more target nucleic acids, and a specific partial sequence relating to a mutation in these target nucleic acids is to be detected. Hereinafter, a series of steps for detecting one mutation for one kind of target nucleic acid will be described. However, the following steps are also applied to a case where a plurality of mutations in a plurality of target nucleic acids are detected simultaneously. In addition, the following steps are applied to individual mutations when detecting a plurality of mutations in one target nucleic acid.

  1A and 1B show an outline of the detection method of the present invention, FIG. 2 shows an outline of an example of the probe set used in the present invention, and FIG. 3 shows another probe set used in the present invention. An outline of an example is shown, and FIG. 4 is an explanatory diagram showing the melting temperature of the tag sequence and the capture probe. In addition, FIGS. 1-3 is an example about the detection method and kit for detecting SNP. Hereinafter, first, a probe used in the detection method of the present invention will be described, and then each step will be described.

(Query probe)
The query probe has a query sequence on the 3 ′ side, and can have a complementary tag sequence on the 5 ′ side so as to be hybridizable to the capture probe.

(Query array)
As shown in FIGS. 1 to 3, the query probe includes a query sequence complementary to a base sequence adjacent to a specific mutation site to be detected in the target nucleic acid. More specifically, the query sequence is complementary to at least a part of the base sequence adjacent to the 3 ′ side of the mutation site in the target nucleic acid. These query sequences do not contain mutation sites that may be present in the target nucleic acid and are directed to regions conserved in the target nucleic acid. Therefore, the query sequence is common for one mutation to be detected.

  The query probe also includes a query sequence so that the nucleotide species complementary to the nucleotide species adjacent to the mutation site in the target nucleic acid (hereinafter also simply referred to as the adjacent nucleotide species) is the 3 'end. In the 3 'end of the query probe, that is, the 3' end of the query sequence, a labeled nucleotide is linked in the linkage step described later.

  The query sequence is designed by calculating, for example, continuous match length, melting temperature prediction by Nearest-Neighbor method, Hamming distance, and secondary structure prediction. The length of the query sequence is not particularly limited, but is preferably 10 bases or longer, for example.

(Tag array)
As shown in FIGS. 1 to 3, the tag sequence is composed of a complementary base sequence capable of hybridizing with a capture probe immobilized on a solid phase carrier.

  The tag sequence has two forms in relation to the mutated base. One form is a form in which the tag sequence is prepared to hybridize with a different capture probe for each mutated base, as shown in FIGS. 1A and 2. At this time, since the query probe for the mutation to be detected has the same query sequence and a different tag sequence for each mutation constituent base, the query probe is prepared according to the type of the mutation constituent base. Become. As a result, the linking molecule obtained for the mutation to be detected hybridizes with different capture probes determined in advance for each mutation base on the solid phase carrier. In FIG. 1A, two types of tag sequences for detecting each of two types of mutation-constituting bases are set, but even if there are three or four types of mutation-constituting bases, the corresponding tags The sequence may be set for each mutation base.

  Another form is a form in which the tag sequence is prepared to hybridize with the same capture probe between the mutated bases, as shown in FIGS. 1B and 3. At this time, the query probe for the mutation to be detected has the same query sequence and the same tag sequence, and one query probe common to all the mutation constituent bases is prepared. As a result, the obtained linking molecule is hybridized with one predetermined capture probe on the solid phase carrier. In this case, each mutated base is identified by the labeling element of the labeled nucleotide.

  The capture sequence of the capture probe to which the tag sequence hybridizes is also called an orthonormalized sequence. For example, continuous match length for a DNA sequence of a predetermined base length obtained from a random number, melting temperature prediction by Nearest-Neighbor method, Hamming distance Designed by performing secondary structure prediction calculations. The orthonormalized sequence is a base sequence of nucleic acid having a uniform melting temperature, that is, a sequence designed so that the melting temperature is within a certain range, and the nucleic acid itself is intramolecular. It means a base sequence that does not form a stable hybrid other than a base sequence that is structured in the above and does not inhibit hybridization with a complementary sequence. A sequence included in one orthonormalized sequence group hardly reacts between sequences other than the desired combination and within a self-sequence, or does not generate a reaction. Further, when the orthonormalized sequence is amplified in PCR, the amount of nucleic acid corresponding to the initial amount of the nucleic acid having the orthonormalized sequence is quantitatively determined without being affected by the problems such as the above-described cross hybridization. Has the property of being amplified. The orthonormalization sequence as described above is described in detail in H. Yshida and A. Suyama, “Solution to 3-SAT by breadth first search”, DIMACS Vol. 54 9-20 (2000) and Japanese Patent Application 2003-108126. Has been. Orthonormalized sequences can be designed using the methods described in these references.

  For example, as the capture sequence, the base sequence represented by SEQ ID NOs: 1 to 100 or a base sequence complementary to the base sequence can be used. These base sequences all have the same base length, and have a melting temperature (Tm) of 40 ° C. or higher and 80 ° C. or lower, preferably 50 ° C. or higher and 70 ° C. or lower. Expected to be obtained.

When a tag sequence is prepared for each mutation base, it is preferable that the melting temperature of each tag sequence and the capture probe be close to each other. When exhaustively detecting multiple mutations in multiple target nucleic acids simultaneously, the melting temperatures of multiple tag sequences and capture sequences of multiple query probes used for one target nucleic acid are closest to each other It is preferable that they are combined. For example, as shown in FIG. 4, when tag sequences (capture sequences) are arranged in the order of melting temperature, tag sequences in two or more query probes for one target nucleic acid are converted into two or more types of bases adjacent in the permutation. You can select from the sequence. The tag sequence and capture sequence of the query probe set corresponding to another target nucleic acid may be selected from a series of base sequences immediately adjacent to the already selected base sequence, as shown in FIG. You may select from a series of base sequences. It is preferable to use a series of base sequences in which the melting temperature of the tag sequence and the capture sequence used in all query probe sets corresponding to the target nucleic acid to be detected simultaneously is in the permutation of the melting temperature. The melting temperature calculated by the GC% method, the Wallace method, a method based on Current Protocols in Molecular Biology (described in Shujunsha's Bio-Experiment Illustrated 3 PCRp25, which is really increased), etc. can be adopted. It is preferably calculated by the Nearest-Neighbor method that can take into account the range of melting temperature and base sequence concentration in the invention. The melting temperature by the Nearest-Neighbor method is, for example, software with Visual OMP (manufactured by Tommy Digital Biology Co., Ltd.) or software provided by Japan Genetic Research Institute (http://www.ngrl.co.jp/). (Oligo Calculator; http://www.ngrl.co.jp/tool/ngrl_tool.html). The capture sequences represented by SEQ ID NOs: 1 to 100 are arranged in descending order of melting temperature (0.1 M probe concentration, 50 mM Na ⁺ ion and 1.5 mM Mg ⁺ ion) calculated by Visual OMP.

  An appropriate spacer sequence may be provided between the tag sequence of the query probe and the query sequence. By providing the spacer, steric hindrance during hybridization with the capture probe can be avoided or suppressed. The spacer is not particularly limited, and examples thereof include a natural or non-natural polynucleotide chain, a hydrocarbon chain, a peptide chain, and a peptide nucleic acid sugar chain. When nucleotides or derivatives thereof are used as the spacer, the number of nucleotide residues may be appropriately determined according to the base sequence complementary to the target nucleic acid and the tag sequence, but is preferably 3 to 10 residues.

(Labeled nucleotide)
The labeled nucleotide can have a query nucleotide having a base complementary to the mutated base in the target nucleic acid and can include a labeling element.

  The labeled nucleotide is appropriately selected depending on the type of ligation reaction. For example, when the ligation reaction is performed using a DNA polymerase reaction, the labeled nucleotide is provided with a query nucleotide (triphosphate) corresponding to the mutated base as the query nucleotide at the 5 'end. In this case, the query nucleotide preferably has 3′OH such as deoxyribose as a hydrogen group so that the ligation reaction is terminated without the nucleotide being additionally linked. By using such a labeled nucleotide, it can be encoded and labeled with the shortest linking molecule by a single base extension reaction. Examples of such query nucleotide include dideoxynucleotide (ddNTP), that is, ddATP, ddTTP or ddUTP, ddGTP, ddCTP.

  When the ligation reaction is performed using DNA ligase, the labeled nucleotide has a query nucleotide (monophosphate) corresponding to the mutated base at the 5 'end. When DNA ligase is used, the labeled nucleotide is composed of a base sequence complementary to the corresponding region of the target nucleic acid from several to about 20 on the 3 ′ end side in addition to the query nucleotide (of the nucleotide constituting the target nucleic acid mutation). It is preferably an oligonucleotide comprising a query sequence for a region adjacent to the 5 ′ side.

  A labeled nucleotide is prepared for each mutation base as shown in FIGS. For example, these figures are illustrated as labeled nucleotides when DNA polymerase is used. As shown in FIG. 1A, when the mutation bases are A and G in SNP, ddTTP and ddCTP are respectively queried. Two types of labeled nucleotides (A query labeled nucleotide and G query labeled nucleotide) are prepared.

The labeled nucleotide is provided with a suitable labeling element. The labeling element that can be added to ddNTP is appropriately selected from known labeling substances. For example, labeling substances such as enzymes, fluorescent substances, haptens, antigens, antibodies, radioactive substances, or luminophores can be mentioned. Examples of the enzyme include alkaline phosphatase and peroxidase. Examples of the fluorescent material include fluorescent materials such as FITC, 6-FAM, HEX, R6G, ROX, R110, TET, TAMRA, and Texas Red, in addition to cyanine-based fluorescent dyes such as Cy2, Cy3, and Cy5. Examples of haptens include biotin and digoxigenin. Examples of radioactive substances include ³² P and ³⁵ S. Examples of the luminophore include ruthenium.

  The labeling element in the labeled nucleotide can take two forms with respect to the relationship with the mutated constituent base. That is, as shown in FIG. 1A and FIG. 2, one form is a form in which the labeling elements of the labeled nucleotide are the same among the mutation bases, that is, the same for the mutation to be detected. This form is combined when the tag sequence of the query probe is prepared for each mutation base. The linking molecule obtained from such a query probe has the presence and ratio of the mutated base based on the signal intensity information in the capture probe prepared for each mutated base even if the labeling element is the same between the mutated bases. It can be detected.

  Another form of the labeling element is a form in which the labeling element of the labeled nucleotide is different for each mutated base as shown in FIGS. 1B and 3. That is, the labeling element has a different type of identification label depending on the type of the mutation constituent base of the mutation to be detected. This form is combined when the tag sequence of the query probe is prepared identically among the mutated bases. Linked molecules obtained from such query probes will hybridize to the same capture probe even if the detected mutant bases are different, so each mutant base must be detected by signal intensity information based on different label elements. Because it will not be.

  From the viewpoint of detection accuracy of polymorphism and mutation ratio, it is preferable to use the same labeling element between different mutation bases for one mutation. This is because the use of the same labeling element can suppress variations in signal intensity between the labels and variations due to quality and the like. On the other hand, in order to reduce the number of capture probes, it is preferable to use the same capture probe between different mutated bases for one mutation and to use a different label element for each mutated base.

(Linking process)
In the linking step, the target nucleic acid and the query probe are hybridized for each mutation base, and a labeled nucleotide is added to the 3 ′ end of the query probe to obtain a linking molecule. The linking step is performed for each mutation base. That is, when the probe set shown in FIG. 2 is used, as shown in FIG. 1A, when the mutation base is T, a query probe for detecting T as a mutation base and a nucleotide having adenine as a base A labeled nucleotide having a species and a target nucleic acid are used. That is, a combination configured to detect T is used. When the probe set shown in FIG. 3 is used, when the mutation base is C, as shown in FIG. 1B, a query probe for detecting C as the mutation base (T is detected as the mutation base). The same query probe as in the above case), a labeled nucleotide having a nucleotide species having guanine as a base, and a target nucleic acid.

  By carrying out the ligation step in the presence of the query probe prepared for each mutation base and the labeled nucleotide as described above, it is possible to eliminate the generation of unintentionally linked molecules, and other query nucleotide species By coexisting, variation in the production rate of the intended linking molecule can be suppressed. In particular, when a query probe is prepared for each mutation-constituting base, an unintended linking molecule is generated, making it impossible to detect mutations and the like.

  DNA polymerase or DNA ligase is used to add a labeled nucleotide to the 3 'end of the query probe. The DNA polymerase is preferably a heat-resistant DNA polymerase such as Taq DNA polymerase. By using a heat-resistant DNA polymerase, it is possible to increase the amount of linked molecules generated by repeating the steps of denaturation, annealing, and base extension a plurality of times in a temperature cycle. The DNA ligase is preferably a heat-resistant DNA ligase from the viewpoint of certainty and efficiency.

  By performing the linking step, a linking molecule is obtained. Since the labeled nucleotide is modified so as not to undergo an additional base extension step, by performing the ligation step, the nucleotide species complementary to the mutated base is ligated to the adjacent nucleotide species at the 3 ′ end of the query sequence, thereby extending the labeled nucleotide. The reaction is stopped. As a result, a linking molecule that is associated with the capture probe so as to detect a mutated base in the target nucleic acid and is provided with a labeling element is obtained.

  As shown in FIG. 1A and FIG. 3, one form of the linking molecule has tag sequences (CP1 (−) and CP2 (−) associated with the capture probe for each mutation constituent base, respectively. And a linking molecule to which a cytosine (C) nucleotide complementary to the mutant base G is added, and these linking molecules have the same labeling element. 1B and 2, each of the other forms has a tag sequence (CP3 (−)) associated with the same capture probe among the mutated bases, and the mutated base C And a linking molecule to which a thymine (T) nucleotide complementary to the mutant base A is added. Linking molecule has a different label elements mutually.

  In consideration of simultaneously detecting a plurality of mutations for a plurality of target nucleic acids, the detection forms shown in FIGS. 1A and 3 can be preferably employed. This is because there is no need to prepare a labeling element according to the mutation, and since the mutation of different base species is detected with different capture probes, the detection accuracy is higher. Even when a plurality of mutations are detected for a plurality of target nucleic acids at the same time, the query nucleotides prepared for the plurality of target nucleic acids and the target nucleic acids may be reacted together with the target nucleotides prepared for each mutation base. .

(Contact process)
The contact step is a step of bringing these capture probes and the linking molecule into contact with each other on a solid phase carrier on which two or more types of capture probes having a capture sequence complementary to the tag sequence in the linking molecule are immobilized. is there. According to this step, when the tag sequence in the linking molecule is complementary to the capture sequence in the capture probe so that it can selectively hybridize under certain conditions, they hybridize to form a duplex. To do. After this contact step, a cleaning step may be further included as appropriate. Hybridization conditions are appropriately set according to the capture probe used.

(Solid phase carrier with capture probe immobilized)
In the contact step, a solid phase carrier on which two or more kinds of capture probes are immobilized can be used. Such a solid support may be, for example, a bead or a flat plate, and the material is not particularly limited, but a solid support made of glass or plastic can be used. Preferably, the solid support is a flat plate, and is an array in which two or more types of capture probes are fixed in a fixed arrangement. An array can fix a large number of capture probes, and is convenient for comprehensively detecting various polymorphisms and mutations at the same time. In addition, the array may include a plurality of partitioned individual array regions on a single solid phase carrier. In each of these individual array regions, the same set of capture probes may be immobilized, or different sets of capture probes may be immobilized.

  A preferred solid phase carrier is an array having an arrangement state in which two or more types of capture probes are arranged in an order based on the melting temperature thereof. For example, using such an array, two or more types of capture probes corresponding to each target nucleic acid are assigned in the order of the capture probes, thereby suppressing variations in hybridization resulting from differences in the melting temperatures of the capture sequences. Thus, the specific partial sequence containing the mutated base in the target nucleic acid can be detected with high accuracy. An example of a preferred array is shown in FIG.

  The solid phase carrier used in the contacting step is preferably an array having an arrangement state in which captures used for one target nucleic acid are adjacent to each other. By being adjacent to each other, it is possible to accurately detect a specific partial sequence in a target nucleic acid containing a mutated base by suppressing variation resulting from the position of the hybridization capture probe immobilized on the array. .

  A more preferable solid phase carrier has an arrangement state in which capture probes used for one target nucleic acid are adjacent to each other, and the maximum temperature difference (absolute value) of the melting temperature between these adjacent capture probes is 5 ° C. or less, preferably 0 An array arranged to be 2 ° C. or lower. By using such an array, it is possible to suppress variations in hybridization based on the difference between the capture probe immobilization position and the capture probe melting temperature on the array. Typically, when multiple capture probes are arranged in the order based on the melting temperature, the capture probes arranged in succession are used as two or more types of capture probes for detecting a specific partial sequence of one target nucleic acid. do it.

  The plurality of capture probes immobilized on the solid phase carrier can each have a capture sequence selected from SEQ ID NOs: 1 to 100. These base sequences are orthonormalized sequences designed under certain conditions, as already described.

  The immobilization form of the capture probe is not particularly limited. It may be covalent or non-covalent. The capture probe can be immobilized on the surface of the solid support by various conventionally known methods. An appropriate linker sequence may be provided on the surface of the solid phase carrier. The linker sequence is preferably the same sequence with the same base length between the capture probes.

(Signal intensity information acquisition process)
The signal intensity information acquisition step is a step of acquiring signal intensity information based on the label element on the solid phase carrier after hybridization. Mutant constituent bases in the target nucleic acid are associated in advance with a predetermined labeling element and a predetermined capture probe. Therefore, by acquiring signal intensity information based on a predetermined labeling element at a predetermined capture probe position, the presence or absence of a specific partial sequence in the target nucleic acid, the amount thereof, and the like can be quantified.

  The signal intensity information associated with such a capture probe can be obtained by the number of mutated bases present in the target nucleic acid. That is, even if it is labeled with a different labeling element for each mutation element base, it is the same even if it is identified with a different capture probe and detected with the same labeling element.

  In the signal intensity information acquisition step, a conventionally known method may be appropriately selected and employed according to the form of the solid phase carrier used and the type of labeling substance. Typically, oligonucleotides that have not been hybridized from the solid phase carrier are removed by a washing operation, etc., and then the fluorescence signal of the added labeling substance is detected by an array scanner or the like, or a chemiluminescent reaction to the labeling substance Can be implemented. When beads are used as the solid phase carrier, a detection method using a flow cytometer can be used.

(Detection process)
The detection step is a step of detecting the presence / absence, ratio, etc. of the mutated base based on the signal intensity information about the mutated base associated with the predetermined labeling element and the predetermined capture probe. That is, the presence or absence of mutation, the type of polymorphism, and the mutation rate can be detected by the detection step. According to the method of the present invention, in order to detect a mutation in a target nucleic acid in a single time, a linked molecule labeled with a predetermined labeling element associated with a capture probe can be obtained, so that a mutation can be detected quickly. is there. In particular, even when a plurality of (many) mutations in a plurality of (many) target nucleic acids are detected, a plurality of target nucleic acids can be collectively performed for each mutation base. Therefore, when detecting a plurality of mutations for a plurality of target nucleic acids, a linking molecule can be prepared in the same time as detecting one mutation in one kind of nucleic acid. Therefore, the method of the present invention is particularly preferable for comprehensively detecting gene mutations. The method of the present invention is particularly suitable for efficiently detecting SNPs and mutations with high throughput.

  There are two forms of detecting polymorphisms or mutations in the target nucleic acid. One is the detection mode shown in FIGS. 1A and 2. In this form, a mutation is detected from signal intensity information based on the same labeling element for the capture probes CP1 and CP2 associated with each mutation base. From the ratio of the signal intensity information obtained for the capture probes CP1 and CP2, polymorphic types, for example, polymorphic types such as AA homo, GG homo and AG hetero can be detected. Further, when it is assumed that A is a normal type and G is a mutant type, [signal intensity information obtained for capture probe CP2 / signal intensity information obtained for capture probes CP1 and CP2] is calculated. Thus, the mutation ratio (ratio in which the mutation base is G) can be obtained. In such a detection form, since signal intensity information based on the same labeling element is used, it is possible to perform accurate detection while suppressing variations based on characteristics and quality of the labeling element itself. In particular, for obtaining the mutation ratio, it is preferable to use a different capture probe for each mutation base.

  Another form is the detection form shown in FIGS. 1B and 3. In this form, a mutation is detected from signal intensity information based on different labeling elements associated with each mutation constituent base for the same capture CP3. Polymorphic types (CC homo, TT homo and CT hetero) are detected from the ratio of signal intensity information for two types of labeling elements obtained for the capture probe CP3 (plotting each signal information on the XY axes) it can. In addition, the mutation ratio can be obtained in the same manner as in the detection mode such as FIG. 1A.

  When detecting the bases of mutations in target nucleic acids with different capture probes, the capture probes may be close in melting temperature, but there may be a bias in the amount of hybridization between the capture probes depending on the melting temperature and other circumstances. However, this can be suppressed or avoided by performing each step as follows.

  For example, for each target capture probe, together with a linking molecule, a correction oligonucleotide prepared with a predetermined tag sequence and a labeling substance different from that used for the target nucleic acid and prepared at the same concentration is placed on the solid phase carrier. The signal intensity information about the target nucleic acid and the correction oligonucleotide is acquired in the signal intensity information acquisition step. By comparing the magnitude of the correction signal intensity information between the capture probes, it is possible to detect the presence or absence of a difference in hybridization efficiency (amount) between the capture probes. If a difference occurs in the signal intensity obtained between the capture probes, it is corrected that such a difference in signal intensity is also present in the linking molecule. Such correction using the correction signal intensity information can be applied to obtain the presence or absence of polymorphism (type), the presence or absence of mutation, and the mutation rate, and is used in combination with correction based on reference signal intensity information described later. be able to.

  When detecting a gene mutation and quantifying the mutation rate, for example, for a reference oligonucleotide prepared in place of a target nucleic acid and having a mutation-constituting base at a predetermined ratio for the mutation to be detected, a contact step, a linking step, The detection step can be performed using the reference signal intensity information acquired for the reference oligonucleotide by performing the contact step and the signal intensity information acquisition step. Preferably, a calibration curve is prepared based on the reference signal intensity information acquired for the reference oligonucleotide prepared at two or more mutation ratios, and the polymorphism in the target nucleic acid in the sample is obtained using this calibration curve. Mutation rates can be detected and the mutation rate can be quantified. By using a calibration curve, highly accurate detection and quantification are possible. Note that acquisition of reference signal intensity information and preparation of a calibration curve may be performed in advance, or may be performed at the same time as detecting a mutation in the target nucleic acid.

  Note that the reference oligonucleotide and the correction oligonucleotide used for correction do not necessarily have to be prepared in a single strand, and may be in a double strand.

(Probe set)
The probe set of the present invention is a probe set used for detecting at least one mutation in a target nucleic acid using a capture probe immobilized on a solid phase carrier. The probe set of the present invention includes a capture probe and a labeled nucleotide for each mutation base that may be detected at a specific site in a target nucleic acid. The capture probe and the labeled nucleotide are as described above.
By using the capture probe and the labeled nucleotide already described, mutations in the target nucleic acid can be rapidly detected. The probe set of the present invention may be kitted with a solid phase carrier such as an array on which such capture probes are immobilized.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, a following example does not limit this invention. In the following examples, gene mutation (SNP) was detected by the following procedure using the detection method of the present invention. Hereinafter, it demonstrates according to these order.
(1) Preparation of DNA microarray for detecting mutant genes (2) Amplification of sample genes (3) Ligation (including labeling) reaction (4) Detection using DNA microarray (5) Data analysis

(1) Preparation of DNA microarray for detecting mutant genes (SNP genes) An aqueous solution in which a synthetic oligo DNA (manufactured by Nippon Genetic Laboratory Co., Ltd.) with a 3 'end modified with an amino group was dissolved in a commercially available plastic substrate As a capture probe, spotting was performed at NGK Corporation using a GENESHOT spotter. The synthetic oligo DNA sequences used were D1_52, D1_71, D1_93, D1_74, D1_41, D1_92, D1_79, D1_78 in descending order of Tm among D1_1 to D1_100 described in Supplementary Table 1 of the literature (Analytical Biochemistry 364 (2007) 78-85). , D1_65 (Table 1). After the spot, baking was performed at 80 ° C. for 1 hour. Furthermore, the synthetic oligo DNA was immobilized by the procedure described below.

  The synthetic oligo DNA was immobilized by the following procedure. After baking, wash with 2 × SSC / 0.2% SDS for 15 minutes, wash with 95 ° C 2 × SSC / 0.2% SDS for 5 minutes, and then wash with sterilized water (10 shaking up and down) 3 times Then, the solution was removed by centrifugation (1000 rpm × 3 minutes).

(2) Preparation and amplification of mutant gene sample The base sequence of the mutant gene to be detected has the sequence around the mutant gene shown in the following table. Both are artificially synthesized genes of about 150 bp. The four types of mutant gene samples were composed of five types of the following synthetic oligo DNAs of wild type and mutant type 1 to 4 (ratio of wild type: mutant type: 100: 0, 95: 5, 90:10). , 75:25, 50:50).

The sample amplification procedure was as follows. As a sample amplification reagent, TaKaRa Ex Taq ^(R) (Catalog # RR001B) manufactured by TaKaRa Bio was used. As a thermal cycler, Applied Biosystems GeneAmp PCR System 9700 was used. The PCR primer was designed to be in the range of Tm = 58.90 ± 0.75 ° C. Each primer sequence is shown in Table 3.

  Mutant gene sample amplification was performed as follows. In this example, each mutant gene sample was amplified individually. First, the following reagents were prepared for each mutant gene sample.

  Next, a thermal cycle reaction was performed. Specifically, 40 cycles of 94 ° C. for 30 seconds, 94 ° C. for 40 seconds, 66 ° C. for 40 seconds, and 72 ° C. for 2 minutes were performed, and then the temperature was lowered to 4 ° C. Individually amplified products (total 4 variants) are mixed in tubes for each mutant gene ratio to make PCR products, and the wild gene: mutant gene ratio is 100: 0, 95: 5, 90:10, 75:25, 50: Fifty 5 tubes were prepared. As described above, 5 kinds of PCR products containing the target mutant gene were obtained.

(3) Ligation (labeling) reaction The ligation (labeling) reaction procedure of the amplified sample was performed as follows. TaKaRa Bio TaKaRa Ex Taq ^(R) (RR001A) was used for the reaction. GeneAmp PCR System 9700 (Applied Biosystems) was used as a thermal cycler for heating. The query probe sequences and reagents are shown in the following table.

  First, the reagent was prepared. Reagents were prepared for each different mutant gene ratio in the mutant gene sample. The combinations of Cy3-labeled ddNTP and query probe were as shown in the following table. In the table below, Y represents the number of query probes.

  The ligation reaction (single base extension reaction) was performed as follows. That is, after 1 minute at 92 ° C, 15 minutes at 30 ° C, 10 minutes at 75 ° C, and then cooled to 10 ° C. Next, a mixture of 4 types of products obtained for each of the 5 mutant gene ratios was used as a product (5 types) for each mutant gene ratio.

(4) Detection using DNA microarray The reaction to the DNA microarray using the labeled sample and the detection procedure were as follows. The composition of the labeled sample solution for hybridization is shown in the following table.

(Hybridization)
The prepared labeled sample solution was heated at 90 ° C. for 1 minute using GeneAmp PCR System 9700 from Applied Biosystems, and then heated at 80 ° C. for 1 minute using a heat block (TATUC DTU-N). Next, 9 μl each of the above sample solution was applied to the spot area of the DNA microarray, and the hybridization reaction was carried out by allowing it to stand at 37 ° C. for 60 minutes using a comfort / plus thermoblock Slide (eppendorf) to prevent drying. went.

(Washing)
Washing was performed as follows. That is, the cleaning liquid having the composition shown below was transferred to a glass dyeing vat, and then the glass substrate after completion of the hybridization reaction was immersed and shaken up and down for 5 minutes. Thereafter, the glass substrate was transferred to a glass staining vat containing sterilized water, shaken up and down for 1 minute, and further centrifuged and dried at 2000 rpm for 1 minute to remove moisture remaining on the glass substrate surface.

(Fluorescence detection by scanner)
Using an Appleied Precision ArrayWoRx, the exposure time was appropriately adjusted to obtain a fluorescence image. Furthermore, using GenePix Pro, the fluorescence signal of the obtained image was digitized.

(5) Data analysis In order to confirm the presence or absence of the difference in hybridization efficiency (amount) between captures, the fluorescence signal of hybrid control hybridized simultaneously was measured. The following table shows the measurement results of the spot for detecting mutation 1 gene (D1_52 and D1_71).

  From the results of Table 10, the fluorescence signal ratio between the two spots for detecting the mutation 1 gene is almost 1, and it is considered that there is no significant difference in the amount of hybridized adsorption between the two spots. Therefore, it was determined that correction of the spot for detecting mutation 1 gene was unnecessary.

  Next, fluorescence signal detection (D1_52 and D1_71) was performed for the mutation 1 gene using a scanner. The results are shown in the table below.

  Based on the results in Table 11, a calibration curve was prepared by plotting the mutant gene ratio on the horizontal axis and B / (A + B) on the vertical axis. The results are shown in FIG.

  According to these results, it was found that the presence of the mutant type in the mutant gene can be easily detected, and can be easily detected even at a low mutation rate. It was also found that the wild type: mutant type ratio could be easily detected.

  As described above, according to the present invention, it has become possible to simultaneously perform the conventional linking / coding process and labeling process. As a result, the time required for carrying out the linking and labeling is 1.5 hours, and the time required for carrying out the process can be shortened to 1/3 or less of the conventional 5 hours. It became. In spite of such shortening of the process time, it was found that the process time could be shortened without degrading the detection accuracy because the detection was possible with high accuracy even in the case of a low mutation rate.

It is a figure which shows typically the outline | summary of an example of the detection form of this invention. It is a figure which shows typically the outline | summary of another example of the detection form of this invention. It is a figure which shows an example of the combination of the query probe and labeled nucleotide which are used for this invention. It is a figure which shows another example of the combination of the query probe used for this invention, and a labeled nucleotide. It is a figure which shows an example of the tag arrangement | sequence and capture arrangement | sequence used for this invention. It is a figure which shows the calibration curve created in the Example. It is a figure which shows the SNP detection form by a prior art.

SEQ ID NOs: 1 to 109: capture probes, SEQ ID NOs: 110 to 117: artificially synthesized genes, SEQ ID NOs: 118 to 125: primers, SEQ ID NOs: 126 to 133: synthetic probes

Claims (10)

A method for detecting a mutation in a target nucleic acid using a plurality of types of capture probes immobilized on a solid phase carrier,
The following (a) and (b) for each mutated base that may be detected at a specific site for the mutation in the target nucleic acid:
(A) a capture sequence that hybridizes with the capture probe and a complementary query sequence to a base sequence adjacent to a specific site in the target nucleic acid, and a nucleotide species adjacent to the specific site in the target nucleic acid A query probe comprising at the 3 ′ end a nucleotide species to be
(B) a labeled nucleotide having a query nucleotide that complements the nucleotide species at the specific site in the target nucleic acid and comprising a labeling element;
The following steps (1) to (3):
(1) A linking step for hybridizing the target nucleic acid and the query probe for each of the mutated bases, and linking a labeled nucleotide to the 3 ′ end of the query probe to obtain a linking molecule;
(2) a contact step in which the linking molecule obtained for each of the mutation-constituting bases and the capture probe are brought into contact with each other so as to be hybridizable on the solid phase carrier;
(3) a signal information acquisition step of acquiring signal information based on the labeling element for each capture probe on the solid phase carrier;
A detection method is carried out.   The detection method according to claim 1, wherein the labeled nucleotide has only the query nucleotide as a nucleotide species, and is formed so that a ligation reaction is terminated by the query nucleotide. For the mutation,
The query probe comprises a capture sequence that hybridizes with a different capture probe for each of the mutated bases,
The labeled nucleotides comprise mutually identical labeling elements,
The detection method according to claim 1 or 2. The presence or absence of the mutation or the ratio of the constitutive mutant base is expressed by the following formula:
[Ratio of signal intensity obtained for a capture probe hybridized with a linking molecule obtained for one of the mutation-constituting bases] / [Signal obtained for a capture probe hybridized with all ligation molecules obtained for the mutation Sum of strength]
The detection method according to claim 3, wherein the detection is performed based on. For the mutation,
The query probe comprises a capture sequence that hybridizes with the same capture probe between the mutated bases,
The labeled nucleotide comprises a different labeling element for each of the mutated constituent bases,
The detection method according to claim 1 or 2.   The detection method according to claim 1, wherein the contacting step includes supplying a mixture of each linking molecule in the linking step performed for each of the mutated bases onto one solid phase carrier. .   The shift according to claim 1, wherein each of the steps (1) to (3) is collectively performed for detecting a plurality of mutations for one or more types of target nucleic acids. The detection method of crab.   The detection method according to claim 7, wherein the plurality of target nucleic acids are prepared by multiplex PCR with respect to nucleic acids in a specimen. A kit for detecting a mutation in a target nucleic acid using a plurality of types of capture probes immobilized on a solid phase carrier,
The following (a) and (b) for each mutated base that may be detected at a specific site for the mutation in the target nucleic acid:
(A) a capture sequence that hybridizes with the capture probe and a complementary query sequence to a base sequence adjacent to a specific site in the target nucleic acid, and a nucleotide species adjacent to the specific site in the target nucleic acid A query probe comprising at the 3 ′ end a nucleotide species to be
(B) a labeled nucleotide having a query nucleotide that complements the nucleotide species at the specific site in the target nucleic acid and comprising a labeling element;
A kit comprising: A kit used to detect a mutation in a target nucleic acid,
A solid support comprising a plurality of capture probes;
The following (a) and (b) for each mutated base that may be detected at a specific site for the mutation in the target nucleic acid:
(A) a capture sequence that hybridizes with the capture probe and a complementary query sequence to a base sequence adjacent to a specific site in the target nucleic acid, and a nucleotide species adjacent to the specific site in the target nucleic acid A query probe comprising at the 3 ′ end a nucleotide species to be
(B) a labeled nucleotide having a query nucleotide that complements the nucleotide species at the specific site in the target nucleic acid and comprising a labeling element;
A kit comprising: