JP5165933B2 - Method and array for detecting specific partial sequence in target nucleic acid - Google Patents

Description

  The present invention relates to a technique for detecting and quantifying a target nucleic acid having a specific base sequence such as a single nucleotide polymorphism or a mutation on a gene.

  It has been found that SNPs and gene mutations 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. 5, two types of 5 ′ query probes having a tag sequence for labeling and a query sequence complementary to a partial sequence containing a specific base of SNP, and a common portion adjacent to the partial sequence One 3 ′ query probe having a sequence and an identifying tag sequence complementary to the capture is used. 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.

  In this method, a 5 'query probe and a 3' query probe are linked (encoded) by a hybridization reaction and a ligation reaction between these probes and a target nucleic acid. Next, labeling is performed by PCR using the tag sequence for labeling of the obtained linking molecule. 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 with an array in which capture probes complementary to the identification tag sequence are immobilized.

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, since the SNP type discrimination is based on the signal intensity plot, the accuracy of the SNP type discrimination may not be sufficient when there are not enough connected molecules or when the number of repeats is small. In addition, since the detection of the specific partial sequence in the target nucleic acid is performed using different labeling substances (two types), if there is a difference in characteristics such as fading between the labeling substances, the SNP type determination is sufficient. May not be able to ensure accurate accuracy. Furthermore, from such circumstances, it may be difficult to detect gene mutations richer in variation than SNP and quantify the mutation rate.

  Therefore, an object of the present invention is to provide a method for detecting or quantifying specific partial sequences such as polymorphisms and mutations in a target nucleic acid with high accuracy, and an array therefor. Another object of the present invention is to provide a method for easily detecting or quantifying a mutation in a target nucleic acid and its mutation rate and an array therefor.

  In detecting a specific partial sequence in a target nucleic acid, the present inventors correspond to a query probe linking molecule with multiple types of labeling substances according to the polymorphism or mutation type to be detected in the target nucleic acid. Rather than letting it form, multiple types of identification tag sequences are made to correspond according to the type of polymorphism, etc., and the linking molecules are hybridized to different capture probes based on these identification tag sequences, so that universal The present inventors have found that polymorphs and the like can be detected with high accuracy by using a solid phase carrier while suppressing errors based on the difference in properties of the labeling substance. That is, according to the present invention, the following means are provided.

  According to the present invention, there is provided a method for detecting a polymorphism or mutation in one or more target nucleic acids in a sample, the polymorphism or mutation to be detected in at least one target nucleic acid. A first query consisting of two or more types of first query probes comprising a query sequence complementary to a specific partial sequence comprising at least one base constituting the base and an identification tag sequence capable of identifying the specific partial sequence A target query nucleic acid is hybridized with a probe set, a second query probe comprising a query sequence complementary to a partial sequence adjacent to the specific partial sequence of the target nucleic acid, and a tag sequence for labeling for adding a label. A first contacting step for allowing soy contact, and the first query probe and the second query probe hybridized to the one target nucleic acid to link these linked molecules. Complementary to the linking step to acquire, a labeling step to label the linking molecule with a single labeling substance using the labeling tag sequence provided in the linking molecule to obtain a labeled linking molecule, and the identification tag sequence These capture probes and the labeled linking molecule are brought into contact with each other on a solid phase carrier on which two or more types of capture probes associated with the specific partial sequence having a specific or homologous capture sequence are immobilized. A second contact step, a signal intensity information acquisition step for acquiring two or more types of signal intensity information for the one target nucleic acid based on the labeling substance on the solid phase carrier after hybridization, and the two types And a detection step of detecting the polymorphism or the mutation in the one target nucleic acid based on the signal intensity information described above. It is.

  In this method, the detection step is obtained corresponding to a capture probe associated with each specific partial sequence via each identification tag sequence of each first query probe in the first query probe set. It may be a step of detecting the presence or absence of the polymorphism or mutation to be detected based on the ratio of signal intensities.

  Further, in this method, the detection step is obtained corresponding to the capture probe associated with the specific partial sequence via the identification tag sequence of one first query probe in the first query probe set. Sum of signal intensities obtained corresponding to all the capture probes associated with the specific partial sequence via the identification tag sequence of all the first query probes in the first query probe set with respect to the obtained signal intensities It is good also as the process of calculating | requiring the type discrimination | determination of the said polymorphism to be detected or the ratio of the said variation | mutation based on the ratio.

  In addition, for one or two or more reference oligonucleotides having the polymorphic type to be detected prepared in advance, the first contact step, the linking step, the labeling step, the second contact The detection step can be performed using the reference signal intensity information acquired for the reference oligonucleotide by performing the step and the signal intensity information acquisition step.

  Further, for one or more reference oligonucleotides prepared in advance and having a predetermined ratio for the mutation to be detected, the first contact step, the linking step, the labeling step, the second step The detection step can also 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.

  Furthermore, the evaluation step may be performed using a calibration curve based on reference signal intensity information acquired for two or more types of reference oligonucleotides.

  In the second contacting step, each of the identification tag sequence of each of the first query probes in the first query probe set and the single labeling substance are different from each other together with the labeled linking molecule. And a correction oligonucleotide prepared at the same concentration with each other so as to be able to hybridize with the capture probe on the solid phase carrier. The signal intensity information for one target nucleic acid is acquired and the signal intensity information for correction based on the other labeling substance for the correction oligonucleotide is acquired, and the detection step includes the signal intensity information for correction It is good also as a process including using.

  Furthermore, the polymorphism or mutation to be detected is a single nucleotide polymorphism, and the first query probe set includes a query sequence complementary to a specific partial sequence containing one base of the single nucleotide polymorphism, and the A first query probe having an identification tag sequence capable of identifying a specific partial sequence, and a query sequence complementary to the specific partial sequence including the other base of the single nucleotide polymorphism can be distinguished from the specific partial sequence A first query probe having another identification tag sequence, and the polymorphism or mutation to be detected is a mutation, and the first query probe set relates to the mutation. A first query probe having a query sequence complementary to a specific partial sequence containing one or more normal bases and an identification tag sequence capable of identifying the specific partial sequence; and 1 or 2 abnormal with respect to the mutation Including at least two types of first query probes, a query sequence complementary to a specific partial sequence including the above bases and a first query probe having an identification tag sequence capable of identifying the specific partial sequence. You can also.

  The solid phase carrier may be an array having an arrangement state in which the two or more types of capture probes for the at least one type of the target nucleic acid are adjacent to each other, and the solid phase carrier may be the at least one type of the target. The two or more types of capture probes for nucleic acids are arranged adjacent to each other, and the maximum temperature difference (absolute value) of melting temperature between these adjacent capture probes is 5 ° C. or less, preferably 0.2 ° C. or less The array may be arranged so that

  According to the present invention, there is provided a solid phase carrier on which a capture probe used in any of the detection methods described above is immobilized, wherein the two or more types of capture probes for the at least one target nucleic acid have melting temperatures thereof. There is provided a solid support which is arranged in an order based on

  According to the present invention, there is provided a solid phase carrier on which a capture probe for detecting a polymorphism or mutation in one or more target nucleic acids in a sample is immobilized, and the two capture probes are It has the capture sequence of the same base length set irrespective of the base sequence of the polymorphism or the mutation to be detected of the target nucleic acid, and has a melting temperature of 40 ° C. or higher and 80 ° C. or lower, preferably There is provided a solid support that is 50 ° C. or higher and 70 ° C. or lower and arranged in an order based on its melting temperature. In this solid phase carrier, the plurality of capture probes preferably have orthonormalized sequences, for example, capture base sequences each selected from SEQ ID NO: 1 to SEQ ID NO: 100.

  According to the present invention, a probe set for detecting a target nucleic acid in a sample is complementary to a specific partial sequence containing at least one base constituting a polymorphism or mutation to be detected in the target nucleic acid. A first query probe set comprising two or more types of first query probes, each of which includes a simple query sequence and an identification tag sequence capable of identifying the specific partial sequence, and a portion adjacent to the specific partial sequence of the target nucleic acid A probe set is provided, comprising: a second query probe comprising a query sequence complementary to the sequence and a tag sequence for labeling for adding a label.

  The present invention relates to a method for detecting a target nucleic acid and an array for detecting the target nucleic acid. According to the method for detecting a target nucleic acid of the present invention, a target nucleic acid is obtained by hybridizing and linking the first query probe and the second query probe in the first query probe set as described above to the target nucleic acid. On a solid phase carrier on which a linking molecule having an identification tag sequence corresponding to the type of a specific partial sequence is obtained, this linking molecule is labeled, and a capture probe complementary to the identification tag sequence is immobilized By detecting the amount of hybridization of different linking molecules to different capture probes by hybridizing with, it is possible to detect the presence and amount of a specific partial sequence without discrimination by the signal intensity ratio of different labeling substances it can. For this reason, the uncertainty based on using a some labeling substance can be excluded, and the detection accuracy fall by it can be avoided. Therefore, polymorphisms and mutations can be detected with high accuracy, and the ratio of mutations can be easily detected with high accuracy.

  Further, according to this method, by using one first query probe for each specific partial sequence that may be present in the target nucleic acid, it exists by the first contact step, the linking step, and the labeling step. Depending on the specific partial sequence and the abundance thereof, a labeled linking molecule can be obtained. Since the obtained linking molecule is associated with the capture probe 1: 1 through the identification tag sequence, the presence of the specific partial sequence and its presence are detected by the second contact step, the signal intensity information acquisition step, and the detection step. The ratio can be accurately detected based on signal intensity information in the capture probe associated with each specific partial sequence.

  Moreover, since the identification tag sequence and the capture sequence can be set independently of the specific partial sequence, any of these sequences can be applied to any target nucleic acid. Therefore, it is possible to ensure the convenience of detecting the polymorphism and the appropriate cost. Moreover, it is suitable for simultaneously detecting polymorphisms or mutations of a large number of target nucleic acids.

  Further, according to the solid phase carrier of the present invention, as described above, two or more of the capture probes have capture base sequences having the same base length set independently of the base sequence to be detected in the target nucleic acid. However, the melting temperature is 40 ° C. or higher and 80 ° C. or lower, preferably 50 ° C. or higher and 70 ° C. or lower, and is arranged in the order based on the melting temperature. When detection is performed with more than one type of capture probe, an error in the amount of hybridization during hybridization due to the melting temperature of the capture probe can be suppressed, and the target nucleic acid can be detected with high accuracy based on the amount of hybridization.

  In addition, by using such a capture probe to detect a target nucleic acid via an identification tag sequence associated with a specific partial sequence in the target nucleic acid, the target nucleic acid can be detected regardless of the target nucleic acid to be detected. It can be used as a universal solid phase carrier. Furthermore, such a solid phase carrier is suitable for simultaneously detecting a large number of polymorphisms or mutations of a target nucleic acid.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate. FIG. 1 is a diagram schematically showing the principle of the detection method of the present invention.

  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. In the present specification, the “target nucleic acid” is an arbitrary nucleic acid having an arbitrary sequence. Typically, it may have a base sequence that serves as a genetic indicator in humans or non-human animals, such as constitution, genetic disease, onset of specific diseases such as cancer, disease diagnosis, treatment prognosis, drug and treatment selection, etc. There may be mentioned nucleic acids with 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, a sample described later or a nucleic acid fraction thereof can be used as it is. However, preferably, all of a plurality of target nucleic acids to be detected are detected by an amplification reaction by PCR, more preferably an amplification reaction by multiplex PCR. It is preferable to use an amplified 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.

  In the present specification, the “polymorphism” is used in a broad sense including a single nucleotide polymorphism and other polymorphisms, and typically includes a single nucleotide polymorphism. 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. In the present specification, the “specific partial sequence” refers to a sequence containing at least one base constituting a polymorphism or mutation. Therefore, for example, in the single nucleotide polymorphism in which A is substituted with G, there are two types of specific partial sequences.

[Method for detecting polymorphism or mutation in target nucleic acid]
The detection method of the present invention includes a first contact step of bringing a first query probe set, a second query probe, and a target nucleic acid into contact with each other, and a step of linking the first query probe and the second query probe. And a labeling step, a second contact step in which the labeled linking molecule and the capture probe are brought into contact with each other, a signal intensity information acquisition step, and a detection step. The detection method of the present invention applies to one or more target nucleic acids, and a specific partial sequence related to polymorphism or mutation in these target nucleic acids is to be detected. Hereinafter, a series of steps for one type of target nucleic acid will be described. However, the following steps are also applied to the case where a plurality of target nucleic acids are detected simultaneously.

  FIG. 1 shows 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 the identification tag sequence and the melting temperature of the capture probe. An explanatory diagram is shown. 1 and 2 are examples of a detection method and probe set for detecting SNP single-base substitution.

<First contact process>
The first contact step is a step of bringing the first query probe set, the second query probe, and the target nucleic acid into contact with each other so as to be capable of hybridizing. First, the first query probe set and the second query probe used in this step will be described. As shown in FIGS. 1 and 2, the first query probe constituting the first query probe set includes a query sequence complementary to a specific partial sequence including at least one base to be detected of the target nucleic acid. And an identification tag sequence capable of identifying the specific partial sequence.

(First query probe set)
The first query probe set is composed of a plurality of first query probes. The first query probe can have a query sequence on its 3 ′ side and can have an identification tag sequence on its 5 ′ side. The arrangement of the query sequence and the identification tag sequence in the first query probe can be appropriately selected in relation to the second query probe.

  The first query probe is prepared according to the number of types of specific partial sequences to be detected. The first query probe is set to have one unique query sequence for each specific partial sequence, depending on the type of the specific partial sequence that may exist. That is, one type of first query probe is prepared for each specific partial sequence that may exist. For example, in the case of a single nucleotide polymorphism in which A is replaced with G as described above, there are two types of specific partial sequences, and two types of first query probes each having two types of query sequences are prepared. The In the case of mutation, two or three or more types of specific partial sequences exist, and two or three or more types of first query probes each having two or three or more types of query sequences are prepared. .

  Each first query probe can have such a query sequence. The query sequence is set complementarily to such an extent that it can hybridize with a specific partial sequence to be detected with high selectivity. Preferably, it is set to be completely complementary. The specific partial sequence is preferably arranged so as to constitute the terminal of the probe. For example, in the example shown in FIGS. 1 and 2, the specific partial sequence is arranged so as to form the 3 ′ end as it is. Preferably it is.

  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 30 bases or more, for example.

  Each of the first query probes has a unique identification tag sequence. The identification tag sequence can be set corresponding to a specific partial sequence that may exist in the target nucleic acid. The first query probe includes the identification tag sequence and the unique query sequence, respectively, so that the specific partial sequence in the target nucleic acid can be replaced with the identification tag sequence. That is, encoding or code conversion can be performed on a preset identification tag array.

  The tag sequence for identification can be set regardless of the base sequence of the target nucleic acid, that is, the specific partial sequence. The identification tag sequence can be made convenient for detection by setting it independently of the specific partial sequence, and any specific partial sequence can be obtained by using a capture probe corresponding to the predetermined identification tag sequence. Even so, it can be detected by a combination of a capture probe and an identification tag sequence.

  The tag sequence for identification and the capture sequence of a capture probe immobilized on a solid phase carrier to be described later are homologous or complementary, preferably a completely homologous sequence or a completely complementary sequence. Whether it is homologous or complementary to the capture sequence may be appropriately determined according to the design of the primer in the labeling step, and the complement that can selectively hybridize to the capture sequence after the labeling step. What is necessary is just to have sex. In the following description, an identification tag sequence having a sequence homologous to the capture sequence of the capture probe is described with (+) added, and an identification tag sequence having a complementary sequence thereto. Shall be described with (−) added. As the tag sequence for identification (+) and the capture sequence, for example, the base sequence described in SEQ ID NO: 1 to SEQ ID NO: 100 or a base sequence complementary to this 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.

The melting temperature of the identification tag sequence (+) and the capture sequence of the plurality of first query probes constituting the first query probe set is preferably as close as possible. More preferably, when comprehensively detecting a plurality of specific partial sequences of a plurality of target nucleic acids simultaneously, a plurality of identification tag sequences of a plurality of first query probes used for one target nucleic acid It is preferable that the melting temperatures of (+) and the capture sequence are combined so as to be closest to each other. For example, as shown in FIG. 3, when the identification tag sequence (+) and the capture sequence are arranged in the order of the melting temperature, the identification tag sequence (+) in two or more first query probes for one target nucleic acid. The capture sequence can be selected from two or more types of base sequences adjacent in the permutation. In addition, the identification tag sequence (+) and the capture sequence of the first query probe set corresponding to other target nucleic acids are selected from a series of base sequences immediately adjacent to the already selected base sequence as shown in FIG. Alternatively, it may be selected from a series of distant base sequences. The identification tag sequence (+) and capture sequence used in all the first query probe sets corresponding to the target nucleic acids to be detected at the same time use a series of base sequences in the permutation of the melting temperature. preferable. In addition, melting temperature is GC% method, Wallace method, Current.
It is possible to adopt a method calculated by a method based on Protocols in Molecular Biology (explained by PCR Jun 25, published by Shujunsha Bio Experiment Illustrated 3), but considering the range of melting temperature and base sequence concentration in the present invention. It is preferable to be calculated by a possible Nearest-Neighbor method. 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). SEQ ID NO: 1 to SEQ ID NO: 100 are Visual
Melting temperature calculated by OMP (0.1 M probe concentration, 50 mM Na ⁺ ion and 1.5 mM
(Mg ⁺ ions) are arranged in descending order.

Such a tag sequence for identification (+) and a capture sequence are also called orthonormalized sequences. For example, a DNA sequence having a predetermined base length obtained from a random number has a continuous match length, a melting temperature prediction by the Nearest-Neighbor method, It is designed by calculating Hamming distance and secondary structure prediction. An orthonormalized sequence is a nucleotide sequence of a 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. Orthonormalized arrays such as those described above are H.264. Yshida
and A. Suyama, "Solution to 3-SAT by breadth first search", DIMACS Vol. 54 9-20 (2000) and Japanese Patent Application No. 2003-108126. Orthonormalized sequences can be designed using the methods described in these references.

  By using the first query probe set as the first query probe set corresponding to each specific partial sequence that may exist in the target nucleic acid, it does not depend on the presence or absence of signals (signal intensity) of different labels. , Specific partial sequences can be detected. That is, in the conventional method, the SNP in the target nucleic acid is detected based on the presence or absence of signals (signal intensity) based on two types of labeling substances in the cell using the capture probe as one cell. According to the present invention, it is possible to individually detect specific partial sequences that can only be detected digitally by using a number of identification tag sequences and capture probes corresponding to specific partial sequences that may exist. And the ratio can be quantified. Further, the specific partial sequence can be detected without being restricted by the type of labeling substance that can be used. Furthermore, by using such a first query probe set, a target nucleic acid having a specific base sequence can be selectively extracted by combination with the second query probe. It contributes to the detection of a large number of target nucleic acids.

  When the polymorphism or mutation to be detected is a single nucleotide polymorphism, for example, the first query probe set can be composed of the following first query probes. That is, a first query probe having a query sequence complementary to a specific partial sequence containing one base of a single nucleotide polymorphism and an identification tag sequence capable of identifying the specific partial sequence, and the other of the single nucleotide polymorphism A query sequence complementary to a specific partial sequence containing the bases of the above and another first query probe having an identification tag sequence that can identify the specific partial sequence can be used. By using such a first query probe set, a single nucleotide polymorphism can be detected with high accuracy.

  When the polymorphism or mutation to be detected is a genetic mutation, for example, the first query probe set can be composed of the following first query probes. That is, a first query probe having a query sequence complementary to a specific partial sequence containing one or more bases normal for mutation and an identification tag sequence that can identify the specific partial sequence, and abnormal for mutation A query sequence complementary to a specific partial sequence containing one or more bases, and at least another first query probe of a first query probe having an identification tag sequence capable of identifying the specific partial sequence Can be used. By using such a first query probe set, mutations on the gene can be detected according to the variation, and the abundance ratios of various mutations can be detected with high accuracy.

(Second query probe)
The second query probe can include a query sequence complementary to a common partial sequence adjacent to a specific partial sequence of the target nucleic acid and a labeling tag sequence for adding a labeling substance. As shown in FIGS. 1 and 2, the second query probe can have a query sequence on its 5 ′ side, and can have a labeling tag sequence for adding a labeling substance on its 3 ′ side. it can. Similar to the first query probe, the arrangement of the query sequence and the identification tag sequence in the second query probe can be selected as appropriate.

  The query sequence that the second query probe has may be a sequence that is complementary to the extent that it can hybridize with high selectivity to the partial sequence adjacent to the specific partial sequence corresponding to the query sequence that the first query probe has. it can. Preferably it can be completely complementary. The query sequence of the second query probe is linked to the first query sequence by a ligase reaction in the linkage step described later. For this reason, it is preferable that the ligation side end such as the 5 'end side of the query sequence is phosphorylated so as to enable ligase reaction. The query sequence possessed by the second query probe does not correspond to a specific base portion such as a polymorphism or mutation that may exist in the target nucleic acid, and is a common base sequence conserved in the target nucleic acid. Is a complementary sequence. The query sequence in the second query probe can be designed in the same manner as the query sequence in the first query probe.

  The second query probe can include a labeling tag sequence for adding a labeling substance. Each has a unique identification tag sequence. The labeling substance is preferably one type (common) for at least one type of target nucleic acid. By making the labeling substance common, errors based on the characteristics of the labeling substance can be eliminated, and it is not necessary to detect the target nucleic acid based on the presence or absence or ratio of signals (signal intensity) based on different labeling substances. The labeling substance can be one kind (common) for two or more kinds of target nucleic acids, and different labeling substances can be appropriately used for different target nucleic acids.

  The tag sequence for labeling is not particularly limited, and a sequence convenient for adding a labeling substance may be appropriately set or selected from known sequences.

  The second query probe has a query sequence complementary to a partial sequence adjacent to the specific partial sequence and a tag sequence for labeling, so that the specific partial sequence in the target nucleic acid is obtained by the first contact step and the linking step described later. Can be selectively extracted and labeled. Also, such a second query probe contributes comprehensively to detecting a plurality of, preferably a large number of target nucleic acids.

  In the first contact step, these query probe and the target nucleic acid are brought into contact with each other so as to be hybridizable. Hybridization conditions can be appropriately set based on the target nucleic acid, a specific partial sequence thereof, and the length thereof. Preferably, a hybridization solution containing all these query probes is prepared in advance, and this hybridization solution is supplied to the target nucleic acid.

  According to the first contact step, when a predetermined specific partial sequence is present in the target nucleic acid by contacting these query probes with the target nucleic acid, the first probe having the query sequence corresponding to the specific partial sequence is provided. The query probe and the second query probe each hybridize to the target nucleic acid in a concatamer form (tandem form). When the predetermined specific partial sequence does not exist in the target nucleic acid, the first query probe does not hybridize to the target nucleic acid, but only the second query probe hybridizes.

(Linking process)
In the linking step, the first query probe and the second query probe hybridized to the target nucleic acid are linked to obtain these linked molecules. As described in the first contact step, when a predetermined specific partial sequence to be detected exists in the target nucleic acid, the first query probe and the second query probe having a query sequence corresponding to the specific partial sequence And hybridize to the target nucleic acid. As shown in FIGS. 1 and 2, in this state, the 5 ′ end of the first query probe and the 3 ′ end of the second query probe are connected.

The connection method of these probes is not particularly limited. An enzymatic reaction using a ligase such as DNA ligase can be used, and a chemical reaction can also be used. Preferably, the ligation reaction of these probes is performed by DNA ligase. Ligases that can be used to link include Thermus Aquaticus (Thermus
aquaticus) Taq DNA ligase, but not limited thereto, any DNA ligase or RNA ligase may be used depending on the type of target nucleic acid or probe.

  According to this linking step, the specific query sequence existing in the target nucleic acid is selectively extracted by linking the first query probe and the second query probe, and associated in advance with the specific query sequence. In addition, a linking molecule that can be replaced with a tag sequence for identification and can be labeled can be obtained according to the presence and abundance of the specific partial sequence. Such a linking molecule can be detected by a capture probe having a capture sequence that has been set in advance together with a tag sequence for identification by applying an appropriate label. That is, as a result, the target nucleic acid can be detected with the capture probe corresponding to the specific partial sequence.

  Therefore, according to the present linking step, the specific base in the target nucleic acid is efficiently obtained by performing the first contact step and the main linking step using the first query probe set and the second query probe. A sequence can be extracted based on its presence and abundance ratio, and a linking molecule capable of individually detecting a specific base sequence can be obtained by a universal capture probe.

  The first contacting step and the main linking step are continuous with the main linking step as a hybridization step prior to the linking step, particularly when the linking step is performed using an enzyme reaction. It can be carried out substantially as one process (also referred to as an encoding process).

  The linked molecule in which the first query probe and the second query probe are linked can be transferred to the next step without collecting only the linked molecule. Further, it can be recovered together with the target nucleic acid by an appropriate method. For example, the linking molecule may be recovered by a purification tool such as a column. Prior to the subsequent labeling step, the linking molecule is dissociated from the target nucleic acid. Dissociation can be achieved, for example, by denaturing processes including chemical denaturation and thermal denaturation. For example, when dissociating oligonucleotides linked by chemical denaturation, a treatment well known to those skilled in the art, such as alkali denaturation, may be performed. When dissociating the ligated oligonucleotide by thermal denaturation, the temperature should be 85 ° C. or higher, preferably 90 ° C. or higher under physiological conditions. Those skilled in the art will select an appropriate dissociation method. be able to.

(Labeling process)
The title step is a step of obtaining a labeled linked molecule by labeling the linked molecule with a single labeling substance using a labeling tag sequence provided in the linked molecule. By labeling the linking molecule in the labeling step, a labeled ligation that can detect the amount of hybridization to an individual capture probe corresponding to the specific base sequence in the detection step described later through the second contact step described later A molecule can be obtained.

  The labeling step can be performed by employing a known method of labeling with a labeling substance using a labeling tag. Preferably, a PCR amplification reaction is used. Primers can be appropriately designed according to the PCR technique employed. Specifically, a PCR amplification reaction is performed with a primer that hybridizes to the identification tag sequence held by the linking molecule and / or a primer that hybridizes to the labeling tag sequence held by the linking molecule and holds the labeling substance. At this time, amplification by normal PCR may be performed, or amplification by asymmetric PCR may be performed as an amplification method for obtaining a single-stranded amplification product. When a labeling substance is applied using a normal PCR amplification reaction, the identification tag sequence (+) derived from the first query probe is complementary to itself and is complementary to the capture sequence. Converted to (-). When asymmetric PCR is used, a labeled product maintaining the same identification tag sequence as before labeling can be obtained by using primers, or a sequence complementary to the identification tag sequence before labeling can be obtained. A converted labeled product can also be obtained. In any case, an identification tag sequence (-) that is a complementary sequence that can be selectively hybridized to the capture sequence after labeling may be obtained as the labeled product.

  As the labeling substance, conventionally known substances can be appropriately selected and used. It may be various dyes such as a fluorescent substance that emits a fluorescent signal when excited by itself, or may be a substance that emits various signals in combination with the second component by an enzyme reaction or an antigen-antibody reaction. Typically, a fluorescent labeling substance such as Cy3, Alexa555, Cy5, Alexa647 can be used.

  Prior to the second contact step in the subsequent stage, it is preferable to separate the labeled linked molecule to which a labeling substance has been added by amplification and to make it a single strand. For the separation, a known method such as using a solid phase support such as a magnetic bead coated with biotin can be adopted, and for the single strand formation, the above-described chemical denaturation method or thermal denaturation method is adopted. it can.

(Second contact step)
In the second contact step, these capture probes are labeled on a solid phase carrier on which two or more types of capture probes having a capture sequence complementary to the identification tag sequence (-) in the labeled linking molecule are immobilized. This is a step of bringing a linking molecule into contact with each other so as to be hybridizable. According to this step, when the identification tag sequence (-) in the labeled linking molecule is complementary to the capture sequence in the capture probe to the extent that it can selectively hybridize under certain conditions, these are hybridized. Soybeans form a duplex at a given capture probe on a solid support. When the labeled linking molecule does not have an identification tag sequence (-) complementary to the capture probe, no duplex is formed on the solid phase carrier. After the first contact step, a cleaning step may be further included as appropriate.

(Solid phase carrier with capture probe immobilized)
In the second contact step, a solid phase carrier on which two or more types 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.

  The solid phase carrier used in the second contact step is preferably an array having an arrangement state in which two or more types of capture probes associated with a specific partial sequence to be detected in the target nucleic acid are adjacent to each other. By being adjacent to each other, it is possible to suppress a variation derived from the position where the hybridization capture probe is immobilized on the array, and to detect a specific partial sequence in the target nucleic acid with high accuracy.

  A preferred solid phase carrier is an array having an array state in which two or more types of capture probes associated with a specific site sequence to be detected in the target nucleic acid are arranged in an order based on the melting temperature. Using such an array, two or more types of capture probes corresponding to each target nucleic acid, in other words, identification tag sequences are assigned in the order of the capture probes, resulting in a difference in the melting temperature of the capture sequences. The specific partial sequence in the target nucleic acid can be detected with high accuracy by suppressing the variation in hybridization. An example of a preferred array is shown in FIG.

  A more preferable solid phase carrier has an arrangement state in which two or more types of capture probes corresponding to a specific partial sequence to be detected in the target nucleic acid are adjacent to each other, and the maximum temperature difference in melting temperature between these adjacent capture probes The array is arranged so that (absolute value) is 5 ° C. or less, preferably 0.2 ° C. or less. 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.

  In addition, a preferred solid phase carrier has two or more capture probes for one target nucleic acid, and a capture sequence having the same base length set independently of the base sequence to be detected in the target nucleic acid is used. And an array having a melting temperature of 40 ° C. or higher and 80 ° C. or lower (preferably 50 ° C. or higher and 70 ° C. or lower, more preferably 55 ° C. or higher and 65 ° C. or lower) and arranged in the order based on the melting temperature. . According to such an array, the specific partial sequence of the target nucleic acid is converted into a tag sequence for identification that is complementary to the extent that it can hybridize with the capture sequence, so that any specific partial sequence can be supported. It can be used as an array. Moreover, by providing two or more, preferably 64 or more, more preferably 100 or more, multiple capture probes, multiple target nucleic acids or a plurality of specific partial sequences in the target nucleic acid can be detected simultaneously. . Furthermore, since these capture probes all have the same base length and a melting temperature of 40 ° C. to 80 ° C. (preferably 50 ° C. to 70 ° C., more preferably 55 ° C. to 65 ° C.), uniform hybridization can be achieved. This can be realized, and a specific partial sequence in the target nucleic acid can be detected and quantified with high accuracy.

  The plurality of capture probes immobilized on the solid phase carrier can each have a capture sequence selected from SEQ ID NO: 1 to SEQ ID NO: 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 about the target nucleic acid based on the labeling substance on the solid phase carrier after hybridization. According to the signal intensity information acquisition process in this case, the identification tag sequence (-) corresponding to the specific partial sequence in the target nucleic acid in the labeled linking molecule is hybridized with the capture probe so that it is associated with the capture probe. Signal intensity information can be obtained based on the labeling substance. The signal intensity information associated with such a capture probe can be obtained by the number of specific partial sequences present in the target nucleic acid.

  Since the specific partial sequence in the target nucleic acid is associated with the identification tag sequence and the capture sequence in advance, by acquiring the signal intensity information associated with the predetermined capture probe, the presence or absence of the specific partial sequence in the target nucleic acid The amount or the like can be quantified.

  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 based on signal intensity information obtained corresponding to each capture probe associated with each specific partial sequence via each identification tag sequence of each first query probe in the first query probe set. , A step of detecting the presence or absence of the polymorphism or the mutation in one target nucleic acid. According to such a detection process, a specific portion in the target nucleic acid is determined based on the signal intensity information based on the same labeling substance for different capture probes, regardless of the presence or intensity of signals based on different labeling substances for the same capture probe. The sequence can be detected. For this reason, it is possible to accurately detect each specific partial sequence, and as a result, it is possible to accurately detect and quantify the presence or absence or ratio of a polymorphism or mutation.

  In order to detect a polymorphism or mutation in the target nucleic acid, for example, it may be a step of detecting the presence or absence of the polymorphism or mutation to be detected based on the ratio of signal intensities obtained for two or more types of capture probes. . As described above, according to this detection method, a capture probe is prepared for each specific partial sequence that constitutes a polymorphism or mutation, and signal intensity information can be obtained for these capture probes. Presence / absence of polymorphism (type) and presence / absence of mutation can be accurately detected based on a simple signal intensity ratio between corresponding capture probes.

  Moreover, as shown in FIG. 1, based on the ratio of the sum of all signal intensities obtained corresponding to two or more capture probes to one signal intensity obtained corresponding to two or more types of capture probes, The polymorphism type discrimination or the mutation ratio in the one target nucleic acid may be obtained. That is, the formula: signal intensity obtained for a capture probe corresponding to one specific partial sequence / (sum of signal intensity obtained for two or more types of capture probes each corresponding to a specific partial sequence that may exist in the target nucleic acid ) Can be used to determine the abundance ratio of the one specific partial sequence in the target nucleic acid. The presence / absence and type of the polymorphism, the presence / absence of the mutation, and the mutation rate can be obtained by this abundance ratio. According to such a detection process, the type of mutation and the mutation rate can be obtained with high accuracy. In addition, even if there are three or more types of specific partial sequences that may exist in one target nucleic acid, if the first query probe having a tag sequence for identification is set according to the number, these can be easily and simultaneously The specific partial sequence can be detected and the ratio thereof can be quantified.

  When the present detection method is to detect a single nucleotide polymorphism, for example, for one or more reference oligonucleotides prepared in advance and having the type of the polymorph to be detected, the first Performing the contact step, the linking step, the labeling step, the second contact step, and the signal intensity information acquisition step, and using the reference signal intensity information acquired for the reference oligonucleotide, the detection step Can be implemented. By doing so, it is possible to correct a bias that may occur in the linking efficiency between specific partial sequences in the linking step, that is, between different first query probes.

  In addition, when the present detection method is for detecting a gene mutation, for example, for one or more reference oligonucleotides prepared in advance and having a predetermined ratio for the mutation to be detected, the first Performing the first contact step, the linking step, the labeling step, the second contact step, and the signal intensity information acquisition step, and using the reference signal intensity information acquired for the reference oligonucleotide, the detection A process can be performed. By doing so, as described above, it is possible to correct a bias that may occur in the coupling efficiency between different first query probes.

  Regarding such bias correction, preferably, a calibration curve is prepared based on reference signal intensity information acquired for two or more kinds of reference oligonucleotides, and a polymorphism in a target nucleic acid in a sample is obtained using the calibration curve. And mutations can be detected and quantified. By using the calibration curve, the above-described bias can be corrected more effectively, and 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 simultaneously with detection of the target nucleic acid.

  A capture probe with a similar melting temperature is used, but there may be a bias in the amount of hybridization between capture probes due to the melting temperature and other circumstances, but by performing each step as follows: This can be suppressed or avoided.

  Further, for example, in the second contact step, together with the labeled linking molecule, each identification tag sequence (−) of each first query probe in the first query probe set and the single labeling substance are Signal intensity information acquisition step, wherein two or more kinds of correction oligonucleotides, which are provided with different different labeling substances and are prepared at the same concentration, are brought into contact with the capture probe so as to be hybridizable on a solid phase carrier. To acquire the signal intensity information about the target nucleic acid, acquire the correction signal intensity information based on the other labeling substance about the correction oligonucleotide, and use the correction signal intensity information in the detection step It may be. For example, when other labeling substances are used at the same concentration and there is a difference in the signal intensity obtained between the capture probes, such a difference in signal intensity is also present in the linking molecule. As a correction. Such correction using the signal intensity information for correction can be applied to obtain both the presence / absence of polymorphism (type), the presence / absence of mutation, and the mutation rate, as well as the correction based on the signal intensity information for reference already described. can do.

  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 for detecting the target nucleic acid in the sample)
The probe set of the present invention comprises a query sequence complementary to a specific partial sequence containing at least one base in a polymorphism or mutation to be detected in a target nucleic acid, and an identification tag sequence capable of identifying the specific partial sequence A first query probe set comprising two or more types of first query probes, and a tag for labeling for adding a query sequence complementary to a partial sequence adjacent to the specific partial sequence of the target nucleic acid and a label A second query probe comprising an array.

  For the first query probe, the second query probe, and the like in the probe set of the present invention, various embodiments in the range described above can be applied as they are.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples. However, the following examples illustrate the present invention and do not limit the scope of the present invention.

Detection of gene mutation (SNP) by the detection method of the present invention was performed by the following procedure. Hereinafter, it demonstrates according to these order.
(1) Preparation of DNA microarray for detecting mutant genes (2) Amplification of sample genes (3) Hybridization and ligation (encoding reaction)
(4) Labeling reaction (5) Detection using DNA microarray (6) Data analysis

(1) Preparation of DNA microarray for detecting gene mutation (SNP) Aqueous solution in which synthetic oligo DNA (manufactured by Nippon Genetic Laboratory Co., Ltd.) in which 5 ′ end is modified with amino group is dissolved in gene slide glass substrate manufactured by Toyo Kohan As a capture probe at NGK Corporation using a GENESHOT (registered trademark) spotter. As shown in Table 1, the synthetic oligo DNA sequences used are supplementary in the literature (Analytical Biochemistry 364 (2007) 78-85).
N1 types D1_52, D1_71, D1_93, D1_74, D1_41, D1_92, D1_79, D1_78, and D1_65 are listed in descending order of Tm from D1_1 to D1_100 shown in Table 1. After the spot, baking was performed at 80 ° C. for 1 hour. In addition, these Tm is the melting temperature (0.1M probe concentration, 50 mM) calculated by Visual OMP.
Na ⁺ ions and 1.5 mM Mg ⁺ ions) are arranged in descending order.

  Furthermore, the synthetic oligo DNA was immobilized by the procedure described below. That is, after washing with 2 × SSC / 0.2% SDS for 15 minutes, washing with 95 ° C. 2 × SSC / 0.2% SDS for 5 minutes, and then washing with sterilized water (shaking up and down 10 times) was repeated three times. Thereafter, it was dehydrated by centrifugation (1000 rpm × 3 minutes).

(2) Preparation and amplification of target nucleic acid having gene mutation The base sequence containing the gene mutation to be detected is about 150 bp which is the peripheral sequence of the mutation shown in Table 2 below, and an artificial gene consisting of these base sequences is selected. The target nucleic acid was used. A total of 4 sets of target nucleic acids were synthesized.

These samples were then amplified as follows. Note that TaKaRa Ex Taq (catalog # RR001B) manufactured by TaKaRa Bio was used as a sample amplification reagent. Applied as a thermal cycler
A Biosystems GeneAmp PCR System 9700 was used. The PCR primer used was designed to have a range of Tm = 58.90 ± 0.75 ° C. Each primer sequence is shown in Table 3. Tm is a software (Oligo) provided by Japan Genetic Research Institute (http://www.ngrl.co.jp/).
Calculator: http://www.ngrl.co.jp/tool/ngrl_tool.html) was used to calculate based on the following formula.
Tm = ((ΔH * 1000 / (ΔS + 41.3))-273.15-21.5971) (wherein the salt concentration is 0.05M, the primer concentration is 250 pmol, and ΔS is the predetermined nearest-entropy change in the hybrid) (A / H represents the sum (kcal / mol) of the predetermined closest entropy change in the hybrid.)

First, the following reagents were prepared for each sample (detection mutant). In addition, sample DNA made the wild gene: mutant gene ratio into five kinds, 100: 0, 95: 5, 90:10, 75:25, 50:50 for every detection variant, and prepared a total of 20 samples ( 5 types x 4 variants = 20 samples in total).
(Reagent preparation)
Primer mix: F, R mix (20 μM, each) 0.5 μl
Ex Taq buffer (10 ×) 1.0μl
Ex Taq polymerase (5 Unit / μl) 0.1μl
dNTP mix (2.5 nM) 0.8 μl
dH ₂ O 7.1μl
Subtotal 9.5μl
Sample DNA (10ng / μl) 0.5μl
Total 10.0μl

  Next, a thermal cycle reaction (after 94 seconds at 94 ° C .; 40 seconds at 94 ° C., 40 seconds at 66 ° C., 40 cycles of 2 minutes at 72 ° C .; then lowered to 4 ° C.) was performed. Each of the individually amplified products (total 4 mutants) was mixed in a tube for each mutant gene ratio to obtain a PCR product. As a result, for each mutant species, five types (5 tubes) of PCR products having wild gene: mutant gene ratios of 100: 0, 95: 5, 90:10, 75:25, and 50:50 were prepared.

(3) Encoding Reaction The encoding reaction for the PCR product of the sample DNA obtained in Example 2 was performed as follows. For the encoding reaction, Taq DNA Ligase from New England Biolabs (Catalog
# M0208S) was used. GeneAmp PCR System 9700 (Applied Biosystems) was used as a thermal cycler for heating. Table 4 shows the sequence of a 5 ′ query probe (first query probe in the method of the present invention) having a complementary query sequence (including a detection base) and an identification tag sequence on the 5 ′ side of the sample DNA. . The 5 ′ query probe has an identification tag sequence that is homologous to the capture probe sequence shown in Table 1 on the 5 ′ side, and a query sequence on the 3 ′ side. Table 5 shows the sequence of a 3 ′ query probe (second query probe in the method of the present invention) comprising a complementary query sequence (not containing a detection base) and a labeling tag sequence on the 3 ′ side of the sample DNA. Show. The 3 ′ query probe has a query sequence adjacent to the query sequence of the 5 ′ query probe on the 5 ′ side, and a tag sequence for labeling on the 3 ′ side. The 5 ′ end of the 3 ′ query probe is phosphorylated.

In the encoding reaction, first, the following reagents were prepared. Reagents were prepared for each PCR product in the ratio of five wild genes: mutant genes for each mutant prepared in Example 2. Next, the encoding reaction was performed by heating. In the encoding reaction, heating was performed at 95 ° C. for 5 minutes, 50 ° C. for 1 minute, and 58 ° C. for 60 minutes, and then lowered to 10 ° C. Five ligation products were obtained for each variant.
(Reagent preparation)
2 types of 5 ′ query probes / 3 ′ query probes Mix (2.5 μM, each)
0.300μl
Taq DNA ligase buffer (10x) 1.500μl
dH ₂ O 1.075μl
Taq DNA ligase (40 Units / μl) 0.125 μl
Subtotal 3.000μl
PCR product (prepared for each mutant gene ratio) 12.000 μl
Total 15.000μl

(4) Labeling reaction The labeling reaction of the ligation product obtained in Example 3 was performed as follows. For the labeling reaction, TaKaRa Bio's TaKaRa Ex Taq (Catalog #
RR001B) was used. A GeneAmp PCR System 9700 from Applied Biosystems was used as the thermal cycler. Asymmetry PCR was used for labeling, and various D1 primers having the same sequence as shown in Table 1 and primers complementary to the labeling tag sequence and labeled with Alexa 555 on the 5 ′ side (Table 6) were used. .

  In the labeling reaction, first, the following reagents were prepared for 5 types of ligation products per 4 types of variants. Subsequently, thermal cycling reaction was performed about the prepared reagent. That is, after 1 minute at 95 ° C .; 30 cycles of 95 ° C. for 30 seconds, 55 ° C. for 6 minutes and 72 ° C. for 30 seconds; Five labeled products were obtained per four variants.

(Reagent preparation)
D1_Primer Mix (50 μM, each) 0.12 μl
Alexa555-ED-1 (200 μM) 0.24 μl
Ex Taq buffer (10 ×) 1.20μl
dNTP mix (2.5 nM) 0.96μl
Ex Taq polymerase (5 Unit / μl) 0.12μl
Milli-Q water 8.36μl
Subtotal 11.00μl
Ligation product 1.00μl
Total 12.00μl

(5) Detection using DNA microarray The reaction to the DNA microarray using the labeled product and the detection procedure were as follows.

(Preparation of labeled sample solution for hybridization)
First, a hybridization correction solution and a hybridization solution were prepared with the following compositions.

(Composition of labeled sample solution for hybridization)
Hybridization correction solution 1.5μl
Hybridization solution 9.0μl
Subtotal 10.5μl
7.5 μl of labeled product
18.0μl in total

(Hybridization correction liquid composition (2.5nM, each))
Alexa647-rD1_52 (100nM) 10μl
Alexa647-rD1_71 (100nM) 10μl
Alexa647-rD1_93 (100nM) 10μl
Alexa647-rD1_74 (100nM) 10μl
Alexa647-rD1_41 (100nM) 10μl
Alexa647-rD1_92 (100nM) 10μl
Alexa647-rD1_79 (100nM) 10μl
Alexa647-rD1_78 (100nM) 10μl
Alexa647-rD1_65 (100nM) 10μl
Alexa555-rD1_65 (100nM) 10μl
TE (pH 8.0) 300μl
400μl

  The Alexa555 or 647-labeled oligo DNA used in the hybridization correction solution was labeled with Alexa555 or 647 at the 5 'end of the sequence complementary to the capture probe sequence shown in Table 1.

(Liquid composition for hybridization (× 2))
20 x SSC 2.0ml
10% SDS 0.8ml
100% Formamide 12.0ml
100 mM EDTA 0.8ml
Milli-Q water 24.4ml
Total 40.0ml

(Hybridization)
Next, the prepared labeled sample solution was heated at 90 ° C. for 1 minute using Gene Bio PCR System 9700 from Applied Biosystems, and then heated at 80 ° C. for 1 minute using a heat block (TAITEC DTU-N). Apply 9 μl each of the sample solution to the spot area of the DNA microarray, and use a comfort / plus thermoblock slide (manufactured by Eppendorf) to prevent drying, and leave it at 37 ° C. for 60 minutes to carry out a hybridization reaction. It was.

(Washing)
After hybridization, the glass substrate after completion of the hybridization reaction is immersed in a glass staining vat filled with a cleaning solution having the following composition, shaken up and down for 5 minutes, and transferred to a glass staining vat containing sterilized water. It was shaken up and down for 1 minute, and then dried by centrifugation at 2000 rpm for 1 minute to remove moisture remaining on the glass substrate surface.
(Composition of cleaning solution)
Milli-Q water 188.0ml
20 x SSC 10.0ml
10% SDS 2.0ml
Total 200.0ml

(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.

(6) Data analysis In order to confirm the presence or absence of the difference in hybridization efficiency (amount) between captures, the fluorescence signal of the hybridization correction solution hybridized simultaneously was measured. Table 7 shows the measurement results (Alexa647) of the spots for detecting mutation 1 gene (D1_52 and D1_71).

  As shown in Table 7, 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 great difference in the amount of hybridized adsorption between these 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 measurement results (Alexa555) are shown in Table 8. Further, based on the results in Table 8, the horizontal axis is the mutant gene ratio, and the vertical axis is plotted as B / (A + B) to create a calibration curve. A calibration curve is shown in FIG.

  As shown in Table 8 and FIG. 4, it was found that the presence of the mutant type in the mutant gene can be easily detected, and it can be easily detected with high accuracy even at a low mutation rate. It was also found that the ratio of wild type: mutant type could be detected easily and accurately. Furthermore, it was found that by using such a calibration curve, mutations can be detected more accurately and the ratios can be quantified. Moreover, according to the present invention, one type of first query probe is prepared for each type of specific partial sequence that may be present in the target nucleic acid, and for each of these first query probes. It was found that the presence of the specific partial sequence and the existence ratio thereof can be detected with high accuracy by preparing the capture probe.

It is a figure which shows typically the outline | summary of the detection method of this invention. It is a figure explaining the 1st query probe set and 2nd query probe used for this invention. It is a figure which shows an example of the tag sequence for identification used in this invention, and a capture arrangement | sequence. 10 is a graph showing a calibration curve created in Example 6. FIG. It is a figure which shows an example of the conventional mutation detection method.

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: 5 ′ query probes, SEQ ID NOs: 134 to 137: 3 ′ query probes, sequences Number 138: Primer

Claims (13)

A method for detecting a polymorphism or mutation in one or more target nucleic acids in a sample, comprising:
A tag sequence for identification capable of discriminating the specific partial sequence from a query sequence complementary to the specific partial sequence containing at least one base constituting the polymorphism or the mutation to be detected in at least one target nucleic acid A first query probe set comprising two or more types of first query probes, and a label for adding a query sequence complementary to a partial sequence adjacent to the specific partial sequence of the target nucleic acid and a label A first contact step in which a second query probe comprising a tag sequence and a target nucleic acid are brought into contact in a hybridizable manner;
A linking step of linking the first query probe and the second query probe hybridized to the one type of target nucleic acid to obtain these linking molecules;
A labeling step of obtaining a labeled linked molecule by labeling the linked molecule with a single labeling substance using the labeling tag sequence provided in the linked molecule;
These capture probes and the labeled linking molecule are immobilized on a solid phase carrier having a capture sequence complementary or homologous to the identification tag sequence and having two or more types of capture probes associated with the specific partial sequence immobilized thereon. A second contacting step in which the contact is made in a hybridizable manner,
A signal intensity information acquisition step of acquiring two or more types of signal intensity information for the one target nucleic acid based on the labeling substance on the solid phase carrier after hybridization;
A detection step of detecting the polymorphism or the mutation in the one type of target nucleic acid based on the two or more types of signal intensity information;
With
In the second contacting step, each of the identification tag sequence of each of the first query probes in the first query probe set and the single labeling substance are different from each other together with the labeled linking molecule. A correction oligonucleotide comprising a labeling substance and prepared at the same concentration with each other, and a step of contacting the capture probe on the solid phase carrier so as to be capable of hybridizing,
The signal intensity information acquisition step is a step of acquiring signal intensity information for the one target nucleic acid and acquiring correction signal intensity information based on the other labeling substance for the correction oligonucleotide,
In the detection step, the first signal with respect to the signal intensity obtained corresponding to the capture probe associated with the specific partial sequence via the identification tag sequence of one first query probe in the first query probe set. Based on the ratio of the sum of the signal intensities obtained corresponding to all the capture probes associated with the specific partial sequence via the identification tag sequences of all the first query probes in one query probe set, and Using the correction signal intensity information, the step of determining the type of the polymorphism in the one target nucleic acid or the ratio of the mutation,
The solid phase carrier has two or more capture probes, has a capture sequence of the same base length set independently of the base sequence of the polymorphism or the mutation of the target nucleic acid, and has a melting temperature of 50 ℃ ≧ 70 ℃,
The capture sequence has a base sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100, respectively .   For one or more reference oligonucleotides that are prepared in advance and have the polymorphic type to be detected, the first contact step, the linking step, the labeling step, the second contact step, and The method according to claim 1, wherein the signal intensity information acquisition step is performed, and the detection step is performed using reference signal intensity information acquired for the reference oligonucleotide.   The first contact step, the linking step, the labeling step, and the second contact step for one or more reference oligonucleotides prepared in advance and having a predetermined ratio for the mutation to be detected 3. The method according to claim 2, wherein the signal intensity information acquisition step is performed, and the detection step is performed using the reference signal intensity information acquired for the reference oligonucleotide.   The method according to claim 2 or 3, wherein the detection step is carried out using a calibration curve based on reference signal intensity information acquired for two or more types of reference oligonucleotides.   The polymorphism or mutation to be detected is a single nucleotide polymorphism, and the first query probe set includes a query sequence complementary to a specific partial sequence containing one base of the single nucleotide polymorphism and the specific portion. A first query probe having an identification tag sequence capable of identifying a sequence, a query sequence complementary to a specific partial sequence including the other base of a single nucleotide polymorphism, and an identification capable of identifying the specific partial sequence The other 1st query probe which has a tag sequence for use, The method in any one of Claims 1-4.   The polymorphism or mutation to be detected is a mutation, and the first query probe set includes a query sequence complementary to a specific partial sequence containing one or more bases normal to the mutation and the specific portion. A first query probe having an identification tag sequence that can identify a sequence, a query sequence complementary to a specific partial sequence containing one or more bases abnormal for the mutation, and the specific partial sequence can be identified The method according to claim 1, comprising at least two types of first query probes and a first query probe having an identification tag sequence.   The method according to claim 1, wherein the solid phase carrier is an array having an arrangement state in which the two or more types of capture probes for the at least one type of the target nucleic acid are adjacent to each other.   The solid phase carrier is an array having an arrangement state in which the two or more types of capture probes for the at least one target nucleic acid are arranged in an order based on the melting temperature thereof. The method described.   The solid phase carrier has an arrangement state in which two or more types of capture probes corresponding to a specific partial sequence to be detected in the target nucleic acid are adjacent to each other, and a maximum temperature difference in melting temperature between these adjacent capture probes The method according to claim 1, wherein (absolute value) is arranged to be 5 degrees or less. A solid phase carrier to which a capture probe is immobilized, which is used in the detection method according to claim 1,
The solid phase carrier is a solid phase carrier in which the two or more types of capture probes for the at least one target nucleic acid are arranged in an order based on the melting temperature thereof. The solid phase carrier has two or more capture probes, each having a capture sequence of the same base length set independently of the base sequence of the polymorphism or the mutation of the target nucleic acid, and having a melting temperature The solid phase carrier according to claim 10, which is 50 ° C or higher and 70 ° C or lower .   The solid-phase carrier according to claim 10 or 11, wherein each of the capture probes has a capture base sequence selected from SEQ ID NO: 1 to SEQ ID NO: 100. A set of probes and the like used in the method for detecting a target nucleic acid in a sample according to any one of claims 1 to 9 ,
Two or more types comprising a query sequence complementary to a specific partial sequence containing at least one base constituting a polymorphism or mutation to be detected in the target nucleic acid, and an identification tag sequence capable of identifying the specific partial sequence A first query probe set comprising:
A second query probe comprising a query sequence complementary to a partial sequence adjacent to the specific partial sequence of the target nucleic acid and a tag sequence for labeling for adding a label;
For correction using the identification tag sequence of each of the first query probes in the first query probe set and another labeling substance different from the single labeling substance and prepared at the same concentration. An oligonucleotide;
A probe set comprising: