JP2007075021A - Genetic polymorphism detection method using mass spectrometry - Google Patents

Abstract

The present invention provides a method for detecting a gene polymorphism by a simple operation with a high degree of freedom at a detection site and a remarkably low risk of erroneous recognition.
A specific sequence (consisting of a first region, a second region, and a third region, the first region is located downstream of the second region, and the second region is composed of polymorphic bases to be detected. Complementary to the first nucleic acid (sequence complementary to the third region, sequence complementary to the second region, and specific sequence) to the specific nucleic acid in the sample having a sequence located downstream of the three regions) A three-stranded structure is formed by hybridizing an oligonucleotide having a non-flapable flap sequence) and a second oligonucleotide (a sequence complementary to the first region and an oligonucleotide having an arbitrary sequence) A polymorphism is confirmed by releasing a nucleic acid fragment containing a flap sequence with a flap endonuclease and detecting the nucleic acid fragment using a mass spectrometer.
[Selection] Figure 6

Description

The present invention relates to a method for detecting a target nucleic acid that can easily detect SNP and the like, and a method for diagnosing drug administration, disease morbidity, and the like using the results obtained by the method.
This gene polymorphism detection method can be used in research of gene analysis and clinical fields.

  It is known that a flap endonuclease (FEN-1) recognizes a three-base structure called a double FLAP structure, and an oligonucleotide called a flap (FLAP) is released (for example, Harrington). JJ and Lieber MR, Journal of Biological Chemistry, 1995 Mar 3; 270 (9): 4503-4508. See Fig. 5.B).

  On the other hand, many methods have been reported for so-called typing for determining the base of the SNP site. Typical examples include the invader method, tackman PCR method, molecular beacon method, primer extension method and the like (for example, Kirk BW, Feinsod M, Favis R, Kliman RM and Barany F, Nucleic Acids Research, 2002 Aug) 1; 30 (15): 3295-3311).

The invader method is a technique for measuring a fluorescent substance released from a flap (FLAP) probe by a two-stage enzyme reaction.
Specifically, the invader method forms a double flap structure in which a flap probe and an invader probe are hybridized to a template DNA, and the enzyme cleavage (cleavase) recognizes this structure and cleaves the flap probe. The step of releasing is a first step; the step of releasing the quencher and the fluorescent material in the fret by combining the released flap with the fret and cleaving again with a chestnut base is included as the second step. As a result, SNP allele determination is performed by detecting the generated fluorescence.
This method is characterized in that it is devised so that the second-stage reaction can be performed in common by making the fret (FRET) sequence common (see, for example, JP-T-2002-515737). ).

  The advantages of the invader method are as follows. 1) It is possible to design probes over a wide range on the genome, and the degree of freedom thereof is relatively high; 2) it is not always necessary to amplify the region including the gene mutation portion in advance by PCR; and 3) By using FRET, which is expensive to synthesize, as a common sequence, the probe setting cost for each SNP can be reduced.

  Tackman PCR method, molecular beacon method, primer extension method, allele-specific competitive PCR method and the like are PCR-based methods. In addition to this, as a polymorphism detection method using PCR, there is a mass array (MASSARAY) system reported by Sequenom (see Japanese translations of PCT publication No. 2002-507883).

  Matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is generally used for proteins in general and has little application to DNA, but a mass array system is a MALDI-TOF. It is a SNP analysis method that also uses MS. In the mass array system, oligonucleotides with different mass numbers are generated by single base extension using a mixed reaction of dideoxynucleotide and deoxynucleotide, and the generated oligonucleotides are detected by MALDI-TOF MS to detect SNP. Make a decision.

Kirk BW, Feinsod M, Fabis R, Kliman RM, and Barany F, long-term association with complex diseases Single nucleotide polymorphism seeking long term association with complex disease, “Nucleic Acids Research”, Vol. 30, No. 15, p. 3295-3411, August 1, 2002 Harrington JJ and Lieber MR, DNA structural elements required for FEN-1 binding, “Journal of Biological Chemistry (Journal of Biological Chemistry) Biological Chemistry), Vol. 270, No. 9, p. 4503-4508, 5th. Figure B, March 3, 1995 JP-T-2002-515737 Japanese translation of PCT publication No. 2002-507883

The conventional method has the following problems, for example.
The invader method requires a dedicated kit including chestnut base and a plate reader for fluorescence detection. Fluorescent labels such as those used in the invader method are generally expensive.

  General problems of the method based on the PCR reaction include the following. For example, in a primer extension or competitive PCR, when a region containing a gene mutation site is amplified by PCR and then reacted with another primer, it is necessary to remove in advance the primer and dNTP used for the first amplification. In the case of the primer extension, it is necessary to completely remove dNTP in order to stop extension by dideoxy. In addition, a PCR product purification step is necessarily required. For example, in a mass array system, nucleic acid amplification is performed first, but it is necessary to remove in advance the primer and dNTP used for the first amplification. For this reason, work is complicated and costly.

  General problems of the method using the MALDI-TOF MS including the mass array system include the following. For example, depending on the arrangement of the probes, decomposition may occur during ionization, and peak detection may be difficult. Moreover, since a difference of about one base (about 300-400 Da) is compared, it is necessary to adjust the mass spectrometer with high accuracy, and it may be difficult to distinguish it from an unreacted oligonucleotide probe.

  Therefore, an object of the present invention is to provide a method for detecting a gene polymorphism with high sensitivity by a simple operation.

The present invention includes the following inventions.
In the following (1), the specific nucleic acid, the first oligonucleotide and the second oligonucleotide form a double flap structure, the flap endonuclease recognizes the structure and the nucleic acid fragment (referred to as a flap fragment in the present specification). In some cases, and the released nucleic acid fragments are detected by mass spectrometry.

(1) As a specific arrangement, an arrangement comprising a first area, a second area, and a third area, wherein the first area is located downstream adjacent to the second area, and the second area is detected. A specific nucleic acid in a sample having a specific sequence that is composed of a base of a polymorphic power and is located downstream and adjacent to the third region;
As the first oligonucleotide, an oligonucleotide having a sequence complementary to the third region, a sequence complementary to the second region, and a flap sequence not complementary to the specific sequence, the third region A sequence complementary to the second region and adjacent to the sequence complementary to the second region, and a sequence complementary to the second region and an oligonucleotide located downstream adjacent to the flap sequence;
As the second oligonucleotide, an oligonucleotide having a sequence complementary to the first region and an arbitrary sequence, wherein the arbitrary sequence is an oligonucleotide located downstream adjacent to the sequence complementary to the first region Forming a triple stranded structure by hybridizing with nucleotides;
Releasing a nucleic acid fragment containing the flap sequence by allowing a flap endonuclease to recognize a triple-stranded portion of the triple-stranded structure;
A genetic polymorphism detection method for confirming the polymorphism by detecting the released nucleic acid fragment using a mass spectrometer.

The following (2) relates to a specific example of a mass spectrometer.
(2) The gene polymorphism detection method according to (1), wherein the mass spectrometer is matrix-assisted laser desorption / ionization (MALDI) / time-of-flight (TOF) mass spectrometry.

The following (3) to (5) relate to the design of the flap arrangement.
(3) The genetic polymorphism detection according to (1) or (2), wherein an oligonucleotide designed to have a different flap sequence depending on each of the polymorphic alleles is used as the first oligonucleotide. Method.

(4) There are a plurality of polymorphic sites to be detected in the sample, and the first oligonucleotide is designed to have a different flap sequence depending on each of the plurality of polymorphic sites to be detected. The method for detecting a gene polymorphism according to any one of (1) to (3), wherein an oligonucleotide is used.

(5) The gene polymorphism detection method according to (3) or (4), wherein the mutually different flap sequences differ in length from each other.

The following relates to the object of detection.
The genetic polymorphism detection method according to any one of (1) to (5), wherein the specific nucleic acid has a nucleic acid sequence selected from cDNA, a genomic gene of a living organism, and a gene of a microorganism.
Microorganisms include bacteria and viruses.

The gene polymorphism detection method described above, wherein the gene is a DNA or RNA gene.
The genetic polymorphism detection method according to any one of (1) to (5), wherein the polymorphism is selected from a single nucleotide polymorphism, an insertion polymorphism, and a deletion polymorphism.

The following relates to a form in which a specific nucleic acid is previously amplified.
The method for detecting a gene polymorphism according to any one of (1) to (5), wherein the region containing the specific sequence of the specific nucleic acid is amplified in advance.

  The gene polymorphism detection method described above, wherein the amplification is performed by PCR.

  The gene polymorphism detection method described above, wherein the amplification is performed in a reaction solution having a pH of 8.5 to 9.5 at 25 ° C.

  According to the present invention, it is possible to provide a method for detecting a gene polymorphism simply and with high sensitivity.

That is, according to the present invention, since the flap sequence of the oligonucleotide probe can be arbitrarily set, for example, in length, it is possible to easily distinguish the detected flap fragment from the unreacted oligonucleotide probe. . Further, since the flap sequence can be arbitrarily set, for example, in length, between two types of oligonucleotide probes corresponding to each allele, it is easy to discriminate the flap fragment corresponding to each allele to be detected. be able to. Furthermore, since the flap sequence of the oligonucleotide probe can be a sequence that is not easily degraded even under ionization conditions, such as Poly-T, peak detection can always be facilitated. Therefore, sensitive detection can be performed easily. Moreover, such sensitive detection can be performed without requiring special accuracy in the mass spectrometer.
In addition, since the present invention eliminates the need for expensive labeling for oligonucleotide probes, for example, gene polymorphism detection can be carried out at a lower cost compared to gene polymorphism detection methods using the conventional invader method.

  Furthermore, according to the present invention, when a region containing a polymorphism to be detected in advance is amplified by PCR, an extension reaction is not performed again after the amplification, so that it is not necessary to remove excess primers and dNTPs. For this reason, for example, gene polymorphism detection can be easily performed compared with the conventional gene polymorphism detection method based on PCR reaction.

  The present invention uses a specific nucleic acid having a polymorphism to be detected as a template nucleic acid, and hybridizes a first oligonucleotide having a flap sequence and a second oligonucleotide to the template nucleic acid to thereby form a triple-stranded structure. A polymorphism is detected by forming a body, releasing a nucleic acid fragment containing a flap sequence with a flap endonuclease, and confirming the released nucleic acid fragment with a mass spectrometer.

  The target of detection in the present invention is not particularly limited, and examples thereof include cDNA, genomic genes of various organisms including humans, genes of various microorganisms including viruses and bacteria, and the like. Accordingly, a template nucleic acid having these nucleic acid sequences can be used. The gene to be detected may be a DNA gene or an RNA gene.

  The sample containing the template nucleic acid is not particularly limited. That is, polymorphism detection can be performed on biological samples, biological samples, extracted nucleic acid samples, and the like. Here, a biological sample or a biological sample is a sample in a form in which a nucleic acid inclusion body (for example, a membrane structure containing nucleic acid inside, such as cells, fungi, bacteria, and viruses) is maintained without performing a nucleic acid extraction operation. is there. Examples of biological samples include organs, tissues, body fluids, excreta and the like. Body fluids include blood samples, spinal fluid, saliva, milk and the like. The blood sample includes whole blood, plasma, serum and the like. Excrements include urine and feces. A biological sample refers to a sample obtained by recovering a nucleic acid inclusion from a biological sample. The method for recovering the nucleic acid inclusion body is not particularly limited, and examples thereof include centrifugation / ultracentrifugation, a method using a coprecipitation agent such as polyethylene glycol, and an adsorption carrier. The extracted nucleic acid sample is a sample subjected to nucleic acid extraction operation, and may be a sample obtained by further purifying nucleic acid after extraction. Examples of extraction methods include methods using enzymes, surfactants, chaotropic agents, etc., and purification methods include phenol / chloroform, ion exchange resin, glass filters, glass beads, magnetic beads, and a reagent having a protein aggregation action. The method using etc. is mentioned.

  The polymorphisms that can be detected in the present invention are not particularly limited, and include both so-called single nucleotide polymorphisms (SNPs) and polymorphisms spanning multiple nucleotide sequences. In the present invention, the polymorphism further includes a mutation. Specifically, examples of the polymorphism that can be detected in the present invention include a single nucleotide polymorphism, an insertion polymorphism, and a deletion polymorphism.

An example of the three-stranded structure of the present invention is shown in FIG.
In the present invention, a template nucleic acid that is a specific nucleic acid includes a specific sequence composed of a first region, a second region, and a third region. The first region is located downstream adjacent to the second region, and the second region is located downstream adjacent to the third region. Of these, the second region is a region composed of polymorphic bases to be detected.

  The first oligonucleotide has a flap sequence and is specific to the second region to the third region. The flap sequence is a sequence that is not complementary to a specific sequence (specifically, a sequence that is not complementary to the first region). That is, the first oligonucleotide has a sequence complementary to the third region, a sequence complementary to the second region, and a flap sequence that is not complementary to the specific sequence. The sequence complementary to the third region is located downstream adjacent to the sequence complementary to the second region, and the sequence complementary to the second region is located downstream adjacent to the flap sequence. Furthermore, the first oligonucleotide has a sequence that is not complementary to the template nucleic acid upstream of the sequence complementary to the third region (specifically, a downstream sequence adjacent to the specific sequence in the template nucleic acid). A sequence having no complementarity).

  The second oligonucleotide is an oligonucleotide specific for the first region. The second oligonucleotide has a sequence complementary to the first region and an arbitrary sequence. The arbitrary sequence is located downstream adjacent to the sequence complementary to the first region. Furthermore, the second oligonucleotide has a sequence that is not complementary to the template nucleic acid downstream of the sequence complementary to the first region (specifically, an upstream sequence adjacent to the specific sequence in the template nucleic acid). A sequence having no complementarity).

  As long as the flap endonuclease recognizes a special structure of nucleic acid, that is, a triple-stranded structure (double-flap structure) and can release a nucleic acid fragment containing a flap sequence by cleaving the first oligonucleotide. There is no particular limitation. Specifically, any enzyme that recognizes the triple chain portion and cleaves the first oligonucleotide when the template nucleic acid, the first oligonucleotide, and the second oligonucleotide form a triple-stranded structure may be used. For example, chestnut (cleavase) and heat resistant flap endonuclease described in JP-A-11-75849 can be mentioned. For example, when the polymorphism is SNP and chestnut base is used, as shown in FIG. 2, the structure of the SNP site of the three-stranded structure is recognized by chestnut base (FIG. 2 (a)), and the base 3 corresponding to the SNP site The first oligonucleotide is cleaved on the ′ side, and a nucleic acid fragment having a base corresponding to the flap sequence and the SNP site (hereinafter referred to as a flap fragment) is released (FIG. 2B).

The first oligonucleotide usually has one polymorphic site in a sample and is designed according to each polymorphic allele. For example, as the first oligonucleotide, two types of allele-specific oligonucleotides can be used. And the flap part of each 1st oligonucleotide can be set so that the flap fragment which arises from one and / or the other of these allele-specific 1st oligonucleotides can be identified in a mass spectrum. The flap array is preferably an array that is not easily decomposed under ionization conditions in mass spectrometry. Examples of such a sequence include a sequence consisting only of T (poly-T).
Each of the first oligonucleotides can be designed to be different depending on the allele in the sequence of the flap portion so that it is possible to distinguish which allele was detected in the mass spectrum.

  In gene polymorphism detection methods, a plurality of polymorphic sites in a sample are often used as detection targets. In this case, the first oligonucleotide is designed according to each of a plurality of polymorphic sites to be detected. For example, as the first oligonucleotide, a set of two allele-specific first oligonucleotides can be used as many as the number of types of polymorphic sites. And the flap part of each 1st oligonucleotide can be set so that each of the flap fragment produced from the 1st oligonucleotide designed according to these polymorphic site | parts can be identified in a mass spectrum. Each of the first oligonucleotides can be designed to be different depending on the polymorphic site in the sequence of the flap portion so that it is possible to distinguish which site of the polymorphism is detected in the mass spectrum. .

  In the first oligonucleotides having different flap sequences, it is preferable that the flap sequences have different lengths. For example, the flap sequences can consist of a single base and differ from each other only in length.

  The lengths of the first oligonucleotide and the flap sequence are not particularly limited, and can be appropriately set by those skilled in the art in consideration of the resolution of the mass spectrometer used in the present invention. Each length may be set so that the unreacted first oligonucleotide, the flap fragment corresponding to one allele, and the flap fragment corresponding to the other allele can be distinguished in the mass spectrum. For example, the first oligonucleotide can be set to a length of 25 to 140 mer, for example. Of this set length, the flap sequence can be set to a length of 10 to 50 mer, for example. Furthermore, between different first oligonucleotides, the flap sequences may be set with a difference in length of 1 to 20 mer. Moreover, among the above set lengths, the sequence complementary to the second and third regions can be set to a length of 15 to 80 mer, for example.

  Although it does not specifically limit as length of a 2nd oligonucleotide, For example, it can set to 15-60mer length. Of this set length, the arbitrary sequence in the second oligonucleotide can be the same length as the sequence constituting the polymorphism, for example. For example, when detecting SNP, arbitrary arrangement | sequences are good to set it as 1mer. Among the above set lengths, the sequence complementary to the first region can be set to a length of 9 to 59 mer, for example.

  The reaction in the formation of the triple-stranded structure and the release of the flap fragment can be performed, for example, under the following conditions. Depending on the flap endonuclease used, the reaction can be carried out under isothermal conditions of 55 to 75 ° C. and 5 minutes to 18 hours. When using chestnut base, the reaction can be carried out at 55 to 68 ° C. for 5 minutes to 18 hours. For example, the reaction is preferably carried out at 63 ° C. for about 30 minutes. When nucleic acid amplification is performed prior to this reaction (the nucleic acid amplification will be described later), it is not necessary to remove reagents such as the used primers.

  Moreover, it is preferable to use the conditions for making it a single strand immediately before this reaction. For this reason, the reaction solution can be held at 90 ° C. or more for 5 minutes or more, for example.

  After the reaction, the reaction solution may be desalted, mixed with the matrix, and applied to the measurement plate. A desalting column may be used for the desalting treatment. Examples of the desalting column include ZipTip (manufactured by Millipore). As the desalting column, a filter-shaped chip or the like can be used.

  As a matrix, 2,4,6-trihydroxyacetophenone (2,4,6-trihydroxyacetophenone), 2,3,4-trihydroxyacetophenone (2,3,4-trihydroxyacetophenone), 3-hydroxypicolinic acid (3- It can be selected from hydroxypicolinic acid (3-HPA), α-cyano-4-hydroxycinnamic acid methyl ester, and the like. These matrices are used, for example, as a solution prepared by dissolving in an appropriate solvent. Examples of such a solvent include an acetonitrile-trifluoroacetic acid aqueous solution. The composition of the matrix solution can be appropriately determined by those skilled in the art.

  The measurement plate is introduced into the mass spectrometer and measurement is performed according to a normal method. In the present invention, since the flap sequence in the first oligonucleotide can be arbitrarily set, the mass spectrometer to be used is not particularly limited, and an existing mass spectrometer can be used. The mass spectrometer preferably has a matrix-assisted laser desorption / ionization (MALDI) ion source. The MALDI ion source is more preferably used in combination with a time-of-flight (TOF) mass spectrometer. Examples of MALDI-TOF MS include AXIMA (manufactured by Shimadzu Corporation) and ABI Voyager (manufactured by Perceptive Biosystems).

  In the present invention, steps up to desalting of the sample, mixing of the sample and the matrix, and preparation of the MS detection plate may be performed automatically. When automated in this way, it is possible to efficiently detect genetic polymorphism for multiple specimens.

  In the mass spectrum, the polymorphism can be detected by confirming the presence of the flap fragment based on the set mass number of the flap sequence. It is preferable to confirm that the peak of the first oligonucleotide detected before the cleavage reaction by the flap endonuclease disappears after the cleavage reaction and that the peak of the flap fragment is detected.

  When the base corresponding to the second region (ie, polymorphic base) in the oligonucleotide designed as the first oligonucleotide matches the polymorphic site assumed in the template nucleic acid, a triple-stranded structure is formed (FIG. 2). (A)) When the chestnut base recognizes this structure, the first oligonucleotide is cleaved, and the flap fragment is released (FIG. 2 (b)). Therefore, as a result of mass spectrometry, the first oligonucleotide is not detected, and the flap sequence is detected.

  On the other hand, when the base corresponding to the polymorphic base in the oligonucleotide designed as the first oligonucleotide does not match the polymorphic site assumed in the template nucleic acid, a triple-stranded structure that can be recognized by Chrybase is formed. As a result, the first oligonucleotide is not cleaved and the flap fragment is not released (FIG. 3). Therefore, as a result of mass spectrometry, the first oligonucleotide is detected and the flap fragment is not detected.

  For example, when a certain SNP site can take T or C, a T oligonucleotide and a C oligonucleotide are prepared as the first oligonucleotide. The T oligonucleotide and the C oligonucleotide have A and G as bases complementary to the second region (ie, SNP), respectively, and are designed so that the flap sequences are different from each other. The flap sequences are preferably designed so that the lengths of the T oligonucleotide and the C oligonucleotide are different from each other.

For example, when the flap sequence of the oligonucleotide for T is Poly-T12 (that is, a sequence composed of 12 T), if the reaction schematically shown in FIG. 4 occurs, the length of Poly-T12 is increased. SNPs can be detected by detecting a flap fragment having a flap sequence. On the other hand, when the flap sequence of the oligonucleotide for C is Poly-T15 (that is, a sequence composed of 15 T), if the reaction schematically shown in FIG. 5 occurs, the length of Poly-T15 SNPs can be detected by detecting a flap fragment having a flap sequence. As a result of the detection, if a flap fragment having the flap sequence Poly-T12 is detected and no flap fragment having the flap sequence Poly-T15 is detected, typing that the SNP is a T / T homozygote is performed. be able to. Further, when both a flap fragment having the flap sequence Poly-T12 and a flap fragment having the flap sequence Poly-T15 are detected, it can be typed that the SNP is a T / C heterozygote. Further, when a flap fragment having the flap sequence Poly-T12 is not detected and a flap fragment having the flap sequence Poly-T15 is detected, typing that the SNP is a C / C homozygote can be performed. it can.
Other polymorphisms can be similarly typed.

  In the present invention, a triple-strand structure formation and a flap endonuclease cleavage reaction may be performed directly from the genome, or a region containing a specific sequence containing a polymorphism to be detected may be amplified in advance. Although it does not specifically limit as a method for amplifying the area | region containing a specific sequence, For example, PCR can be performed. When detecting a plurality of polymorphisms in a sample, multiplex PCR can be performed on a plurality of corresponding specific sequences.

  In nucleic acid amplification, it is preferable to use a hot start method. By using the hot start method, primer misannealing and oligomerization can be prevented, and undesirable nucleic acid amplification can be prevented. Further, when the hot start method is used, a large number of regions can be amplified simultaneously, so that polymorphism can be detected efficiently.

  In the amplification of the genome, in order to amplify one polymorphic site, a pair of primers that bind across the polymorphic site are used. When there are a plurality of polymorphisms to be detected in the target sample and the polymorphic sites to be detected are located at positions separated from each other, a primer twice the number of types of polymorphic sites is used. However, for example, when a plurality of polymorphisms, for example, two polymorphisms are close to each other, amplification may be performed by binding a primer across each of the two polymorphism sites. Amplification may be carried out by binding a pair of primers to both ends of the two polymorphic sites without binding the primer.

The PCR reaction solution contains a pH buffer solution, salts such as MgCl ₂ and KCl, primers, deoxyribonucleotides, and a thermostable synthase. In addition, substances such as surfactants and proteins can be added as necessary.

  As the pH buffer solution, a combination of tris (hydroxymethyl) aminomethane and a mineral acid such as hydrochloric acid, nitric acid, sulfuric acid, and various other pH buffer solutions can be used. The pH-adjusted buffer is preferably used at a concentration between 10 mM and 100 mM in the PCR reaction solution.

A primer refers to an oligonucleotide that serves as a starting point for nucleic acid synthesis by a PCR reaction. The primer may be synthesized or isolated from the biological world.
The thermostable synthase is an enzyme for nucleic acid synthesis by adding a primer and includes a chemical synthesis system. Suitable synthetic enzymes include E. coli DNA polymerase, Klenow fragment of E. coli DNA polymerase, T4 DNA polymerase, Taq DNA polymerase, T. litoralis DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Hot Start Taq polymerase, KOD DNA polymerase, Examples include, but are not limited to, EX Taq DNA polymerase and reverse transcriptase. The thermal stability means a property of a compound that retains its activity at high temperature, preferably 65 to 95 ° C.

  Other conditions in the amplification reaction are not particularly limited, and the reaction can be performed based on conditions in a known method. When a biological sample or a biological sample is used as the sample, the amplification reaction is preferably performed in a reaction solution having a pH of 8.5 to 9.5 at 25 ° C. By doing so, it is also possible to react by directly acting a nucleic acid amplification reaction solution on a biological sample or a biological sample.

Suitable kits for carrying out the method of the present invention include the following.
As a specific sequence, a sequence composed of a first region, a second region and a third region, wherein the first region is located downstream adjacent to the second region, and the second region is a polymorphism to be detected A gene polymorphism detection kit for detecting a base polymorphism of a specific nucleic acid in a sample having a specific sequence located downstream and adjacent to the third region.
<1> An oligonucleotide having a sequence complementary to the third region, a sequence complementary to the second region, and a flap sequence not complementary to the specific sequence, as the first oligonucleotide, A sequence complementary to the third region is located downstream adjacent to the sequence complementary to the second region, a sequence complementary to the second region is located downstream adjacent to the flap sequence, and Oligonucleotides designed so that the flap sequence is different for each of the polymorphic alleles;
<2> As a second oligonucleotide, an oligonucleotide having a sequence complementary to the first region and an arbitrary sequence, wherein the arbitrary sequence is located downstream of the sequence complementary to the first region. A positioned oligonucleotide; and
<3> flap endonuclease,
<4> A genetic polymorphism detection kit comprising a matrix and / or a desalting column.

The oligonucleotide sequences used in the examples and comparative examples are listed below. The base marked with * on the right shoulder is a base assumed as a polymorphic site, or a complementary base or a non-complementary base corresponding to the base.
Inv_A: AGTCTGTACTAATTGCACATG ^* TATTGTTGGGGATTTTCCAT (SEQ ID NO: 1)
Inv_A40merA: AGTCTGTACTAATTGCACATG ^* TATTGTTGGGGAT (SEQ ID NO: 2)
Inv_Cw1: TTTTTTTTTTTTC ^* ATGTGCAATTAGTACAGACT (SEQ ID NO: 3)
Inv_Cm: TTTTTTTTTTTTTTTA ^* ATGTGCAATTAGTACAGACT (SEQ ID NO: 4)
Inv_B1: ATGGAAAATCCCCAACAATAG ^* (SEQ ID NO: 5)

<Example 1>
1. Preparation of oligonucleotide As template nucleic acid, oligonucleotide Inv_A having the base sequence shown in SEQ ID NO: 1, the first oligonucleotide, oligonucleotide Inv_Cw1 having the base sequence shown in SEQ ID NO: 3, and the second oligonucleotide, The oligonucleotide Inv_B1 having the sequence shown in SEQ ID NO: 5 was synthesized by Proligo Japan Co., Ltd., and each synthesized oligonucleotide was prepared to 100 pmol / ml with DW (distilled water).

  Here, the 21st base of the template nucleic acid Inv_A is assumed as the SNP. The base sequence (SEQ ID NO: 3) of the first oligonucleotide Inv_Cw1 is composed of a base sequence complementary to the 1st to 21st base sequences of the base sequence (SEQ ID NO: 1) of the template nucleic acid and a flap sequence (Poly-T12). And have. The base sequence (SEQ ID NO: 5) of the second oligonucleotide Inv_B1 is a base sequence complementary to the 22nd to 32nd base sequences of the base sequence (SEQ ID NO: 1) of the template nucleic acid and a sequence unrelated to the template nucleic acid. Have.

2. Formation of double-flap structure and cutting of flap A reaction solution containing 7 μl of the following components in DW was prepared.
Inv_A (template nucleic acid) aqueous solution 0.5μl
Inv_Cw1 (first oligonucleotide) aqueous solution 1μl
Inv_B1 (second oligonucleotide) aqueous solution 1μl
Enzyme Mix (Flap Endonuclease) 0.3μl
Buffer (0.8g / ml PEG800, 0.2mmol / ml MOPS) 0.3μl
In addition, the Enzyme Mix used what was contained in Kit of Invader DNA Assay (THIRD WAVE TECHNOLOGIES).
7 μl of the reaction solution was placed in a 0.5 ml microtube, and mineral oil was overlaid on the reaction solution to prevent evaporation. A microtube was set on the thermal cycler, and the reaction was carried out at 95 ° C for 3 minutes and at 60 ° C for 60 minutes.

3. Confirmation of flap fragment by mass spectrometer After completion of the reaction, 2 μl was taken out from the reaction solution in the microtube, desalted with ZipTipC18, and used as a measurement sample. The measurement sample was eluted on a MS plate with 0.5 μl of 50 v / v% acetonitrile aqueous solution. Further, 0.5 μl of a 3-HPA (3-hydroxypicolinic acid) solution (a 50 v / v% solution of 0.3 M 3-HPA-0.07 M ammonium phosphate aqueous solution and acetonitrile) was overlaid as a matrix solution. The measurement was performed in Linear-Negative mode using AXIMA-CFR (manufactured by Shimadzu Corporation).

  The obtained mass spectrum is shown in FIG. In FIG. 6, the horizontal axis represents mass / charge, and the vertical axis represents relative intensity (% Int.). As shown in FIG. 6A, a 13mer peak and a 20mer peak were detected. This is because by cutting the first oligonucleotide Inv_Cw1, the 13-mer flap fragment of (Poly-T12) + (1 base: G) and the remaining 20-mer oligonucleotide fragment are separated from the first oligonucleotide Inv_Cw1. It is thought that it was generated. Further, in the spectrum shown in FIG. 6A, the mass / charge range around 33mer corresponding to the first oligonucleotide Inv_Cw1 is not shown, but the 33mer peak disappears. From this, it is considered that the double flap structure was recognized and the flap was cut.

<Comparative Example 1>
As a template nucleic acid, an oligonucleotide Inv_A40merA having the base sequence shown in SEQ ID NO: 2 and as a first oligonucleotide, an oligonucleotide Inv_Cm having the base sequence shown in SEQ ID NO: 4 (both are outsourced to Proligo Japan Co., Ltd.) The same operation as in the method of Example 1 was performed except that Here, the base sequence (SEQ ID NO: 4) of the first oligonucleotide Inv_Cm is a base sequence complementary to the 1st to 20th base sequences of the base sequence (SEQ ID NO: 2) of the template nucleic acid and the 21st base. And a flap sequence (Poly-T15).

  The obtained mass spectrum is shown in FIG. As shown in FIG. 6B, a 16mer peak corresponding to the flap fragment and a 20mer peak corresponding to the remaining oligonucleotide fragment when the flap fragment was released from the first oligonucleotide were not detected. Further, in the spectrum shown in FIG. 6B, the mass / charge range around 36mer corresponding to the first oligonucleotide Inv_Cm is not shown, but a 36mer peak is detected. From this, it is considered that since the 21st base of Inv_A40merA was not complementary to Inv_Cm, a double flap structure could not be formed and the recognition site did not exist, so the enzyme did not work.

  As described above, in Example 1, if SNP is complementary to the probe, a reaction by flap endonuclease occurs and SNP is detected. On the other hand, in Comparative Example 1, if SNP is not complementary to the probe, flap endonuclease. It was confirmed that the reaction by did not occur. Thereby, it can be said that SNP could be discriminated.

It is the schematic diagram which showed an example when the template nucleic acid, 1st oligonucleotide, and 2nd oligonucleotide of this invention formed a triple strand structure. Schematic diagram (a) of the triple-stranded structure of the present invention formed when the oligonucleotide designed as the first oligonucleotide matches the template nucleic acid at the polymorphic site, and the triple-stranded structure is cleaved by flap endonuclease It is a schematic diagram (b) which shows a mode that it received reaction and the flap fragment | piece was released. It is the schematic diagram showing the case where the oligonucleotide designed as a 1st oligonucleotide does not match a template nucleic acid in a polymorphic region. It is a schematic diagram showing the case where C is detected as SNP. It is a schematic diagram showing the case where T is detected as SNP. It is the mass spectrum (a) obtained in Example 1, and the mass spectrum (b) obtained in Comparative Example 1.

SEQ ID NO: 1 is a synthetic oligonucleotide.
SEQ ID NO: 2 is a synthetic oligonucleotide.
SEQ ID NO: 3 is a synthetic oligonucleotide.
SEQ ID NO: 4 is a synthetic oligonucleotide.
SEQ ID NO: 5 is a synthetic oligonucleotide.

Claims (5)

As a specific sequence, a sequence composed of a first region, a second region and a third region, wherein the first region is located downstream adjacent to the second region, and the second region is a polymorphism to be detected A specific nucleic acid in a sample having a specific sequence that is located downstream of and adjacent to the third region.
As the first oligonucleotide, an oligonucleotide having a sequence complementary to the third region, a sequence complementary to the second region, and a flap sequence not complementary to the specific sequence, the third region A sequence complementary to the second region and adjacent to the sequence complementary to the second region, and a sequence complementary to the second region and an oligonucleotide located downstream adjacent to the flap sequence;
As the second oligonucleotide, an oligonucleotide having a sequence complementary to the first region and an arbitrary sequence, wherein the arbitrary sequence is an oligonucleotide located downstream adjacent to the sequence complementary to the first region Forming a triple stranded structure by hybridizing with nucleotides;
Releasing a nucleic acid fragment containing the flap sequence by allowing a flap endonuclease to recognize a triple-stranded portion of the triple-stranded structure;
A genetic polymorphism detection method for confirming the polymorphism by detecting the released nucleic acid fragment using a mass spectrometer.   The genetic polymorphism detection method according to claim 1, wherein the mass spectrometer is matrix-assisted laser desorption / ionization (MALDI) / time-of-flight (TOF) mass spectrometry.   The genetic polymorphism detection method according to claim 1 or 2, wherein an oligonucleotide designed to have a different flap sequence according to each of the polymorphic alleles is used as the first oligonucleotide.   An oligonucleotide designed so that there are a plurality of polymorphic sites to be detected in the sample, and the first oligonucleotide has a different flap sequence depending on each of the plurality of polymorphic sites to be detected The genetic polymorphism detection method according to any one of claims 1 to 3, wherein The genetic polymorphism detection method according to claim 3 or 4, wherein the different flap sequences are different from each other in length.

Images