JPWO2014156513A1 - Mutation detection method - Google Patents

Abstract

A DNA sample that may have a mutation, a first query probe having a first identification sequence for identifying a mutation, and a second query having a second identification sequence for identifying a region adjacent to the mutation A probe is brought into contact with each other, a DNA ligase reaction is performed, and the first identification sequence and the second identification sequence are hybridized specifically with the probe detection sequence corresponding to the type of mutation. A ligation product each having a sequence for labeling and a labeling sequence for imparting a label, the ligation product, a first primer having a sequence complementary to the hybridization sequence, and the labeling sequence An amplification product comprising the labeling sequence and the hybridizing sequence by a DNA polymerase amplification reaction in contact with a second primer having Obtaining a hybridized product by contacting the probe on the solid phase carrier with the amplification product, and the total concentration of the second primer is equal to the total concentration of the first primer. 0.5 times or more and less than 12.5 times.

Description

The present specification relates to a method for detecting a mutation in DNA.
This application is a related application of Japanese Patent Application No. 2013-72897, which is a Japanese application filed on March 29, 2013, and claims priority based on this Japanese application. All contents are incorporated.

  Detection of various mutations such as SNPs in genes is useful in medical diagnosis and the like. Conventionally, as a general method for detecting this type of mutation, there is a method using a DNA array in which a mutation-specific DNA probe is immobilized on a solid phase carrier. In this method, a mutation is detected through a fluorescent signal generated at a unique position on the DNA array by hybridization between a DNA probe on the DNA array and an amplification product possibly containing the mutation obtained from a biological sample.

  Various improvements have also been made in order to make the detection of mutations by such DNA arrays quicker and easier. For example, a method for detecting specific hybridization visually instead of a fluorescent signal is disclosed (Patent Document 1, Non-Patent Document 1). In these methods, the amplification product is labeled with a known coloring material or enzyme reaction substrate for visual detection.

  Furthermore, in order to omit the design and synthesis of mutation-specific DNA probes, a technique has been developed that can detect all SNs with a universal DNA array that is not SNP-specific but has a universal probe immobilized thereon (non-patent literature). 2). In this method, a query probe set for obtaining a ligation product having a base sequence that can specifically hybridize with a previously associated universal probe and specific for a mutation is prepared, and a ligation reaction is performed. Then, a primer set for labeling the ligation product is prepared and an amplification reaction is performed. Then, this labeled amplification product is provided to a DNA array on which a universal probe is immobilized.

International Publication WO2013 / 002185 Pamphlet

JARMAM Vol.4 (1), p.45-50,2003 Determination of drug resistance of clinically isolated M. tuberculosis using DNA microarray Analytical Biochemistry_364_1_2007_78-85

  According to the present inventors, when trying to establish a DNA array method in which visual detection and the use of a universal probe are applied at the same time, the detection sensitivity may decrease depending on conditions, such as a decrease in S / N ratio. all right.

  The present specification provides a method for detecting a mutation that can realize visual detection and use of a universal probe at the same time and can stably ensure good mutation detection ability.

Means for solving the problem

  As a result of various studies on the detection of mutation by visual observation and the use of universal probes, the present inventors have obtained the following knowledge. That is, it was found that there was a decrease in specificity or ligation efficiency in the ligation reaction described above, and a decrease in reaction efficiency in the amplification reaction described above. Furthermore, according to the study by the present inventors, by improving the ligation reaction and amplification reaction, the above-mentioned problems are solved, the S / N ratio is ensured, and good detection sensitivity, detection accuracy and reproducibility are also obtained. It turned out that it can secure. According to the present specification, the following means are provided based on these findings.

(1) A method for detecting mutations,
A DNA sample possibly having the mutation, a first query probe having a first identification sequence for identifying at least a part of the mutation, and a second identification sequence for identifying a region adjacent to the mutation A first query sequence according to the type of mutation, wherein the first query probe and the second query probe are linked by a DNA ligase reaction. And a second identification sequence, a hybridization sequence or a complementary sequence that specifically hybridizes with a detection sequence of a probe associated in advance according to the type of mutation, and a labeling sequence for imparting a label, or Obtaining one or more ligation products each having a complementary sequence in association with the mutation;
The label is obtained by a DNA polymerase amplification reaction by contacting the one or more ligation products, a first primer having a sequence complementary to the hybridization sequence, and a second primer having the labeling sequence. Obtaining one or more amplification products comprising the sequence for use and the sequence for hybridization;
Contacting the probe of the solid phase carrier holding the probe with the one or more amplification products to obtain a hybridization product of the probe and the amplification product;
Detecting the hybridized product based on a label imparted via the labeling sequence;
The total concentration of the second primer is 0.5 times or more and less than 12.5 times the total concentration of the first primer.
(2) The method according to (1), wherein the ligation product acquisition step is performed in the presence of a mismatch binding protein.
(3) The method according to (1) or (2), wherein the ligation product obtaining step is performed by repeating the ligation reaction with the DNA ligase a plurality of times.
(4) The method according to any one of (1) to (3), wherein the total concentration of the second primer is not more than 5 times the total concentration of the first primer.
(5) The method according to any one of (1) to (5), wherein the step of obtaining the hybridized product is performed in a chromatographic form.
(6) The method according to any one of (1) to (5), wherein the second primer holds the label or a label-binding substance capable of binding the label.
(7) The method according to any one of (1) to (6), wherein the label is a visible coloring material.
(8) The method according to any one of (1) to (7), wherein the probe is selected from the group consisting of a base sequence represented by SEQ ID NOs: 1 to 100 and a complementary sequence thereof.
(9) The detection method according to any one of (1) to (8), wherein the solid phase carrier is any one selected from the group consisting of polyethersulfone, nitrocellulose, nylon, polyvinylidene fluoride, and filter paper. .
(10) The detection method according to any one of (1) to (9), wherein the mutation is any one of substitution mutation, insertion mutation, and deletion mutation of one or several bases.
(11) A kit for detecting a mutation,
A first identification sequence that identifies at least a part of the mutation, and a hybridization sequence that specifically hybridizes with a detection sequence of a probe associated in advance according to the type of the mutation, or a complementary sequence thereof. A second query probe set having one query probe, a second identification sequence for identifying a region adjacent to the mutation, and a labeling sequence or a complementary sequence thereof, or the first identification sequence and the labeling A query probe set comprising: a first query probe having a sequence or a complementary sequence thereof; and a second set of query probes having the second identification sequence and the hybridizing sequence or a complementary sequence thereof; ,
A primer set comprising: a first primer having a sequence complementary to the hybridizing sequence; and a second primer having the labeling sequence;
A mismatch binding protein;
A kit comprising:
(12) The kit according to (11), wherein the total concentration of the second primer is 0.05 times or more and less than 12.5 times the total concentration of the first primer.
(13) The kit according to (11) or (12), wherein the solid phase carrier is a chromatographic carrier having liquid permeability or permeability.
(14) A method for producing DNA associated with mutation,
A DNA sample possibly having the mutation, a first query probe having a first identification sequence for identifying at least a part of the mutation, and a second identification sequence for identifying a region adjacent to the mutation A first query sequence according to the type of mutation, wherein the first query probe and the second query probe are linked by a DNA ligase reaction. And a second identification sequence, a hybridization sequence for hybridizing with a detection sequence of a probe associated in advance according to the type of mutation, or a complementary sequence thereof, and a labeling sequence for imparting a label, or Obtaining one or more ligation products each having a complementary sequence in association with the mutation;
The label is obtained by a DNA polymerase amplification reaction by contacting the one or more ligation products, a first primer having a sequence complementary to the hybridization sequence, and a second primer having the labeling sequence. Obtaining one or more amplification products having a sequence for hybridization and a sequence for hybridization;
The method wherein the ligation product obtaining step is performed in the presence of a mismatch binding protein.
(15) The method according to (14), wherein the total concentration of the second primer is 0.5 times or more and less than 12.5 times the total concentration of the first primer.

It is a figure which shows the outline | summary of the detection method disclosed by this specification. It is a figure which shows an example of the query probe set used for the detection method disclosed by this specification. It is a figure which shows an example of the ligation product and the amplification product in the detection method disclosed in this specification. It is a figure which shows the outline | summary of the ligation process in the detection method disclosed by this specification. It is a figure which shows the outline | summary of the amplification process of the detection method disclosed by this specification. It is a figure which shows another example of a query probe set. It is a figure which shows the result in the reference example 1. It is a figure which shows the spot pattern of the probe in the array used in the reference example 2. It is a figure which shows the detection result in the reference example 2 and Example 1. FIG. 6 is a diagram showing a spot pattern of a probe in the chromatography used in Example 2. FIG. It is a figure which shows the detection result in Example 2. FIG. It is a figure which shows the detection evaluation result about SNP1 and SNP2 in Example 3.

  The present specification relates to a method for detecting a mutation. The detection method disclosed in the present specification can ensure a good S / N ratio and detection sensitivity to suppress a decrease in detection accuracy, ensure reproducibility, and detect a mutation with good detection ability. The detection method disclosed in the present specification employs a probe that is set regardless of the mutation sequence to be detected (hereinafter also simply referred to as a universal probe), and employs visual detection using a visible label. Suitable for mutation detection method. Moreover, according to this detection method, a mutation can be easily detected. Furthermore, according to this detection method, the design, production and immobilization operation of the mutation-specific probe can be omitted, and the mutation can be detected easily and rapidly without requiring a special detection device. As a result, it is possible to reduce the cost related to the design and manufacturing time of the apparatus and the array.

  Although this method is not necessarily constrained, the above-described effect of this method is that the total concentration of the second primer for labeling in this method is within a certain range with respect to the total concentration of the first primer. It is considered that the S / N ratio due to mishybridization or the like can be reduced, and good detection sensitivity and strength can be maintained, and a decrease in detection accuracy can be suppressed.

  Further, in this method, by using a mismatch binding protein at the time of ligation, the generation of an erroneous ligation product in the ligation is suppressed, and the S / N ratio is suppressed and the detection sensitivity is maintained.

  In the present specification, the mutation means a characteristic on the base sequence related to a biological or abiotic characteristic that is naturally or artificially imparted to a polynucleotide such as DNA. Therefore, mutations include abnormalities with respect to so-called normal, natural, or dominant DNA base sequences (including sequences of two or more bases and only one base), or It includes differences such as artificial or inferior DNA. In addition, the mutation includes an artificial base sequence such as on DNA associated with some article.

  More specifically, the mutation includes, for example, features such as constitution, genetic disease, onset of specific diseases such as cancer, disease diagnosis, and DNA features associated with treatment prognosis. In addition, the mutation includes a base or base sequence on DNA or the like that serves as a genetic index in organisms such as humans and non-human animals such as selection of drugs and treatments. Typically, polymorphisms such as SNP on DNA and congenital or acquired mutations can be mentioned. In addition, a nucleotide sequence that contributes to identification of microorganisms derived from microorganisms such as pathogenic bacteria and viruses or identification of the type of microorganism is also included in the mutation. Furthermore, the mutation includes a feature on an artificial base sequence such as DNA that can specify the source and distribution channel of a product.

  The mutation can be any one selected from the group consisting of substitution mutations, insertion mutations and deletion mutations of 1 base or 2 bases to 9 bases (preferably 5 bases or less).

  In the present specification, the polynucleotide means a polymer of nucleotides, and the number thereof is not particularly limited. Therefore, the polynucleotide includes an oligonucleotide in which about several tens of nucleotides are linked.

  Hereinafter, representative and non-limiting specific examples of the present disclosure will be described in detail with reference to the drawings as appropriate. This detailed description is intended merely to provide those skilled in the art with details for practicing the preferred embodiments of the present invention and is not intended to limit the scope of the present disclosure. In addition, the additional features and inventions disclosed below can be used separately or together with other features and inventions to provide further improved compositions for the prevention or treatment of diabetes.

  Further, the combinations of features and steps disclosed in the following detailed description are not essential in carrying out the present disclosure in the broadest sense, and are particularly only for explaining representative specific examples of the present disclosure. It is described. Moreover, various features of the representative embodiments described above and below, as well as those described in the independent and dependent claims, are described herein in providing additional and useful embodiments of the present disclosure. They do not have to be combined in the specific examples given or in the order listed.

  All features described in this specification and / or claims, apart from the configuration of the features described in the examples and / or claims, are individually disclosed as limitations on the original disclosure and claimed specific matters. And are intended to be disclosed independently of each other. Further, all numerical ranges and group or group descriptions are intended to disclose intermediate configurations thereof as a limitation to the original disclosure and claimed subject matter.

  Hereinafter, embodiments disclosed in the present specification will be described with reference to the drawings as appropriate. FIG. 1 illustrates an outline of the detection method disclosed in this specification.

(Mutation detection method)
As shown in FIG. 1, the detection method disclosed in this specification includes a step of obtaining a ligation product (ligation step), a step of obtaining an amplification product (labeling step or amplification step), and obtaining a hybridized product. And a step of performing (hybridization step).

(Ligation product acquisition (ligation) process)
The step of obtaining a ligation product includes a DNA sample that may have a mutation, a first query probe having a first identification sequence that identifies the mutation, and a second identification that identifies a region adjacent to the mutation. This is a step of bringing a second query probe having a sequence into contact with each other and linking the first query probe and the second query probe by a DNA ligase reaction. In addition, the ligation product obtaining step includes the first identification sequence, the second identification sequence, the hybridization sequence associated in advance according to the type of mutation, or a complementary sequence thereof, by such ligation, It is a step of obtaining one or more ligation products having a labeling sequence for imparting a label or a complementary sequence thereof.

  According to the ligation product acquisition step, a ligation product can be acquired corresponding to the mutation. That is, if the mutation is an SNP, a ligation product can be obtained according to the type of polymorphism of the SNP (the type of base constituting the polymorphism).

(DNA sample)
If the DNA sample contains DNA that may have a mutation, its origin, preparation method and the like are not particularly limited. Generally, a DNA sample extracted from a sample such as a living body, or a sample obtained by a DNA amplification reaction such as so-called PCR or multiplex PCR with respect to the DNA sample is included. Alternatively, it may be a DNA sample obtained from mRNA via reverse transcription. Furthermore, an ink containing DNA as a component may be used.

(Query probe set)
FIG. 2A shows an example of a query probe set, and FIG. 2B illustrates a ligation product and an amplification product when the query probe set is used. As shown in FIG. 2A, the query probe set is a combination of a first query probe and a second query probe. The first query probe has a first identification sequence, and the second query probe has a second identification sequence. The query probe set is a combination of one or more first query probes and one or more second query probes.

  One query probe set is prepared for one mutation. When two or more mutations are detected in this detection method, two or more query probe sets are prepared according to the number of mutations to be detected.

  The first query probe and the second query probe may constitute the 5 'side or the 3' side in relation to the ligation product to be obtained. The first identification sequence and the second identification sequence of the first query probe and the second query probe are arranged in each query probe so as to be arranged at the center side of the ligation product.

  For example, as shown in FIGS. 2A and 2B, when the first query probe constitutes the 5 'side of the ligation product, the first identification sequence constitutes the 3' end of the first query probe. In that case, the second query probe constitutes the 3 'side of the ligation product, and the second identification sequence constitutes the 5' end of the second query probe.

(First query probe)
(First identification sequence)
As shown in FIGS. 2A and 2B, the first query probe has a first identification sequence for identifying a mutation. The first identification sequence is a base sequence designed to specifically hybridize to a base sequence constituting a mutation (mutation constituent sequence). That is, it is a base sequence designed to hybridize to a mutated component sequence under stringent conditions. The first discriminating sequence is typically configured to be completely complementary to the mutated component sequence.

  As shown in FIG. 2A and FIG. 2B, for example, when the mutation is SNP (single nucleotide polymorphism), two or more kinds of first identification sequences are set according to variations of the bases constituting the polymorphism. . As a result, two or more types of first query probes are also prepared according to the base variation. In addition, when the mutation is a specific single base sequence (base sequence having two or more bases), two or more types of first identification sequences are prepared according to variations of the mutation constituent sequence. Two or more types of query probes are also prepared. FIG. 2A shows the first query probe applied when the SNP is A / T.

(Hybridization sequence)
The first query probe can comprise a hybridizing sequence or a complementary sequence thereof. 2A and 2B exemplify a form in which the first query probe includes a complementary sequence of the hybridization sequence. When the first query probe has a hybridization sequence or a complementary sequence thereof, the hybridization sequence is a base that specifically hybridizes with a detection sequence of a predetermined probe associated in advance according to the variation included in the mutation. Is an array. Therefore, the hybridization sequence is composed of different types of base sequences depending on the number of variations constituting the mutation. The hybridizing sequence typically has a base sequence that is completely complementary to the detection sequence. The hybridizing sequence or the complementary sequence that the first query probe has is directly provided in the ligation product. The probe will be described in detail later.

  For example, as shown in FIG. 2A and FIG. 2B, when one mutation to be detected is SNP and the base type variation is A / T, it has a first identification sequence for identifying type A The first query probe has one hybridizing sequence (or its complementary sequence) associated with one probe. The other first query probe having the first identification sequence for identifying the T-type has another one hybridization sequence (or its complementary sequence) associated with the other one probe.

(Second query probe)
(Second identification array)
As shown in FIGS. 2A and 2B, the second query probe has a second identification sequence for identifying a base sequence (mutant adjacent sequence) adjacent to the mutation to be detected. Mutant flanking sequences do not constitute mutations and do not contain mutations. Therefore, typically, there is one type of mutant flanking sequence for one mutation and one type of second query probe.

  For example, as shown in FIGS. 2A and 2B, when the mutation is a SNP (single nucleotide polymorphism), one kind of second identification sequence is set according to the common sequence adjacent to the mutation constituting the polymorphism. Is done.

  The second identification sequence is a base sequence designed to specifically hybridize to the mutated flanking sequence. That is, it is a base sequence designed to hybridize to a constituent sequence such as a mutation under stringent conditions. The second discriminating sequence is typically configured to be completely complementary to the mutant flanking sequence.

(Labeling array)
The labeling sequence is a sequence for providing a labeling element provided in the second primer used in the amplification step described later. The hybridization sequence is a sequence that specifically hybridizes with a probe used in a hybridization step described later. The second query probe can comprise a labeling sequence or its complementary sequence. When the second query probe comprises a labeling sequence or its complementary sequence, the labeling sequence need only be capable of providing a labeling element that generates a detectable signal. 2A and 2B illustrate a mode in which the second query probe includes a complementary sequence of the labeling sequence. The base sequence may not be different depending on the variation so as to be impartable depending on the variation constituting the mutation. Therefore, it may be common to one or more mutations to be detected. The labeling sequence or its complementary sequence is provided as it is in the ligation product.

  For example, as shown in FIG. 2A and FIG. 2B, when one mutation to be detected is SNP and the base type variation is A / T, the second query probe is one second identification probe. And a sequence complementary to one (common) labeling sequence.

  The distribution of the hybridization sequence or its complementary sequence) and the labeling sequence or its complementary sequence) with respect to the first query probe and the second query probe can be appropriately determined according to the detection form of the hybridized product, etc. is there. In the probe set described in FIGS. 2A to 2D, the first query probe includes a first identification sequence and a hybridization sequence (or a complementary sequence thereof), and the second query probe includes 2 identification sequences and a labeling sequence (or a complementary sequence thereof). This embodiment is preferably used for detecting the hybridization product in the hybridization step based on the positional information, preferably visually. That is, in the probe set, the first query probe may include a first identification sequence and a hybridization sequence, and the second query probe may include a labeling sequence together with the second identification sequence. Good.

  Furthermore, when detecting a hybridization product in the hybridization step based on information based on the labeling element, the probe set shown in FIG. 3 can be preferably used. That is, the first query probe of the probe set includes a first identification sequence and a labeling sequence or a complementary sequence thereof, and the second query probe includes a second identification sequence and a hybridization sequence or a complementary sequence thereof. Can be provided.

  Such a query probe can be obtained by a person skilled in the art according to a normal DNA synthesis method or the like. As already described, the first query probe may be provided with a labeling sequence or its complementary sequence in place of the hybridization sequence or its different sequence, or for labeling the second query probe. Instead of the sequence or its complementary sequence, a labeling sequence or its complementary sequence may be provided.

  In the ligation step, the first query probe and the second query probe are brought into contact with the DNA sample under conditions that allow hybridization, and these are further ligated by DNA ligase. Under conditions that allow the first query probe and the second query probe to hybridize to the DNA sample, the first query probe and the second query probe have the first identification sequence and the second query probe, respectively. It specifically hybridizes with the mutated component sequence and the mutated component sequence by the identification sequence. That is, a mutation is detected. A DNA ligase links such a first query probe and a second query probe.

  In addition, as DNA ligase, well-known DNA ligase can be utilized suitably. Moreover, those skilled in the art can appropriately set hybridization conditions and ligase reaction conditions.

(Mismatch binding protein)
Ligation with DNA ligase can be performed in the presence of mismatch binding proteins with the intention of better ability to detect mutations. FIG. 2C shows an outline of the action of the mismatch binding protein in the ligation step when using the probe set or the like disclosed in FIGS. 2A and 2B. FIG. 2C illustrates the case where the base type of the SNP in the target nucleic acid is T, and the same mode is applied when the base type is A.

  As shown in FIG. 2C, the first query probe and the second query probe hybridize specifically to the mutant constituent sequence and the mutant adjacent sequence, respectively, but depending on the base sequence and conditions, the hybridization specificity May decrease. In that case, mishybridization may occur. The mismatch binding protein detects such mishybridization and binds to the mishybridization site. As a result, the ligation reaction by DNA ligase during mishybridization is suppressed, and as a result, the production of ligation products due to mishybridization can be suppressed.

  As used herein, a mismatch binding protein is a protein that recognizes and binds to a mismatch in a double-stranded nucleic acid. There is no particular limitation as long as such a mismatch can be recognized. The mismatch binding protein may be any of these partial peptides as long as the mismatch binding protein can recognize the mismatch. Furthermore, it may be a binding protein as another protein such as glutathione-S-transferase. Examples of the mismatch binding protein include MutS, MSH2, MSH6, and the like.

  In addition, ligation with DNA ligase can be repeated a plurality of times with the intention of better mutation detection ability. That is, it is possible to set a temperature condition in which the hybridization between the DNA sample, the first query probe, and the second query probe is repeated a plurality of times. By repeating the ligation reaction a plurality of times, it becomes easy to obtain an appropriate ligation product by appropriate hybridization. In particular, it is effective to repeatedly perform the ligation reaction in the presence of a mismatch binding protein.

  A person skilled in the art can appropriately set the temperature condition for repeatedly performing the ligation reaction. The number of repetitions is not particularly limited, but is preferably 10 times or more, more preferably 20 times or more, and still more preferably 30 times or more.

(Label process (amplification process))
The width production step is a step of performing a DNA polymerase amplification reaction by bringing a ligation product, a first primer having a complementary sequence of a hybridization sequence into contact with a second primer having a labeling sequence. . In addition, this step makes it possible to obtain one or more amplification products comprising a labeling sequence and a hybridization sequence.

  That is, this step is a step of obtaining the hybridization strand or the complementary strand thereof as a template strand in preference to obtaining the strand for hybridization thereof. For this purpose, the primer is designed so that the hybridization strand can be obtained more preferentially using the ligation product or its complementary strand as a template strand. Thereby, according to this process, the single-stranded amplification product for hybridization to be efficiently used for final detection can be obtained. As described above, the amplification product includes a hybridization sequence that specifically hybridizes with the detection sequence, and a labeling sequence that can include a labeling element. For this reason, it can hybridize with the probe on the solid phase carrier, and at the same time, a labeling element is added via the labeling sequence.

(Primer set)
The first primer and the second primer are a hybridizing sequence or a complementary sequence thereof and a labeling sequence or a complementary sequence thereof in a ligation product that may be intended by the first query probe and the second query probe. And designed according to.

  2B and 2D illustrate primer sets when the query probe set illustrated in FIG. 2A is used. In this primer set, a labeling element is provided in advance for the second primer. FIG. 2D illustrates the case where the base type of the SNP in the target nucleic acid is T, and the same mode is applied when the base type is A.

  In this detection method, one query probe set is prepared for one mutation (mutation types can be 1 or 2 or more), and one or a plurality of ligation products are generated. For these ligation products, One primer set is prepared. When two or more mutations are detected simultaneously, two or more query probe sets are prepared, a plurality of ligation product sets are generated, and a plurality of primer sets are prepared for these sets.

(First primer)
The first primer has a sequence complementary to the hybridizing sequence regardless of the form of the ligation product. As a result, the priming product or its complementary strand is used as a template strand so that an amplification product comprising a hybridization strand derived from the second primer, that is, a labeling sequence and a hybridization sequence, can be obtained from the first primer. Designed to amplify as. The first primer thus has a complementary sequence to the hybridizing sequence.

(Second primer)
The second primer has a labeling sequence. The second primer is preliminarily provided with a labeling element or is capable of binding the labeling element.

(Signal element)
The second primer can be provided with a labeling element in advance. The labeling element is for detecting the amplification product bound to the probe on the solid phase. As a labeling element, a conventionally known element can be appropriately selected and used. The label is not particularly limited, and a known light emitting substance such as a fluorescent substance or a coloring substance can be used. Preferably, the labeling element uses a luminescent material or a chromogenic material that exhibits luminescence or color development that can be detected visually (with the naked eye). This is because it is useful for simple and rapid detection.

  In addition to labeling elements using enzyme (eg, peroxidase, alkaline phosphatase, etc.) reaction, phosphorescence, chemiluminescence, coloring, etc., the labeling element is a substance that emits various coloring signals in combination with the second component by antigen-antibody reaction. Is included.

  Examples of the labeling element include various colorants such as various pigments and dyes. In addition to this, in addition to noble metals such as gold and silver, various metals or alloys such as copper, or organic compounds containing the metal (may be complex compounds) may be used. In addition, inorganic compounds such as mica, which are similar to the colorant, can be used.

  This kind of labeling substance typically includes various dyes, various pigments, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxene, aryl imidazoles, lucigenin, luciferin and eclion. A chemiluminescent substance is mentioned. Moreover, particles such as latex particles labeled with such a labeling substance are also included. Furthermore, colloids or sols including gold colloids or sols or silver colloids or sols can be mentioned. Furthermore, a metal particle, an inorganic particle, etc. are mentioned.

  As described above, the labeling element may include particles in a part thereof. The average particle diameter of particles such as latex particles constituting a part of the labeling substance is not particularly limited.

  The labeling sequence may include a molecule or substance (hereinafter also referred to as a labeling element binding substance) capable of binding these so that they can be finally identified by the labeling element. As such substances, protein-protein interaction, low molecular compound-protein interaction, and the like can be used. For example, antibodies in antigen-antibody reaction, biotin in avidin (streptavidin) -biotin system, digoxigenin in anti-digoxigenin (DIG) -digoxigenin (DIG) system, or haptens represented by FITC in anti-FITC-FITC system, etc. Is mentioned. In this case, the labeling element used for the final detection is the other molecule or substance that interacts with the labeling element binding substance (eg, antigen, ie streptavidin, anti-FITC, etc.) and the labeling element binding substance. It is modified so as to be provided as a site for binding. When the amplification product comprises a labeling element binding substance, a label comprising a labeling element binding substance of the amplification product and a site that binds to the labeling element binding substance in the hybridization step, prior to or after this step A complex with the element can be formed and the amplification product detected by the label element.

  Such labeling elements and label-binding substances are commercially available, and the production of labeling elements and labeling element-binding substances and methods for labeling labeling element substances and the like are also known. It can be obtained by using it. Further, the labeling element or the particle labeled with the labeling element or the labeling element substance-binding substance and the oligonucleotide such as DNA can be appropriately bound via a functional group such as an amino group as such. Well known in the art.

  The second primer is labeled by labeling a probe having a labeling element to a labeling sequence or another sequence by hybridization in a subsequent hybridization step or a subsequent step. Also good.

  As shown in FIG. 2B, the first primer and the second primer are, for example, when the mutation is SNP and the base variation is A / T, the possible ligation products correspond to each base type 2. It is a kind. In the case shown in FIG. 2B, the first primer is a first primer having a hybridization sequence corresponding to the probe associated with the type of base A for detecting a ligation product having a base species of A. When the base type is T, a first primer having a hybridization sequence corresponding to a probe associated in advance and one second primer corresponding to one labeling sequence are prepared.

  Each concentration of the first primer and the second primer is not particularly limited. When good detection ability is intended, it is useful to adjust the total concentration of the first primer and the second primer used in the amplification step. For example, the total concentration of the second primer used in the labeling step is the total concentration of the first primer used in the same labeling step (second primer total concentration (total amount) / first primer total concentration (total amount)). It is preferably 0.5 times or more and less than 12.5 times. If this ratio is less than 0.5 times, the intended amplification product cannot be obtained effectively. As a result, the color intensity or the detection sensitivity is low, and the S / N ratio is less than 5 times, and the detection accuracy is also lowered. Moreover, even if it is 12.5 times or more, the intended amplification product cannot be obtained effectively. As a result, the color intensity or the detection sensitivity is low, and the S / N ratio is less than 5 times, and the detection accuracy is also lowered. More preferably, it is 10 times or less, More preferably, it is 5 times or less, More preferably, it is 2 times or less, More preferably, it is 1 time or less.

  The total concentration of the first primer is the total concentration of various first primers used in the labeling step. Therefore, it contains at least all of the first primers for the mutation to be detected. The total concentration of the second primer is the total concentration of various second primers used in the labeling step. Instead of the first primer total concentration, the first primer concentration and the second primer total concentration corresponding to the type of mutation to be detected can be correlated. In this case, the total concentration of the second primer / each concentration of the first primer is preferably 16 times or more and less than 400 times. If this ratio is less than 16 times, the intended amplification product cannot be obtained effectively. As a result, the color intensity or the detection sensitivity is low, and the S / N ratio is less than 5 times, and the detection accuracy is also lowered. Moreover, even if it is 400 times or more, the intended amplification product cannot be obtained effectively. As a result, the color intensity or the detection sensitivity is low, and the S / N ratio is less than 5 times, and the detection accuracy is also lowered. Preferably it is 160 times or less, More preferably, it is 64 times or less, More preferably, it is 32 times or less.

  As shown in FIGS. 2B and 2D, according to such an amplification step, one set of amplification products can be obtained by one primer set. The set of amplification products includes the types of amplification products according to the presence or absence of mutations and variations of mutations.

(Hybridization product acquisition (hybridization) process)
The hybridization step is a step of obtaining a hybridization product of the probe and the amplification product by bringing the amplification product into contact with the probe on a solid phase carrier on which a probe having a detection sequence is held. In this step, a hybridized product is obtained by performing hybridization between the probe immobilized on the solid phase carrier and the amplification product on the solid phase carrier.

  According to the present hybridization step, an amplification product having a specific hybridization sequence that can be preferentially hybridized with the probe is preferentially obtained in the amplification step. For this reason, many hybridized products can be obtained in a state where mishybridization is suppressed.

  The hybridization step is performed by supplying a hybridization solution to the entire solid phase body (immersion-type hybridization) and supplying a hybridization solution that is also a mobile phase to a part of the solid phase body. Any form of affinity chromatography (development type hybridization) in which a hybridization solution is developed in a predetermined direction with respect to the phase body may be used.

  Hereinafter, the probe and the solid phase carrier on which the probe is immobilized will be described.

(probe)
Each probe has a detection sequence which is a unique base sequence for probing. Such a detection sequence can be set independently of the sequence characteristic of the target nucleic acid, that is, the target sequence. By setting the detection sequence independent of the target sequence, the detection sequence of the detection probe can suppress or avoid non-specific binding between a plurality of detection probes, and is suitable for hybridization at a suitable temperature and time. Can be set in consideration of the hybridization conditions. In addition, the same detection probe can always be used regardless of the type of target nucleic acid. Such a probe can be used as a universal probe capable of dealing with all mutations.

  The length of the detection sequence is not particularly limited, but is preferably 10 bases or more and 50 bases or less. This is because within this range, hybridization efficiency can be ensured while ensuring the specificity of each detection sequence. For example, such a base length detection sequence is a 46 base length sequence obtained by combining two base sequences each having a base length of 23 bases each selected from SEQ ID NOs: 1 to 100 and a complementary sequence thereof, or the combined base sequence. Can be obtained by appropriately adding or deleting a base. More preferably, it is 10 bases or more and 25 bases or less. For example, such a base length detection sequence can be obtained by appropriately adding or deleting bases to the 23 base length sequences of SEQ ID NOS: 1 to 100 and their complementary sequences or these base sequences. it can. Since the tag sequence in the first primer is a base sequence that is paired with the detection sequence, the base length of the tag sequence is preferably 10 bases or more and 50 bases or less, as in the detection sequence. Preferably, it is 10 bases or more and 25 bases or less.

  As such a probe detection 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.

  The detection sequence in such a probe is also referred to as an orthonormalization sequence. For example, a DNA sequence having a predetermined base length obtained from a random number has a continuous match length, melting temperature prediction by Nearest-Neighbor method, Hamming distance, secondary order Designed by performing 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 affected without being affected by the problems such as the above-mentioned cross-hybridization. Has the property of being amplified. Details of the orthonormalized sequence as described above are described in H. Yoshida and A. Suyama, “Solution to 3-SAT by breadth first search”, DIMACS Vl. 54, 9-20 (2000). Orthonormalized sequences can be designed using the methods described in these references.

(Solid phase carrier)
A solid phase carrier can be used as the solid phase carrier. For example, the carrier may be plastic or glass, and the material is not particularly limited. Moreover, porous bodies, such as a cellulose, a nitrocellulose, nylon, polyether sulfone, a polyvinylidene fluoride, and filter paper, may be sufficient. This type of porous carrier is particularly suitable for hybridizing a probe immobilized on a solid phase carrier and an amplified fragment by development-type hybridization.

  The shape of the carrier may be a flat plate shape, but may be a bead shape, and the shape is not particularly limited. The solid phase is preferably an array (particularly a microarray) in which the carrier has a solid plate shape and a plurality of probes are fixed in a fixed arrangement. The array can fix a large number of detection probes 4 and is convenient for comprehensively detecting various target nucleic acids at the same time. Further, the solid phase body may include a plurality of partitioned array regions on the carrier. In the plurality of array regions, a set of probes each having the same combination may be fixed, or a set of probes each having a different combination may be fixed. If different sets of probes are immobilized in multiple array regions, individual array regions can be assigned for detection of target nucleic acids in different genes.

  The shape of the carrier can also be set in consideration of the form of hybridization. For example, when hybridization is performed in a microtube such as Eppendorf tube (trademark), which is widely used for examinations and research, in the case of immersion type hybridization, the hybridization solution contained in the tube is used. Preferably, the size and shape of the array area of the carrier is immersed.

  Further, in the embodiment implemented by the development type hybridization, at least the end of the carrier can be immersed in a hybridization solution supplied to a microtube such as Eppendorf tube (trademark), which is widely used for testing and research. It is preferable to have various sizes (width direction) and shapes. Preferably, it is a long body provided with a part that can be accommodated from the vicinity of the bottom of this type of tube to the upper end.

  The immobilization form of the probe is not particularly limited. The 3 'end of the probe may be bound to a carrier, or the 5' end may be bound. It may be covalent or non-covalent. The probe can be immobilized on the surface of the carrier by various known methods. Moreover, the probe may be provided with a suitable linker sequence for the surface of the carrier. The linker sequence is preferably the same sequence with the same base length between the probes.

  The probe is supplied to the solid phase carrier in a predetermined pattern according to the form of hybridization described later. In the case of immersion type hybridization, typically, a pattern in which dots corresponding to individual detection probes are arranged. In the case of development-type hybridization, typically, a stream (band) corresponding to each detection probe is a pattern arranged at one or more development positions along the development direction.

  The conditions for the hybridization step can be appropriately set according to the base sequence of the probe and the hybridization mode.

  In the amplification step, when no substantial label is given by the second primer, in the amplification product given by the second primer in the hybridization step, prior to the step, or in the detection step after the step A label or a label-binding substance is appropriately supplied via a sequence that specifically hybridizes with the labeling sequence or a label-binding substance bound thereto.

(Detection process)
The detection step is a step of detecting the hybridization product based on the label imparted via the second primer, thereby detecting the presence or type of mutation. Various forms of labeling are known and can be used as appropriate in this detection method.

  In this detection step, it is preferable to use a label that can be visually recognized. By detecting a mutation based on such a label, the mutation can be detected anytime and anywhere without a special device for detection. As described above, the label is passed through the second primer at any time of the amplification step, the post-amplification step, the hybridization step, the hybridization step, the post-hybridization step, the detection step, or the detection step. Is granted. The label is given based on the second primer or a sequence that specifically hybridizes to the labeling sequence held by the amplification product derived from the second primer.

  For example, in immersion type hybridization, a biotin-labeled amplification product obtained using a second primer labeled with biotin is subjected to hybridization on a solid phase carrier, and after washing, the hybridized product, biotin HRP, and streptavidin And contact. As a result, a complex is formed, and further, a color development reaction via peroxidase is performed, whereby the hybridized product can be detected with the color developing substance.

  In development-type hybridization, the biotin-labeled amplification product obtained using the second primer labeled with biotin is developed with a developing solution containing colored latex beads coated with streptavidin, so that the hybridization product Can be detected with colored latex beads.

(kit)
According to this specification, the kit for a mutation detection containing the above-mentioned query probe set and primer set is provided. The kit may comprise a mismatch binding protein. In addition, this kit has a total concentration (total amount) of the second primer prepared for labeling of 0.05 times or more of the total concentration (total amount) of the first primer applied at the time of labeling. As such, the first primer and the second primer may be provided. That is, it is preferable that the first primer and the second primer are contained so as to have the above concentration ratio or at the above concentration ratio. Further, the kit may include a solid phase carrier on which an associated probe is immobilized. This solid phase carrier may be a chromatographic carrier having permeability or permeability. In addition, the said ratio can take a suitable range with the aspect similar to the detection method of this variation already demonstrated. Also, the total concentration of the second primer / each concentration of the first primer can take the range as described above.

(Method for producing DNA associated with mutation)
According to the present disclosure, a method for producing DNA associated with mutation is also provided. That is, the present production method comprises a DNA sample that may have the mutation, a first query probe having a first identification sequence that identifies at least a part of the mutation, and a region adjacent to the mutation. A second query probe having a second identification sequence for identification, and contacting the first query probe and the second query probe by a DNA ligase reaction, and depending on the type of mutation The first identification sequence and the second identification sequence, a hybridization sequence for hybridizing with the detection sequence of the probe associated in advance according to the type of the mutation, or a complementary sequence thereof, and a label are provided. A step of obtaining one or more ligation products each having a labeling sequence or a complementary sequence to be associated with the mutation, and the one or more ligation products An hybridization product, a first primer having a complementary sequence of the hybridization sequence, and a second primer having the labeling sequence are brought into contact with each other to hybridize with the labeling sequence by a DNA polymerase amplification reaction. The step of obtaining one or more amplification products having a sequence for use and the step of obtaining the ligation product are carried out in the presence of a mismatch binding protein.

  According to this production method, an amplification product associated with mutation can be efficiently obtained from a DNA sample. Also in this method, it is preferable for efficient amplification that the total concentration of the first primer and the total concentration of the second primer used in the labeling step have the ratios already described. Also, the total concentration of the second primer / each concentration of the first primer can take the range as described above.

  Hereinafter, the present disclosure will be specifically described with reference examples for comparison in addition to the examples. However, the following examples illustrate the present disclosure and do not limit the scope of the present disclosure. .

Reference example 1

  In this reference example, SNP analysis was performed by an existing method (based on Prior Art Document 3).

(1) Preparation of DNA microarray Capture an aqueous solution in which synthetic oligo DNA (manufactured by Nippon Genetic Laboratory Co., Ltd.) in which the 5 ′ end is modified with an amino group is dissolved in a genelide glass substrate manufactured by Toyo Kohan Co., Ltd. The probe was spotted with GENSHOT (registered trademark) spotter at NGK Corporation. 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 sequences used for the capture probe were as follows.

(2) Amplification / fluorescence labeling of sample genes Amplification of sample genes and pretreatment such as fluorescence labeling were performed with reference to the technique described in Prior Art Document 3. As samples, 50 types of Japanese-derived B cell lines / genomic DNA (Health Science Research Resources Bank) were used.

  The primer sequences (Table 2) used for amplification, the probe sequences (Table 3) used in the Encoding step, and the primer sequences (Table 4) used in the Labeling step were as follows. In Table 2, the amount of each primer during the reaction was 250 fmol. In Table 3, the amount of each probe during the reaction was 5 fmol / Common, and the probe used was 5'-terminal phosphorylated. In Table 4, ED-1 was labeled with Cy3 and ED-2 was labeled with Cy5 at the 5 'end. The amount of ED-1, 2 as the second primer is 6 pmol each, and the amount of the first primer D1_1 to 32 is 30 fmol, and the second primer total concentration / first primer total concentration (P / C) = 12.5 (P = 12 pmol, C = 0.96 pmol).

(Ligation process)
The ligation reaction procedure is as follows. New England Biolabs Taq DNA Ligase (Catalog # M0208S) was used for the reaction, and GeneAmp PCR System 9700 (Applied Biosystems) was used for the thermal cycler.
(Reagent composition)
Query probe (mixed 6 types-Table 3) / Common probe (mixed 3 types-Table 3) Mix (50nM, each)
0.100 μl
Taq DNA Ligase buffer (10 ×) 1.500 μl
dH ₂ O 10.275 μl
Taq DNA ligase (40 Units / μl) 0.125 μl
(Subtotal) (12.00 μl)
PCR products (*) 3.00 μl
Total 15.00 μl

  After preparing the above reagents, ligation was performed at 95 ° C for 5 minutes, 50 ° C for 1 minute, 58 ° C for 60 minutes, and then 10 ° C.

(Labeling process)
The labeling step was performed as follows. TaKaRa Bio TaKaRa Ex Taq® (Catalog # RR001B) was used for labeling, and GeneAmp PCR System 9700 (Applied Biosystems) was used for the thermal cycler.

(Reagent composition)
D1_i primer (mixed 32 types-Table 4) Mix (0.5 μM, each) 0.06 μl
Cy3-labeled-ED-1 (25 μM) 0.24 μl
Cy5-labeled-ED-1 (25 μM) 0.24 μl
Ex Taq buffer (10 ×) 1.20 μl
dNTP mix (2.5 nM) 0.96 μl
Ex Taq polymerase (5 Unit / μl) 0.12 μl
milliQ 8.18 μl
(Subtotal) (11.00 μl)
ligation products 1.00 μl
total 12.00 μl

  After preparing the above reagents, after 1 minute at 95 ° C, carry out 25 cycles of 95 ° C for 30 seconds, 55 ° C for 6 minutes, 72 ° C for 30 seconds, and then carry out the labeling step under the conditions of 10 ° C did.

(3) Detection using DNA microarray The reaction / washing to the DNA microarray using the labeled sample was performed according to the contents described in Prior Art Document 3.
(Fluorescence detection by scanner)
LaserPower and PMT were adjusted as appropriate using MolecularDevices GenePix4000B, and fluorescence images were acquired.

(4) Data analysis GenePix Pro was used as image digitization software, and the fluorescence signal of the obtained image was digitized. The results are shown in FIG.

  As shown in FIG. 4, regarding the sample used this time, according to the analysis of variance shown in FIG. 4, it was found that heterogeneous and homozygous can be distinguished for each SNP.

Reference example 2

  In this reference example, mutation (SNP) was detected by immersion type hybridization. In this reference example, mutation was detected by the following procedure. The target SNPs were rs10442713 (SNP1), 1229984 (SNP2), and rs671 (SNP3).

(1) Production of Membrane Type DNA Microarray A capture DNA probe solution having the base sequence shown in the following table is discharged on a Hi-Flow Plus membrane sheet (60 mm × 600 mm) manufactured by Merck Millipore as described in JP-A-2003-75305. Spots were placed in the arrangement shown in FIG. 5 using a GENISHOT (registered trademark) spotter manufactured by NGK Corporation using a unit (inkjet method).

  As the probe used, the following 11 types of sequences out of 100 types of D1_1 to D1_100 described in Supplementary Table 1 of Non-Patent Document 2 were used. The probe used was modified at the 5 'end (or 3' end) with albumin (or protein such as avidin). After the spot, the synthetic oligo DNA was immobilized by baking at 45 ° C. for 2 hours.

(2) Sample gene amplification, ligation and labeling The sample gene amplification, ligation step, and labeling step (biotinylation) were performed with reference to the method described in Non-Patent Document 2.

(amplification)
The samples used were 6 types of Japanese-derived B cell lines / genomic DNA (Health Science Research Resources Bank) with known SNP types for each sample.

  The primer sequences used for amplification, ligation and labeling of the sump gene, the first and second query probes (corresponding to the query probe and common probe in the table), and the primer sequences were as follows. Primer ED-1 was labeled with biotin at the 5 'end.

  The amount of each primer during the amplification reaction was 250 fmol. The amount of each probe during the ligation reaction was 5 fmol, and the 5 ′ terminal phosphorylated one was used as the Common probe.

(Ligation)
The ligation reaction procedure was as follows. Taq from New England Biolabs
DNA Ligase (Catalog # M0208S) was used, and GeneAmp PCR System 9700 (Applied Biosystems) was used as the thermal cycler. Specifically, the following reagents were prepared, and ligation was carried out under the conditions of 10 ° C after 5 minutes at 95 ° C, 1 minute at 50 ° C, and 60 minutes at 58 ° C. GeneAmp PCR System 9700 (Applied Biosystems) was used as the thermal cycler.

(Reagent composition)
Query probe / Common probe Mix (50 nM, each)
0.100 μl
Taq DNA Ligase buffer (10x)
1.500 μl
dH ₂ O 10.275 μl
Taq DNA ligase (40 Units / μl)
0.125 μl
Subtotal 12.000 μl
PCR products (*) 3.000 μl
total 15.000 μl

(Signs)
For labeling, the following reagents are prepared first, followed by 25 cycles of 95 ° C for 1 minute, 95 ° C for 30 seconds, 55 ° C for 6 minutes, and 72 ° C for 30 seconds, followed by 10 ° C PCR was performed under the conditions of PC. The obtained product was labeled as Labeled products. GeneAmp PCR System 9700 (Applied Biosystems) was used for the thermal cycler and TaKaRa Ex Taq (R) (Catalog # RR001B) of TaKaRa Bio was used for PCR. In the following reagent composition, the amount of biotin-labeled ED-1 as the second primer is 12 pmol, the primer Mix (D1_1 to 32) as the first primer is 30 fmol each, and the total amount is 0.96 pmol. It was. As a result, the second primer total concentration / first primer total concentration was 12.5 (P = 12 pmol, C = 0.96 pmol).

(Reagent composition)
D1_i primer Mix (0.5 μM, each) 0.06 μl
Biotin-labeled-ED-1 (25 μM) 0.48 μl
Ex Taq buffer (10 ×) 1.20 μl
dNTP mix (2.5 nM) 0.96 μl
ExTaqpolymerase (5Unit / μl) 0.12 μl
milliQ 8.18 μl
Subtotal 11.00 μl
ligation products 1.00 μl
total 12.00 μl

(3) Detection using DNA microarray The reaction to the DNA microarray and its detection procedure were as follows. That is, the following hybridized samples were prepared, 200 μl of each hybridized sample was placed in a 0.2 ml tube and reacted at a heat block temperature of 90 ° C. for 1 minute and at 80 ° C. for 1 minute. Thereafter, the DNA microarray was cut into a size that could fit into a 0.2 ml tube, set in the tube, and reacted at a heat block temperature of 37 ° C. for 30 minutes. As a control, oligo DNA (25 μM) labeled with biotin at the 5 ′ end of the sequence complementary to the D1-100 sequence was used.

(Composition of hybridized sample)
Hybrid Solution (0.5% Tween 20-1% BSA-PBS)
200.0 μl
Control 4.0 μl
Amplified sample 4.0 μl
total 208.0 μl

  After completion of the reaction, the DNA microarray was transferred to a 0.2 ml tube containing a washing solution (0.1% Tween 20-1 mM EDTA-TBS) and washed in a 37 ° C. heat block (37 ° C. × 1 min, 37 ° C. × 10 min, 37 ° C. × 1 min).

  Next, the washed array was transferred to a 0.2 ml tube containing a mixed solution of biotin-HRP and streptavidin, and reacted at room temperature for 20 minutes. After completion of the reaction, the DNA microarray was transferred to a 0.2 ml tube containing a washing solution (0.1% Tween 20-1 mM EDTA-TBS) and washed (room temperature × 1 min, room temperature × 10 min, room temperature × 1 min).

  The washed array was subjected to a color reaction for about 5 minutes at room temperature using Vector Laboratories TMB Peroxidase Substrate Kit, 3, 3 ', 5, 5'-tetramethylbenzidine. The presence or absence of color development after drying of the array was visually confirmed. The results are shown in FIG. 6 together with Example 1.

  Hereinafter, according to Reference Example 1, the detection method disclosed in this specification was performed. Hereinafter, only the parts (ligation and labeling) that are different from the reference example 2 will be described. Other operations were performed according to the reference example. The results are shown in FIG.

  Concerning ligation, in this example, the same procedure as in Reference Example 2 was performed except that mismatch binding protein (Nippon Gene's TaqMutS (316-04011)) was used and different thermal cycle conditions were employed for the ligation reagent. did. The reagent composition used for ligation is shown below. The thermal cycle conditions were 95 ° C. for 5 minutes, 95 ° C. for 0.5 minute, 50 ° C. for 1 minute, and 58 ° C. for 4 minutes for one cycle. That is, in Reference Example 2, ligation was performed only for one cycle, but in this example, a short ligation cycle was performed 30 times.

(Reagent composition)
Query probe / Common probe Mix (50 nM, each)
0.100 μl
Taq DNA Ligase buffer (10x)
1.500 μl
dH ₂ O 8.275 μl

Taq DNA ligase (40 Units / μl)
0.125 μl
10 × Taq MutS buffer 1.500 μl
Taq MutS (1 μg / μl) 0.500 μl
Subtotal 12.000 μl
PCR products (*) 3.000 μl
total 15.000 μl

  Regarding labeling, in this example, the same procedure as in Reference Example 2 was performed except that the following composition was used for the reagent at the time of labeling. The reagent composition used for labeling is shown below. In this example, the total concentration of the following primer Mix (first primer) was higher than that in Reference Example 2 than in Reference Example 2. That is, the second primer (biotin-labeled-ED-1 was maintained at 12 pmol, and the total concentration of D1_i primer Mix was 625 fmol each, ie, 20 pmol. In other words, the second primer total concentration / first primer total concentration was 0. 6.

(Reagent composition)
D1_i Primer Mix (0.5 μM, each) 1.25 μl
Biotin-labeled-ED-1 (25 μM) 0.48 μl
Ex Taq buffer (10 ×) 1.20 μl
dNTP mix (2.5 nM) 0.96 μl
ExTaqpolymerase (5Unit / μl)
0.12 μl
milliQ 6.99 μl
Subtotal 11.00 μl
ligation products 1.00 μl
total 12.00 μl

  As shown in FIG. 6, in the reference example, the color intensity of the spots to be colored was significantly weaker than that of the control, and spots that should not be colored were colored to the same intensity in a mishybridized manner. As a result, in Reference Example 2, it was difficult to determine the SNP type for each sample. On the other hand, in Example 1, the spot to be colored was colored more strongly than Reference Example 2, and was almost equivalent to the control. In addition, spots that should not be colored hardly developed color. As a result, in Example 1, the type of SNP could be clearly determined for each sample. That is, according to Example 1, it was possible to ensure better detection sensitivity and accuracy as compared with Reference Example 2.

  Also, the obtained image was digitized and the S / N ratio was calculated (S: intensity of sample reaction site in capture DNA region on membrane, N: intensity of region without spot on membrane). In Example 2, the result was generally less than 5, whereas in Example 1, a result exceeding approximately 50 was confirmed, and it was found that the visibility was improved at least 10 times by the present invention.

  From the above results, it was found that a high color intensity and a good S / N ratio can be obtained by using a mismatch binding protein during ligation. Further, it was found that a high color intensity and a good S / N ratio can be obtained by performing ligation a plurality of times in a short cycle. Further, in the labeling element, the total concentration of the first primer relative to the total concentration of the second primer in the reagent is increased so that the second primer total concentration / first primer total concentration is less than 12.5, and 0 It was found that a high color intensity and a good S / N ratio can be obtained by setting the ratio to about .6.

  In this example, SNP was detected by developmental hybridization. The SNP to be detected in this example was rs10442713 (SNP1).

(1) Production of membrane type DNA chromatography body A capture DNA probe solution comprising a base sequence shown in the following table on a Hi-Flow Plus membrane sheet (60 mm x 600 mm) manufactured by Merck Millipore is described in JP-A-2003-75305. Spots were placed in the arrangement shown in FIG. 7 using a GESHOT (registered trademark) spotter using NGK Corporation using a discharge unit (inkjet method).

  The DNA sequences of the probes used were the following 8 types out of 100 types D1_1 to D1_100 described in Supplementary Table 1 of Non-Patent Document 2, and the probes used for the spots were those whose 5 ′ end was modified with albumin used. After the spot, the probe was immobilized by baking at 45 ° C. for 2 hours.

(2) Sample gene amplification, ligation, and labeling Sample gene amplification, ligation, and biotin labeling were performed as follows.

  There are two types of samples obtained from Japanese-derived B cell lines and genomic DNA (Human Science Promotion Foundation: Health Science Research Resources Bank), and the SNP type to be detected is known in advance for each sample. I used something.

  The primer sequences used for the amplification of the sample gene, the probes used for ligation, and the primer sequences used for the label were as follows. ED-1 was labeled with biotin at the 5 'end. The amount of each primer used for amplification was 250 fmol, the amount of each query probe was 5 fmol, and the common probe was phosphorylated at the 5 end. Each D1 primer for labeling was 625 fmol (total concentration was 20 pmol), and the ED-1 primer was 12 pmol with the 5 'end labeled with biotin.

(Ligation)
The ligation reaction procedure was performed under the same conditions using the same reagents as in Example 1 except that the above-described query probe was used. A mismatch binding protein is used as the ligation reagent.

(Signs)
The above PCR product was labeled by performing PCR under the same conditions as in Example 1. In the reagent, the total concentration of the second primer / the total concentration of the first primer was 0.6.

(3) Detection using chromatography main body Hybridization reaction by nucleic acid chromatography and its detection were performed. That is, the amplification reaction solution was reacted in advance at a heat block temperature of 90 ° C. for 1 minute and at 80 ° C. for 1 minute, and then immediately cooled on ice to prepare a developing solution having the following composition. The latex stock solution was prepared by coating PBS with polystyrene latex beads containing a blue colorant at a predetermined concentration using PBS.

(Composition of developing solution)
PBS 30.0 μl
Latex solution 5.0μl
Amplification reaction solution 10.0 μl
Millipore water 5.0μl
Total 50.0μl

(Hybridization)
50 μl of the developing solution for each sample was added to a 0.2 ml tube, and the lower end of each chromatography body was inserted to start a hybridization reaction by chromatography. All the developing solution was sucked up in about 20 minutes, and the hybridization reaction by chromatography was completed. After completion of the reaction, the chromatography main body was air-dried, and then the reaction site was visually confirmed and an image was taken.

(Detection process)
The presence or absence of color development after drying of the array was visually confirmed. The results are shown in FIG. As shown in FIG. 8, by performing the same ligation and labeling as in Example 1, it was possible to confirm the result that SNP discrimination was sufficiently possible even in the developmental hybridization.

  From the above results, it was found that a high color intensity and a good S / N ratio can be obtained by using a mismatch binding protein during ligation. Further, it was found that a high color intensity and a good S / N ratio can be obtained by performing ligation a plurality of times in a short cycle. Further, at the time of labeling, the total concentration of the first primer set for detecting each mutation relative to the total concentration of the second primer in the reagent is increased, and the second primer total concentration / the total concentration of the first primer is set. It was found that a high color development intensity and a good S / N ratio can be obtained even in the development-type hybridization by setting it to about 0.6.

  Furthermore, according to the present example, it was found that the hybridization process can be completed within about 0.3 hours by performing in the form of chromatography, and the time required for the entire mutation detection can be greatly shortened.

  In this example, in the labeling step, the total concentration of the first primer (D1_i primer in this example) and the second primer (labeling primers (Cy3, Cy5 labeled ED-1, The influence of the ratio of the total concentration of 2 primers) on the detection process was examined.

In this example, only sample 1 (SNP1 (G / G) and SNP2 (A / A)) in Table 6 was used as a sample, and the other steps were performed according to Reference Example 1 except for the labeling step. While fixing the primer concentration, the concentration of the first primer (D1_i primer) was varied during the composition of the labeling reagent, and Table 15 and FIG.

  As shown in FIG. 9, it is preferable to make it smaller than the second primer total concentration / first primer = 12.5 (condition 3) of the conventional primer amount ratio, more preferably smaller than the same 5 (condition 4). By doing so, the detection performance of the Homo type was further improved, and the best result was obtained in the same 0.5 (condition 7). That is, the second primer total concentration / first primer total concentration is within a certain range (0.05 or more and less than 12.5, preferably 10 or less, more preferably 5 or less, further preferably 2 or less, more preferably It was confirmed that the discriminating ability was increased by adjusting to about 1 or less. On the other hand, it was also confirmed that when the second primer total concentration / first primer total concentration was further reduced, the signal intensity itself was significantly reduced.

  In the above-described embodiment, the second primer total concentration (P) is constant. However, depending on the sample detection sensitivity, the second primer total concentration may be increased or decreased. Is preferable.

SEQ ID NOs: 1 to 129: synthetic DNA

Claims (15)

A method for detecting a mutation, comprising:
A DNA sample possibly having the mutation, a first query probe having a first identification sequence for identifying at least a part of the mutation, and a second identification sequence for identifying a region adjacent to the mutation A first query sequence according to the type of mutation, wherein the first query probe and the second query probe are linked by a DNA ligase reaction. And a second identification sequence, a hybridization sequence or a complementary sequence that specifically hybridizes with a detection sequence of a probe associated in advance according to the type of mutation, and a labeling sequence for imparting a label, or Obtaining one or more ligation products each having a complementary sequence in association with the mutation;
The label is obtained by a DNA polymerase amplification reaction by contacting the one or more ligation products, a first primer having a sequence complementary to the hybridization sequence, and a second primer having the labeling sequence. Obtaining one or more amplification products comprising the sequence for use and the sequence for hybridization;
Contacting the probe of the solid phase carrier holding the probe with the one or more amplification products to obtain a hybridization product of the probe and the amplification product;
Detecting the hybridized product based on a label imparted via the labeling sequence;
The total concentration of the second primer is 0.5 times or more and less than 12.5 times the total concentration of the first primer.   The method according to claim 1, wherein the ligation product obtaining step is performed in the presence of a mismatch binding protein.   The method according to claim 1 or 2, wherein the ligation product obtaining step is performed by repeating the ligation reaction with the DNA ligase a plurality of times.   The method according to claim 1, wherein the total concentration of the second primer is 5 times or less than the total concentration of the first primer.   The method according to claim 1, wherein the step of obtaining a hybridized product is performed in a chromatographic form.   The method according to claim 1, wherein the second primer holds the label or a label-binding substance capable of binding the label.   The method according to any one of claims 1 to 6, wherein the marker is visually recognizable.   The method according to any one of claims 1 to 7, wherein the probe detection sequence is selected from the group consisting of a base sequence represented by SEQ ID NOs: 1 to 100 and a complementary sequence thereof.   The detection method according to claim 1, wherein the solid phase carrier is any one selected from the group consisting of polyethersulfone, nitrocellulose, nylon, polyvinylidene fluoride, and filter paper.   The detection method according to claim 1, wherein the mutation is one or several base substitution mutations, insertion mutations, or deletion mutations. A kit for detecting mutations,
A first identification sequence that identifies at least a part of the mutation, and a hybridization sequence that specifically hybridizes with a detection sequence of a probe associated in advance according to the type of the mutation, or a complementary sequence thereof. A second query probe set having one query probe, a second identification sequence for identifying a region adjacent to the mutation, and a labeling sequence or a complementary sequence thereof, or the first identification sequence and the labeling A query probe set comprising: a first query probe having a sequence or a complementary sequence thereof; and a second set of query probes having the second identification sequence and the hybridizing sequence or a complementary sequence thereof; ,
A primer set comprising: a first primer having a sequence complementary to the hybridizing sequence; and a second primer having the labeling sequence;
A mismatch binding protein;
A kit comprising:   The kit according to claim 11, wherein the total concentration of the second primer is 0.05 times or more and less than 12.5 times the total concentration of the first primer.   The kit according to claim 11 or 12, wherein the solid phase carrier is a chromatographic carrier having liquid permeability or permeability. A method for producing DNA associated with a mutation, comprising:
A DNA sample possibly having the mutation, a first query probe having a first identification sequence for identifying at least a part of the mutation, and a second identification sequence for identifying a region adjacent to the mutation A first query sequence according to the type of mutation, wherein the first query probe and the second query probe are linked by a DNA ligase reaction. And a second identification sequence, a hybridization sequence for hybridizing with a detection sequence of a probe associated in advance according to the type of mutation, or a complementary sequence thereof, and a labeling sequence for imparting a label, or Obtaining one or more ligation products each having a complementary sequence in association with the mutation;
The label is obtained by a DNA polymerase amplification reaction by contacting the one or more ligation products, a first primer having a sequence complementary to the hybridization sequence, and a second primer having the labeling sequence. Obtaining one or more amplification products having a sequence for hybridization and a sequence for hybridization;
The method wherein the ligation product obtaining step is performed in the presence of a mismatch binding protein.   The method according to claim 14, wherein the total concentration of the second primer is 0.5 times or more and less than 12.5 times the total concentration of the first primer.