JP2008048705A - Method for analyzing base sequence and primer for analyzing base sequence - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method for analysis with high analytical accuracy in a method for analyzing a base sequence in a specific position in a target nucleic acid. <P>SOLUTION: A primer for analyzing the base sequence having a base sequence complementary to the base sequence adjacent to the 3'-side of the base sequence in the specific position in the target nucleic acid and a tag sequence which is a base sequence without binding to the natural type nucleic acid is used. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for analyzing a base sequence at a specific position in a target nucleic acid and a base sequence analysis primer used in the method.

  In addition to single nucleotide polymorphisms (SNPs), gene polymorphisms include microsatellite and VNTR (variable number of tandem repeat), which have different repeat base sequences with two to tens of bases as one unit. Or AnTn having different numbers of consecutive base sequences of A and T.

  Human SNPs are genetic polymorphisms found with a frequency of about one in several hundred bases. These mutations are widely scattered in the genome regardless of coding region or non-coding region, and insertions and deletions are seen as well as base substitutions. Since the size of the human genome is 3 billion base pairs, there will be 3 million SNPs even if there is one such polymorphism per 1000 bases. At present, it is considered that the number of SNPs related to one drug or disease susceptibility needs to be typed at several hundreds and several tens at most. In addition, when trying to detect microsatellite, VNTR, and AnTn at the same time, there is a method of typing using a DNA sequencer by the Sanger method. However, in order to perform this type of typing, the reagents and equipment for sequencing reaction are expensive. Since it takes time and effort, it is desirable to simplify the operation.

  The method combining the single base extension method and the DNA chip is excellent in that a large number of gene polymorphisms can be analyzed in one reaction. However, for detection, the primer labeled according to the polymorphism was merely captured using a probe having a base sequence complementary to the base sequence of the target nucleic acid to be detected. For this reason, the optimal hybridization conditions of each probe are not constant, and false positive hybridization is likely to occur, which has a problem in the accuracy of analysis. In addition, conventionally, if the type of target nucleic acid to be detected changes, the complementary base sequence of those nucleic acids must be re-immobilized, so it can be applied to clinical tests in which a large number of nucleic acids are targeted for detection. In addition, there was a problem that versatility was not sufficient.

  In order to solve these problems, each base sequence of the target nucleic acid to be detected has an arbitrarily designed tag sequence, that is, a base that has approximately the same length and approximately the same dissociation temperature and does not bind to the target nucleic acid. A method for capturing a polynucleotide fragment to which a sequence has been added as a primer has been disclosed (Non-patent Document 1). However, even in this method, when analyzing a plurality of polymorphisms at the same time, each specificity between tag sequences must be taken into consideration.

As a method for designing a tag sequence, for example, a method has been proposed in which a base sequence of a genetically distant species such as a virus, which is not similar to a mammal, is used as a capture probe sequence when analyzing a polymorphism in a mammal. (See Patent Documents 1 and 2). However, all the tag sequences must have the same length and the same dissociation temperature, and at the same time satisfy the condition of maintaining specificity, so that complex calculation is still required to design the tag sequence. there were.
J-B. Fan (JB. Fan), Genome Research (Genome Res.), 10, 853-860, 2000 Japanese translation of PCT publication No. 2002-539849 Special table 2003-522527 gazette

  As described above, in the single base extension method, the design of the tag sequence greatly affects the accuracy of the analysis. Therefore, when designing a plurality of tag sequences, the dissociation temperature should be the same, and the base that does not bind to the target nucleic acid. Since it is necessary to consider the fact that it is a sequence and that all tag sequences maintain specificity, it is very difficult to design multiple tag sequences, and therefore the actual usable nucleotide sequences are limited. There was a problem of being.

  This invention solves the said subject in the method of analyzing the base sequence of the specific position in a target nucleic acid.

That is, the present invention is for base sequence analysis having a base sequence complementary to the base sequence adjacent to the 3 ′ side of the base sequence at a specific position in the target nucleic acid and a tag sequence that is a base sequence that does not bind to a natural nucleic acid. A method for analyzing a base sequence, characterized by using a primer,
A first step of hybridizing the target nucleic acid and the base sequence analysis primer under appropriate conditions to form a complex of the base sequence analysis primer and the target nucleic acid;
A second step of obtaining a labeled product by performing a base extension reaction in which a labeled nucleotide complementary to the base sequence at the specific position is added to the base sequence analysis primer in the complex obtained in the first step;
A third step of specifically capturing the labeled product obtained in the second step via the tag sequence;
A fourth step of measuring the presence or signal intensity of the label contained in the labeled product and analyzing the base sequence at a specific position using the presence or signal intensity of the label as an index;
A method for analyzing a base sequence is provided.

  The present invention also provides a base sequence complementary to a base sequence adjacent to the 3 ′ side of a base sequence at a specific position in a target nucleic acid and a base sequence that does not bind to a natural nucleic acid, which is used in the base sequence analysis method described above. A primer for base sequence analysis having a tag sequence is provided.

  Furthermore, the present invention provides a biochip on which a tag capture probe having a base sequence capable of selective binding to the tag sequence in the base sequence analysis primer is immobilized.

  One preferred form of the primer for base sequence analysis of the present invention is such that the tag sequence is a base sequence having a non-natural nucleotide. In a more preferred embodiment, the non-natural nucleotide has a non-natural sugar such as L-type ribose or L-type deoxyribose.

  By using the present invention, it is possible to design a tag sequence very simply and to analyze the base sequence at a specific position of the target nucleic acid with high accuracy. The present invention can be widely applied to base sequence analysis such as single base sequence analysis and detection of the number of repetitive base sequence repeats.

The present invention is a method for analyzing a base sequence at a specific position in a target nucleic acid, and does not bind to a base sequence complementary to a base sequence adjacent to the 3 ′ side of the base sequence at the specific position and a natural nucleic acid. Using a primer for base sequence analysis having a tag sequence that is a base sequence,
A first step of hybridizing the target nucleic acid and the base sequence analysis primer to form a complex of the base sequence analysis primer and the target nucleic acid;
A second step of obtaining a labeled product by performing a base extension reaction in which a labeled nucleotide complementary to the base sequence at the specific position is added to the base sequence analysis primer in the complex obtained in the first step;
A third step of specifically capturing the labeled product obtained in the second step via the tag sequence;
A fourth step of measuring the presence or signal intensity of the label contained in the labeled product and analyzing the base sequence at a specific position using the presence or signal intensity of the label as an index;
Is a method for analyzing a base sequence.

  The base sequence analysis method of the present invention will be specifically described in the order of each step.

  The first step is a step of hybridizing a target nucleic acid and a base sequence analysis primer to form a complex of the base sequence analysis primer and the target nucleic acid.

  The primer for base sequence analysis used in the base sequence analysis method of the present invention is a tag that does not bind to a base sequence complementary to the base sequence adjacent to the 3 ′ side of the base sequence at a specific position in the target nucleic acid and the natural nucleic acid. A primer for base sequence analysis having a sequence. FIG. 1 shows a schematic diagram for explaining a primer for base sequence analysis. The target nucleic acid has a base sequence complementary to the base sequence adjacent to the 3 'side of the base sequence at a specific position to be analyzed, and a tag sequence that does not bind to the natural nucleic acid is added to the 5' end. The length of the complementary base sequence is usually preferably 5 to 100 bases, and more preferably 10 to 60 bases in consideration of hybridization specificity and synthesis cost.

  The primer for base sequence analysis of the present invention is a polynucleotide having a tag sequence that does not bind to a natural nucleic acid. The base sequence that does not bind to the natural nucleic acid is not particularly limited as long as it does not hybridize with the natural nucleic acid. As the nucleotide in the base sequence, a method using a nucleotide in which either a sugar part or a base part is modified so as not to bind to a natural nucleic acid can be used. Among these, when the sugar part is modified, examples thereof include a method of introducing an appropriate functional group into the hydroxyl group of the sugar and a method of using a non-natural sugar as the sugar itself. Of these, nucleotides having unnatural L-type sugars (hereinafter sometimes referred to as L-type nucleotides) are preferably used, and nucleotides having L-type ribose or L-type deoxyribose non-natural sugars are more preferred. Preferably used. Since the sugar of double-stranded natural DNA including the genome as the target nucleic acid is D-type, it does not hybridize with a polynucleotide having L-type sugar (hereinafter sometimes referred to as L-type polynucleotide). Furthermore, it may be further modified to L-type ribose, and for example, 2'-fluororibose, 2'-chlororibose, 2'-o-methylribose and the like can be used. Examples of nucleotides modified in the nucleobase include 5-propynyluracil, 2-aminoadenine and the like.

  Moreover, when using a plurality of primers for base sequence analysis, the base sequences of the tag sequences have the same dissociation temperature between the tag sequences, and each has specificity. A base sequence is preferred. Such a base sequence is publicly available or available as a DNA chip for expression analysis in a natural base sequence, and a modification that does not bind to a natural nucleic acid, for example, a non-natural L-type sugar is introduced. Even when a base sequence to be selected is selected, these can be used as the base sequence to be converted into a non-natural base sequence and used.

  The length of the base sequence of the tag sequence is not particularly limited, but usually 5 to 100 bases are preferable, and 10 to 60 bases are more preferable in consideration of hybridization specificity and synthesis cost.

  L-type nucleotides used as tag sequences that do not bind to natural nucleic acids can be synthesized by known methods. Also, for example, commercially available Beta-L-deoxy Adenosine (n-bz) CED phosphoramidite, Beta-L-deoxy Cytidine (n-bz) CED phosphoramidite, Beta-L-deoxy Guanosine (n-ibu) CED phosphoramidite, Beta-L-deoxy Thymidine CED phosphoramidite (ChemGenes Corporation) etc. can be used as it is.

  The L-type nucleotide can be used to synthesize the nucleotide sequence analysis primer of the present invention using an ordinary DNA / RNA automatic synthesizer, for example, an Applied Biosystems 392 DNA / RNA automatic synthesizer. Here, the 3 ′ terminal amination of the synthesized oligonucleotide is carried out by, for example, a DNA / RNA automatic synthesizer using a glass bead support for 3 ′ amination modification (3′-PT Amino-Modifer C6 CPG) manufactured by GLEN RESEARCH. It can be performed by using.

  In the primer for base sequence analysis of the present invention, a spacer is preferably provided between the base sequence complementary to the target nucleic acid and the tag sequence. By providing a spacer, especially in the third step of capturing specifically via a tag sequence, the steric congestion due to the presence of the complementary base sequence and the tag sequence is eliminated, and for base sequence analysis Hybridization efficiency between the primer and the tag capture probe is increased.

  Specific examples of the spacer include natural or non-natural polynucleotide chains, hydrocarbon chains, peptide chains, peptide nucleic acid sugar chains, etc. Among these, non-natural polynucleotide chains are preferable. As the non-natural nucleotide used here, a sugar having a non-natural sugar, for example, an L-nucleotide comprising a sugar consisting of L-type ribose or L-type deoxyribose may be used. L-type polythymidine is preferably used as the L-type polynucleotide. The thymidine residue has a property that it is difficult to take a higher-order structure from its electronic configuration, so it functions as a spacer for the analysis primer, and each of the base sequence portion complementary to the target nucleic acid and the tag sequence portion can be freely hybridized. There is an effect to make it. When a non-natural polynucleotide chain is used as a spacer, the number of residues is not limited, and may be appropriately determined according to the base sequence complementary to the target nucleic acid of the primer for base sequence analysis and the tag sequence. 3 to 10 residues.

  The hybridization reaction can be performed under normal conditions in which an annealing reaction is performed by polymerase chain reaction (PCR). The temperature of the hybridization reaction may be in a range where the specificity of the primer for base sequence analysis to be used is maintained, and is preferably set according to the heat resistance of the enzyme, the salt concentration of the solution, and the like. Usually, it can carry out at 30 to 70 degreeC.

  The second step is a step of obtaining a labeled product by performing a base extension reaction in which a labeled nucleotide complementary to the base sequence at a specific position is added to the base sequence analysis primer in the complex obtained in the first step. is there.

  The base extension reaction may be performed under the conditions used in ordinary PCR. The temperature of the base extension reaction may be within the optimum activity range of the enzyme while maintaining the specificity of the primer for base sequence analysis to be used, but is preferably set according to the heat resistance of the enzyme, the salt concentration of the solution, etc. . Usually, it can carry out at 30 to 70 degreeC.

  The labeled nucleotide to be added is a dideoxynucleotide (ddNTP), ie, ddATP, ddTTP or ddUTP, ddGTP, ddCTP, or a deoxynucleotide (dNTP), ie, dATP, dTTP or dUTP, dGTP, dCTP. The dideoxynucleotide is a substance in which the hydroxyl group at the 3 'position of deoxyribose of the deoxynucleotide is a hydrogen group. When ddNTP is incorporated instead of dNTP, the complementary strand is not synthesized any more and plays a role as a specific terminator. Fulfill.

  When the base sequence at a specific position in the target nucleic acid to be analyzed is a single base sequence, only four types of ddNTPs are used as shown in FIG. 2, and at least one of these is an identifiable label. DdNTP to which is added is used. The extension reaction proceeds from the primer for base sequence analysis, ddNTP complementary to the base at a specific position (ddGTP in FIG. 2) is incorporated, and the base extension reaction stops at one base. The base sequence of the specific position in the target nucleic acid can be analyzed by identifying the base type of the ddNTP incorporated at this time by confirming the presence or absence of the label. If a plurality of mutually distinguishable labels are added to ddNTP during the base extension reaction, one to four simultaneous base sequence analyzes can be performed by measuring the presence of each incorporated label. Yes (in Fig. 2 all four ddNTPs are labeled).

  The base sequence at a specific position in the target nucleic acid to be analyzed is a repetitive base sequence composed of 1 to 3 bases selected from the group consisting of adenine, guanine, cytosine and thymine or uracil, such as a repetitive base sequence such as a microsatellite. In some cases, as shown in FIG. 3, all deoxynucleotides (dNTP) complementary to the bases included in the repetitive base sequence and the bases included in the repetitive base sequence are added with labels that can be distinguished from each other. All dideoxynucleotides (ddNTPs) that are not complementary to are used. A base extension reaction proceeds from the primer for base sequence analysis, and complementary labeled dNTPs (dCTP and dATP in FIG. 3) are incorporated at a specific position. Eventually, when a base that does not exist in the repetitive base sequence following the repetitive base sequence appears, ddNTP (ddTTP or ddGTP in FIG. 3) is taken in and the base extension reaction stops. By measuring the signal intensity of dNTP to which a distinguishable label is added, the number of repetitions in a repetitive base sequence at a specific position can be calibrated.

  As the identifiable label to be added to ddNTP or dNTP used in the second step, a known label can be used.

Examples of the distinguishable label include labels such as enzymes, fluorescent substances, haptens, antigens, antibodies, radioactive substances, and luminophores. Examples of the enzyme include alkaline phosphatase and peroxidase. Examples of the fluorescent substance include cyanine fluorescent dyes such as Cy2, Cy3, and Cy5, and fluorescent substances such as FITC, 6-FAM, HEX, R6G, ROX, R110, TET, TAMRA, and Texas Red. Examples of haptens include biotin and digoxigenin. Examples of radioactive substances include ³² P and ³⁵ S. Examples of the luminophore include ruthenium. Among these, it is preferable to use a cyanine fluorescent dye or semiconductor fine particles in order to make the incorporation efficiency into the enzyme constant between the labels.

As cyanine-based fluorescent dyes, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7 and the like having different excitation and fluorescence wavelengths are commercially available (GE Healthcare Bioscience Co., Ltd.). Cy2 is represented by X = O, n = 1 in the general formula 1, Cy3 is represented by X = C (CH ₃ ) ₂ , n = 1 in the general formula 1, and Cy5 is represented by X = C (CH ₃ ) ₂ , n = 2, Cy7 is represented by General Formula 1, X = C (CH ₃ ) ₂ , n = 3, and Cy3.5 is represented by General Formula 2, where n = 1 Cy5.5 is represented by n = 2 in general formula 2, and Cy3B has a chemical structure represented by general formula 3. Here, in the general formulas 1 to 3, R and R ′ are functional groups that are appropriately selected and used when binding to nucleotides. Cyanine fluorescent dyes can be preferably used because they can suppress variations in enzyme uptake efficiency. In the base sequence analysis method of the present invention, it is particularly preferable to use Cy2, Cy3, and Cy5 in consideration of excitation, fluorescence wavelength, and fluorescence signal decay.

  Semiconductor fine particles are semiconductors composed of III-V group atoms or II-IV group atoms, and are also called quantum dots. It is known that optical properties change depending on the quantum size, and is used as a multicolor fluorescent label. For example, ZnSe, CdS, CdSe, CdTe, InAs, InP, PbS, Si and the like can be mentioned, but are not limited thereto. CdSe is preferably used because it can generate an arbitrary visible light wavelength from blue to red by simply changing the crystal diameter. Further, ZnSe, CdS, CdSe, CdTe, InAs, InP, PbS, Si and the like are preferably coated with ZnS and CdS because fluorescence quenching can be suppressed.

  The label may be bound to any position of ddNTP or dNTP as long as it does not affect the base extension reaction of the primer. For example, in the case of adenine or guanine, the purine skeleton is linked to the 7th or 8th position, cytosine is linked to the 5th or 6th position of the pyrimidine skeleton, thymine is linked to the 6th position of the pyrimidine skeleton, or the 5th or 6th position of the uracil pyrimidine skeleton. Can do. The labeling method is not particularly limited, and the labeling substance can be bound by a general method as described in, for example, “CyDye catalog” (page 22) of GE Healthcare Biosciences. . In addition, commercially available Cy3 labeled ddNTP, Cy5 labeled ddNTP (Perkin Elmer Japan Co., Ltd.), Cy3 labeled dNTP, Cy5 labeled dNTP (Perkin Elmer Japan Co., Ltd.) and other known or known labeled substances are combined. It is also possible to use ddNTP or dNTP.

  A polymerase is used for the base extension reaction, but preferably a heat-resistant DNA polymerase is used. By using a heat-resistant DNA polymerase, the incorporation efficiency can be improved by repeating the steps of denaturation, annealing, and base extension a plurality of times in a temperature cycle. More preferably, Thermo Sequenase DNA Polymerase (GE Healthcare Bioscience Co., Ltd.) or Sequenase Version 2.0 T7 DNA Polymerase (GE Healthcare Bioscience Co., Ltd.) is used because of its high dideoxynucleotide incorporation efficiency.

  The third step is a step of specifically capturing the labeled product obtained in the second step via the tag sequence of the base sequence analysis primer contained in the labeled product.

  The labeled product can be captured by using a tag capture probe having a base sequence that can selectively bind to the tag sequence in the primer for base sequence analysis. As shown in FIG. 4, a base sequence analysis primer is specifically captured via a tag sequence using a tag capture probe having a complementary base sequence that specifically binds to the tag sequence. This tag capture probe can be immobilized on a support and used as a biochip. That is, by using a biochip on which a tag capture probe having a base sequence capable of selective binding to a tag sequence in a primer for base sequence analysis is used, a labeled product can be easily captured.

  In the present invention, a biochip is a general term for tools for analyzing genetic information of organisms, and includes, but is not limited to, microwells, microarrays, DNA chips, and the like.

  The biochip on which the tag capture probe of the present invention is immobilized is not particularly limited in structure, but has the effect of reducing noise and increasing hybridization efficiency. A biochip having a support and using a support having a fine concavo-convex structure in an immobilization region as shown in FIG. 10 and a tag capturing probe immobilized on the upper surface of the convex part of the concavo-convex structure is preferable.

  The material of the support is not particularly limited. For example, inorganic materials such as gold, glass, ceramics, and silicon, polymethyl methacrylate (PMMA), polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polydimethylsiloxane, silicon rubber, Examples thereof include polymers such as gels, beads, fibers, and hollow fibers. Preferably, it is a synthetic polymer that can be easily molded, and PMMA is suitably used in combination with the fine uneven structure shown in FIG.

  As the molding of the support, a method such as an injection molding method or a hot embossing method can be used in the case of a polymer, and a sand blasting method or the like can be used in the case of glass or ceramic. In the case of silicon, a known method used in a semiconductor process can be used.

  When the support is made of a polymer, the tag capture probe is immobilized on the upper surface of the convex portion of the support by chemically treating the upper surface of the convex portion and capable of binding to the tag capture probe, such as an amino group or a hydroxy group. , A carboxyl group, an aldehyde group, an epoxy group, and the like can be generated and chemically bonded to the probe DNA. For example, when the support is made of PMMA, a carboxy group is generated on the surface of the support by surface treatment with an alkaline solution and / or an acidic solution as illustrated in FIG. 7, and as illustrated in FIG. Immobilization can be achieved by condensing a tag capture probe aminated at the 3 ′ end with a chemical reaction. In addition, such a functional group is introduced by, for example, introducing a polar group by subjecting the surface to plasma treatment or radiation treatment (for example, γ-ray, electron beam, etc.) or further graft polymerization treatment after these treatments. Or by coating with a polycation (for example, poly-L-lysine, a silane coupling agent, etc.).

  In the biochip on which the tag capture probe of the present invention is immobilized, a bead 6 can be included in the recess between the support and the cover (FIG. 10). The presence of the beads 6 enables efficient stirring of the liquid, resulting in a reaction promoting effect. The size of the beads can be appropriately selected according to the shape of the concave portion as long as a plurality of beads can move freely in the concave portion of the support, and has a diameter of several tens to several hundreds μm, for example. The material of the beads is not particularly limited, and examples thereof include glass, ceramics (for example, yttria stabilized zirconia), metals such as stainless steel, and polymers such as nylon and polystyrene. Among these, ceramic beads are preferable because they are chemically stable and have a large specific gravity.

  The biochip on which the tag capture probe of the present invention is immobilized is usually prepared by immobilizing one type of tag capture probe for each immobilization region (spot). For example, as shown in FIG. 5, by immobilizing a plurality of types of tag capture probes on a single support, it is possible to simultaneously analyze a single base sequence and a repeated base sequence.

  The label capture product is specifically captured on the biochip by hybridizing the tag capture probe and the base sequence analysis primer labeled by the base extension reaction on the biochip of the present invention and washing the surplus. can do. In this case, the hybridization is generally performed under stringent conditions. Such conditions include, but are not limited to, for example, hybridization at 30-50 ° C., 3-4 × SSC, 0.1-0.5% SDS for 1-24 hours, followed by 2 × SSC and 0. Washing with a solution containing 1% SDS can be included. Here, 1 × SSC is a solution (pH 7.2) containing 150 mM sodium chloride and 15 mM sodium citrate.

  The fourth step is a step of analyzing the base sequence at a specific position using the presence or signal intensity of the label as an index by measuring the presence or signal intensity of the label contained in the labeled product captured in the third step. is there.

  When the base sequence at a specific position in the target nucleic acid to be analyzed is a single base, the base type at the specific position in the target nucleic acid is identified by identifying the base type of ddNTP by confirming the presence or absence of the label. Sequences can be analyzed. That is, if the presence of a ddNTP label complementary to a base at a specific position, that is, a complementary ddNTP label signal is measured, it can be determined that the base is present at the specific position.

  When the base sequence at a specific position in the target nucleic acid to be analyzed is a repetitive base sequence such as a microsatellite, the repetitive base at the specific position is measured by measuring the signal intensity of dNTPs to which a mutually distinguishable label is added. The number of repeats in the sequence can be calibrated. For example, as shown in FIG. 6, using a calibration curve with the signal intensity on the vertical axis and the number of repetitions of the target nucleic acid on the horizontal axis, the number of repetitions is calculated from the signal intensity of the dNTP added with the label obtained by measurement. Can be calibrated.

  The target nucleic acid to which the base sequence analysis method of the present invention can be applied is not particularly limited as long as it does not bind to a non-natural nucleic acid, and can be applied to any nucleic acid having any natural or artificial base sequence. is there. The nucleic acid referred to here includes all DNA or RNA including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA, and synthetic RNA. Nucleic acids that can serve as markers for diseases and treatments, such as drug metabolism-related genes, genes causing genetic diseases, cancer-related genes, various other disease-related genes or virus-derived nucleic acids, are particularly preferred target nucleic acids.

  Samples of the target nucleic acid include body fluids such as blood, urine, saliva, etc., but any sample other than body fluids can be used. If the sample is solid, it may be dissolved in the liquid by an appropriate method such as enzyme treatment, addition of a surfactant or an organic solvent.

  In addition to the above, the sample of the target nucleic acid to which the method of the present invention is applied can be arbitrarily changed. For example, by preparing a large amount of human genomic DNA necessary for this method by establishing a large number of cells and culturing them in large quantities or by obtaining a large amount of peripheral blood, a direct genomic DNA sample can be obtained. Alternatively, a sample obtained by obtaining a small amount of genomic DNA and amplifying genomic DNA non-specifically by a WGA method (Whole Genome Amplification) such as a reagent kit GenomiPhi of GE Healthcare Biosciences may be used. . A sample obtained by amplifying a specific base sequence using a primer such as a PCR method, a multiplex PCR method, or an asymmetric PCR method may be used. In particular, when genomic DNA or the like is used, a region including a base sequence at a specific position to be analyzed may be amplified in advance by PCR or the like. For example, in the case of a double-stranded sample obtained by the enzymatic amplification method, it is heated to 95 ° C. and then rapidly cooled to 4 ° C. to become a single strand, or up to 95 ° C. in a solution having a very low salt concentration. The sample may be heated and fragmented, or fragmented with ultrasonic waves, or cleaved with a restriction enzyme and subjected to single stranding and / or fragmentation operations.

  The present invention will be described more specifically with reference to the following examples. However, the present invention is not limited to these examples.

  Oligonucleotides containing L-sugar nucleotides used in the following Examples and Comparative Examples were synthesized using an Applied Biosystems 392 DNA / RNA automatic synthesizer.

  As the nucleotide, only a nucleotide of a D sugar (dATP, dGTP, dCTP, dTTP), a nucleotide of an L sugar, or a combination of a nucleotide of a D sugar and a nucleotide of an L sugar was used. Nucleotide of L-type sugar is synthesized by a known method or commercially available, such as Beta-L-deoxy Adenosine (n-bz) CED phosphoramidite, Beta-L-deoxy Cytidine (n-bz) CED phosphoramidite, Beta -L-deoxy Guanosine (n-ibu) CED phosphoramidite and Beta-L-deoxy Thymidine CED phosphoramidite (manufactured by ChemGenes Corporation) were used.

  When an amino linker is added to the 3 ′ end of the synthesized oligonucleotide, a glass bead support for 3 ′ amination modification (3′-PT Amino-Modifer C6 CPG) manufactured by GLEN RESEARCH is used with a DNA synthesizer. It was.

(Example 1)
In the single nucleotide polymorphism of the human IL-6 gene, the base sequence (G / C) present at the -174th position and the base sequence (C) present at the -209th position were simultaneously analyzed.

  The analysis was performed according to the following procedures (1) to (8).

Procedure (1) Preparation of Oligonucleotide As an IL-6 gene amplification primer, an oligonucleotide having the base sequence shown in SEQ ID NO: 1 and an oligonucleotide having the base sequence shown in SEQ ID NO: 2 were synthesized.

  An oligonucleotide having the base sequence shown in SEQ ID NO: 3 as a primer for base sequence analysis at -174th from the transcription start point of the IL-6 gene is shown in SEQ ID NO: 4 as a tag capture probe for the oligonucleotide of SEQ ID NO: 3. An oligonucleotide having a base sequence was synthesized.

  An oligonucleotide having the base sequence shown in SEQ ID NO: 5 is also used as a primer for base sequence analysis for analyzing the base at position −209 from the transcription start point of the IL-6 gene as the base at the specific position of the target nucleic acid. An oligonucleotide having the base sequence shown in SEQ ID NO: 6 was synthesized as a tag capture probe for the oligonucleotide No. 5.

  In the oligonucleotides of SEQ ID NO: 3 and SEQ ID NO: 5, 27 bases from the 5 'end are L-type nucleotides, 28 bases to 3' end are D-type nucleotides, and 22 bases from the 5 'end are tag sequences. In addition, 23 to 27 bases from the 5 'end are arranged with 5 bases of a thymidine residue as a spacer. The D-type polynucleotide region has a base sequence adjacent to the 3 'side of the base sequence at a specific position. That is, the D-type polynucleotide region of the oligonucleotide of SEQ ID NO: 3 is a base sequence complementary to the nucleotide sequence of −173 to −147 from the transcription start point of the IL-6 gene. The D-type polynucleotide region is a base sequence complementary to the base sequence of −208 to −182 from the transcription start point of the IL-6 gene.

  The oligonucleotides of SEQ ID NO: 4 and SEQ ID NO: 6 are all tag capture probes consisting of L-type nucleotides and having base sequences complementary to the tag sequences of the oligonucleotides of SEQ ID NO: 3 and 5, respectively, and an amino linker at the 3 ′ end Have

  The oligonucleotide tag sequences of SEQ ID NO: 3 and SEQ ID NO: 5 had the following characteristic base sequences in order to clarify the selectivity between D-type and L-type nucleotides.

  That is, the tag sequence of the oligonucleotide of SEQ ID NO: 3 has the same base sequence as part of the D-type polynucleotide region of the oligonucleotide of SEQ ID NO: 5, and the tag sequence of the oligonucleotide of SEQ ID NO: 5 is SEQ ID NO: 3 Have the same base sequence as part of the D-type polynucleotide region of the oligonucleotide.

Procedure (2) Production of Support for Immobilizing Tag Capture Probe A support for immobilizing the tag capture probe prepared in Procedure (1) was produced as follows.

  Using a known method, LIGA (Lithographie Galvanforming Abforming) process, a mold for injection molding is prepared, and a support for fixing a tag capturing probe made of PMMA having a shape as described later is obtained by an injection molding method. It was. The average molecular weight of PMMA used in this example is 50,000, carbon black (Mitsubishi Chemical # 3050B) is contained in PMMA at a ratio of 1% by weight, and the support is black. . When the spectral reflectance and the spectral transmittance of this black support were measured, the spectral reflectance was 5% or less at any wavelength in the visible light region (wavelength of 400 nm to 800 nm), and in the same range of wavelengths. The transmittance was 0.5% or less. Neither spectral reflectance nor spectral transmittance had a specific spectral pattern (such as a peak) in the visible light region, and the spectrum was uniformly flat. The spectral reflectance is the value when specularly reflected light from the support is captured using an apparatus (Minolta Camera, CM-2002) equipped with an illumination / light-receiving optical system that conforms to condition C of JIS Z 8722. Spectral reflectance was measured.

  The shape of the support (reference numeral 1 in FIGS. 9 and 10) was 76 mm in length, 26 mm in width, and 1 mm in thickness, and the surface was flat except for the central portion of the support. In the center of the support, a concave portion having a length of 22 mm and a depth of 0.15 mm is provided. In this recess, a convex portion having a diameter of 0.15 mm and a height of 0.15 mm is provided 1296 (36 × 36 ) Locations. When the height difference between the height of the upper surface of the convex portion of the concave and convex portion (the average value of the height of 1296 convex portions) and the flat portion was measured, it was 3 μm or less. In addition, variations in the heights of the top surfaces of 1296 convex portions (the difference between the height of the top surface of the highest convex portion and the height of the top surface of the lowest convex portion), and the average value of the heights of the top surfaces of the convex portions and the flat portions The difference in height of the upper surface was measured and found to be 3 μm or less. Furthermore, the pitch (the distance from the central part of the convex part to the central part of the adjacent convex part) of the convex part of the uneven part was 0.5 mm.

Procedure (3) Immobilization of Tag Capture Probe The PMMA support obtained in Procedure (2) was immersed in a 10N aqueous sodium hydroxide solution at 70 ° C. for 12 hours. This was washed with pure water, 0.1N HCl aqueous solution, and pure water in this order to generate carboxyl groups on the surface of the support. This reaction scheme is shown in FIG.

Tag capture probes (22 bases, 3 ′ terminal amination) having the base sequences shown in SEQ ID NO: 4 and SEQ ID NO: 6 were each dissolved in pure water at a concentration of 0.3 nmol / μl to obtain a stock solution. When spotting on a support, PBS (NaCl 8g, Na ₂ HPO ₄ · 12H ₂ O 2.9g, KCl 0.2g, KH ₂ PO ₄ 0.2g in pure water was made up to 1 liter. The final concentration of probe DNA is adjusted to 0.03 nmol / μl by adding hydrochloric acid for adjusting pH to pH 5.5), and the carboxylic acid on the support surface is condensed with the amino group at the end of the probe DNA. Therefore, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to make the final concentration 50 mg / ml. Then, these mixed solutions were spotted on the entire upper surface of the support convex portion with an arrayer (manufactured by Nippon Laser Electronics; Gene Stamp-II). Next, the support was sealed in a plastic container, incubated at 37 ° C. and humidity of 100% for about 20 hours, and washed with pure water. This reaction scheme is shown in FIG.

Procedure (4) Production and attachment of cover, loading of beads Cover with four through-holes (reference numeral 4 in FIGS. 9 and 10) by injection molding method (reference numeral 2 in FIGS. 9 and 10; overhang on the outer periphery) With structure). PDMS was applied to the support on which the tag capturing probe (reference numeral 5 in FIGS. 9 and 10) obtained in the procedure (3) was immobilized (reference numeral 3 in FIGS. 9 and 10), and the cover 2 produced thereon was applied. And cured at 42 ° C. for 2 hours to produce a biochip on which the tag capture probe was immobilized (FIG. 9). 20 mg of 180 μm diameter zirconia beads (symbol 6 in FIG. 10; manufactured by Tosoh Corporation; TZB180) was loaded into the biochip from the through-hole 4 of the cover (FIG. 10).

Procedure (5) Amplification by PCR of region containing single nucleotide polymorphism to be specified Nucleic acid extraction kit using oligonucleotide having base sequence shown in SEQ ID NO: 1 and oligonucleotide having base sequence shown in SEQ ID NO: 2 A part of IL-6 gene was amplified by PCR from a human genomic DNA sample extracted from cultured cells (derived from HEK293 human fetal kidney) with “QIAmp” (Qiagen). These DNA samples were confirmed in advance by a conventional base sequence determination method to be G for the -174th base sequence and C for the -209th base from the transcription start point of the IL-6 gene. For PCR, KOD-Plus-DNA Polymerase (Toyobo) was used, and 35 cycles of amplification reaction were performed under the conditions specified by the manufacturer. However, since the purpose of this PCR is simply to amplify a specific region of a gene, there are no particular restrictions on the PCR method or the type of enzyme. Thereafter, the residue was purified by GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Bioscience Co., Ltd.) to remove excess dNTPs.

Here, for PCR, 30 μM forward primer (SEQ ID NO: 1) 1 μl, 30 μM reverse primer (SEQ ID NO: 2) 1 μl, 10 × PCR buffer for KOD-Plus-10 μl, 2 mM dNTPs 10 μl, 25 mM MgSO _{4 4} μl, KOD− Plus-DNA Polymerase 2 units and 450 ng of the extracted DNA solution were purely made up to 100 μl. PCR was performed at 94 ° C. for 2 minutes, 94 ° C. for 30 seconds / 55 ° C. for 30 seconds / 68 ° C. for 30 seconds, and stored at 4 ° C. after the reaction.

Procedure (6) Specific ddNTP incorporation reaction The PCR product obtained in Procedure (5) was added to the oligonucleotides of SEQ ID NOS: 3 and 5, ddNTP labeled with Cy3 or Cy5 (Cy3-ddNTP or Cy5-ddNTP), Unlabeled ddNTP, DNA Polymerase, and the like were added to incorporate ddNTP. Since the base species to be detected is G or C this time, Cy3-ddGTP, Cy5-ddCTP, ddTTP (ddUTP is also acceptable), and ddATP were used. As DNA Polymerase, modified T7 DNA Polymerase Thermo Sequenase DNA Polymerase (GE Healthcare Bioscience) widely used for the Sanger method was used.

  Here, for the ddNTP incorporation reaction, 0.3 μMIL-6-174 nucleotide sequence analysis primer (SEQ ID NO: 3) and IL-6-209 nucleotide sequence analysis primer (SEQ ID NO: 5) each 2 μl, Reaction Buffer 2 μl, PCR Product (after column purification) 800 ng, 10 μMCy3-ddGTP 1 μl, 10 μMCy5-ddCTP 1 μl, 10 μMddTTP 1 μl, 10 μMddATP 1 μl, Thermo Sequenase DNA Polymerase 1.0 units was used in a pure 20 μl solution. The ddNTPs uptake reaction was carried out at 95 ° C. for 2 minutes, 95 ° C. for 15 seconds / 60 ° C. for 30 seconds, and stored at 4 ° C. after the reaction.

Procedure (7) Hybridization The final concentration is 1 wt% BSA (bovine serum albumin), 5 × SSC, 0.1 wt% SDS (sodium dodecyl sulfate), 0.01 wt% salmon sperm DNA solution. A stock solution for hybridization was prepared.

  9.3 μl of the uptake reaction solution in the procedure (6) was heat denatured at 95 ° C. for 5 minutes and rapidly cooled in ice. 30.7 μl of the above hybridization stock solution was added thereto, and 40 μl of the hybridization solution was injected from the through hole using a micropipette. Then, the through-hole was closed with Kapton tape (As One 5-5018-01) and set in a chamber (TaKaRa, Takara Hybridization Chamber 5 No. TX711). The chamber was fixed to a shaker (manufactured by EYELA, Multi Shaker MMS), and incubated at 42 ° C. for 16 hours. The rotation speed of the shaker was 230 rpm so that the rotation surface of the shaker and the chamber were in equilibrium.

  After the incubation, the support and the PDMS were detached from the support, and the support was washed and dried.

Procedure (8) Measurement of fluorescence intensity A support for the DNA chip scanner (Axon Instruments' GenePix 4000B) after the treatment of the procedure (7) is set, the laser output is set to 33%, and the voltage setting of the photomultiplier is set to 500 In this state, the fluorescence intensity was measured. Here, the fluorescence intensity is an average value of the fluorescence intensity in the spot, and the background noise is the fluorescence intensity of the convex portion where the probe DNA is not immobilized.

  The measurement result of fluorescence intensity is shown in FIG. The vertical axis represents a value (signal / noise) obtained by dividing the fluorescence intensity of the target spot measured by the scanner by the background noise. Signal / noise of Cy3 and Cy5 of the spot to which the tag capture probe to be analyzed was immobilized was calculated, and for each tag capture probe, the value of Cy3 was indicated by a white bar and the value of Cy5 was indicated by a black bar.

  From FIG. 11, the fluorescence of Cy3 was specifically detected from the tag capture probe of SEQ ID NO: 4, and the fluorescence of Cy5 was specifically detected from the tag capture probe of SEQ ID NO: 6. As a result, it was found that the former incorporated G and the latter incorporated C, and it was confirmed that the base analysis was performed with high accuracy.

(Comparative Example 1)
The procedure of Example 1 except that the oligonucleotides of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20 were used in place of the oligonucleotides of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. The same procedure as in (1) to (8) was performed.

  The results are shown in FIG. The way of viewing the figure is the same as that of FIG. 11 of the first embodiment.

  Cy3 and Cy5 were both detected from the tag capture probe of SEQ ID NO: 4, and both Cy3 and Cy5 were detected from the tag capture probe of SEQ ID NO: 6, making it impossible to perform base sequence analysis.

  Since the tag capture probe of SEQ ID NO: 4 has a complementary base sequence not only to the tag sequence portion of SEQ ID NO: 3 but also to the region other than the tag sequence of SEQ ID NO: 5, it is considered that cross hybridization occurred with both.

  Conversely, since the tag capture probe of SEQ ID NO: 6 is complementary not only to the tag sequence of SEQ ID NO: 5 but also to the region other than the tag sequence of SEQ ID NO: 3 as a base sequence, Conceivable.

  The base sequence analysis primer of Example 1 and Comparative Example 1 and the base sequence of the tag capture probe are exactly the same, but in Example 1, the tag sequence and the tag capture probe are made of L-type nucleotides. In the case of producing a tag sequence and a tag capture probe with a D-type nucleotide as in Comparative Example 1 while confirming that the base sequence analysis can be performed with high accuracy without causing cross-hybridization as in 1, It was shown that it was necessary to concentrate on cross-hybrid.

(Example 2)
AnTn analysis of the human IL-6 gene, the base sequence near the −396th position from the transcription start point, ie, the number of A in the AnTn sequence was analyzed. Among the procedures (1) to (8) of Example 1, the oligonucleotide used in the procedures (1), (3), (5) and (6) is changed, and the labeled nucleotide used in the procedure (6) is changed. The procedure was the same as in Example 1 except that.

Procedure (1) Preparation of Oligonucleotide As an IL-6 gene amplification primer, an oligonucleotide having the sequence shown in SEQ ID NO: 7 and an oligonucleotide having the sequence shown in SEQ ID NO: 8 were synthesized.

  An oligonucleotide having the sequence shown in SEQ ID NO: 9 as a primer for base sequence analysis at the -396th position from the transcription start point of the IL-6 gene, and a sequence shown in SEQ ID NO: 10 as a tag capture probe of the oligonucleotide of SEQ ID NO: 9 Were synthesized.

  The oligonucleotide of SEQ ID NO: 9 has 27 nucleotides from the 5 'end and L nucleotides from 28 to 3' end, and 22 nucleotides from the 5 'end is the tag sequence. In addition, 23 to 27 bases from the 5 'end are arranged with 5 bases of a thymidine residue as a spacer.

  The D-type polynucleotide region has a base sequence adjacent to the 3 'side of the base sequence at a specific position. That is, the D-type polynucleotide region of the oligonucleotide of SEQ ID NO: 9 is a nucleotide sequence complementary to the nucleotide sequence of −395 to −371 from the transcription start point of the IL-6 gene.

  Further, the tag sequence of the oligonucleotide of SEQ ID NO: 9 was made to have the same base sequence as a part of the D-type polynucleotide region of the oligonucleotide of SEQ ID NO: 9.

  The oligonucleotide of SEQ ID NO: 10 is a tag capture probe consisting of all L-type nucleotides and having a base sequence complementary to the tag sequence of the oligonucleotide of SEQ ID NO: 9 and having an amino linker at the 3 'end.

  The tag capture probe of SEQ ID NO: 10 was immobilized on a support in the same manner as in Example 1.

  Procedure (2) Production of support for immobilizing tag capture probe, Procedure (3) Immobilization of tag capture probe, Procedure (4) Production and attachment of cover and loading of beads were carried out in the same manner as in Example 1. .

Procedure (5) Amplification by PCR of the region containing the AnTn sequence to be identified As a primer, the oligonucleotide having the base sequence shown in SEQ ID NO: 7 and the sequence instead of the oligonucleotide of SEQ ID NO: 1 and the oligonucleotide of SEQ ID NO: 2 The same procedure as in Example 1 was performed except that an oligonucleotide having the base sequence shown in No. 8 was used as a primer.

  In these DNA samples, the base sequence of IL-6AnTn has been confirmed in advance by a normal base sequence determination method, and the base sequence of A8T11, that is, the -396th base from the transcription start point of the IL-6 gene is A. .

Procedure (6) Specific ddNTP incorporation reaction The PCR product obtained in procedure (5) was added to the oligonucleotide of SEQ ID NO: 9, ddNTP labeled with Cy3 or Cy5 (Cy3-ddNTP or Cy5-ddNTP), unlabeled ddNTP , DNA Polymerase and the like were added, and ddNTP was taken up. Since the AnTn sequence was determined this time, Cy3-ddATP, Cy5-ddUTP, ddCTP, ddGTP were used. As DNA Polymerase, modified T7 DNA Polymerase “Thermo Sequenase DNA Polymerase” (GE Healthcare Bioscience) widely used in the Sanger method was used.

  Here, for ddNTP incorporation reaction, 0.3 μM base sequence analysis primer (SEQ ID NO: 9) 2 μl, Reaction Buffer 2 μl, PCR product (after column purification) 800 ng, 10 μMCy3-ddATP 1 μl, 10 μMCy5-ddUTP 1 μl, 10 μMddCTP 1 μl, 1 μl of 10 μM ddGTP and Thermo Sequenase DNA Polymerase 1.0 units were used which were purely made up to 20 μl. The ddNTPs uptake reaction was performed in the same manner as in Example 1.

  Procedure (7) Hybridization and (8) Measurement of fluorescence intensity were carried out in the same manner as in Example 1.

  The results are shown in FIG. The way of viewing the figure is the same as that of FIG. 11 of the first embodiment.

  From the tag capture probe of SEQ ID NO: 10, Cy5 light was specifically detected, which revealed that T was incorporated, and it was confirmed that the base analysis was performed with high accuracy.

(Example 3)
A GT repeat base sequence analysis of human Heme oxygenase-1 (HO-1), and a GT repeat base sequence existing in the vicinity of −270 from the transcription start point were analyzed. Among the procedures (1) to (8) of Example 1, the oligonucleotide used in the procedures (1), (3), (5) and (6) is changed, and the labeled nucleotide used in the procedure (6) is changed. The procedure was the same as in Example 1 except that.

Procedure (1) Preparation of oligonucleotide As an HO-1 gene amplification primer, an oligonucleotide having the base sequence shown in SEQ ID NO: 11 and an oligonucleotide having the base sequence shown in SEQ ID NO: 12 were synthesized.

  An oligonucleotide having the base sequence represented by SEQ ID NO: 13 as a primer for base sequence analysis at the -270th position from the transcription start point of the HO-1 gene is represented by SEQ ID NO: 14 as a tag capture probe for the oligonucleotide of SEQ ID NO: 13. An oligonucleotide having a base sequence was synthesized.

  Furthermore, an oligonucleotide having the base sequence represented by SEQ ID NO: 15 as a primer for base sequence analysis at the −270th position from the transcription start point of the HO-1 gene is added to SEQ ID NO: 16 as a tag capture probe for the oligonucleotide of SEQ ID NO: 15. An oligonucleotide having the indicated base sequence was synthesized.

  In the oligonucleotides of SEQ ID NO: 13 and SEQ ID NO: 15, 27 bases from the 5 'end are L-type nucleotides, 28 bases to 3' end are D-type nucleotides, and 22 bases from the 5 'end are tag sequences. In addition, 23 to 27 bases from the 5 'end are arranged with 5 bases of a thymidine residue as a spacer.

  The D-type polynucleotide region has a base sequence adjacent to the 3 'side of the base sequence at a specific position. That is, the D-type polynucleotide region of the oligonucleotides of SEQ ID NO: 13 and SEQ ID NO: 15 is a base sequence complementary to 26 bases downstream of the GT base sequence of the HO-1 gene.

  The oligonucleotides of SEQ ID NO: 14 and SEQ ID NO: 16 are all tag capture probes consisting of L-type nucleotides and having base sequences complementary to the tag sequences of the oligonucleotides of SEQ ID NO: 13 and SEQ ID NO: 15, respectively, at the 3 ′ end. Has an amino linker.

  The tag sequence added to the oligonucleotide of SEQ ID NO: 13 has the same base sequence as part of the D-type polynucleotide region of the oligonucleotide of SEQ ID NO: 13, and the tag sequence added to the oligonucleotide of SEQ ID NO: 15 is SEQ ID NO: 9 Since the nucleotide sequences of the oligonucleotides of SEQ ID NO: 16 and SEQ ID NO: 10 are completely identical, the oligonucleotide of SEQ ID NO: 16 is not synthesized here. .

  The tag capture probes of SEQ ID NO: 14 and SEQ ID NO: 16 were immobilized on a support in the same manner as in Example 1.

  Procedure (2) Production of support for immobilizing tag capture probe, Procedure (3) Immobilization of tag capture probe, Procedure (4) Production and attachment of cover, and loading of beads were carried out in the same manner as in Example 1.

Procedure (5) Amplification by PCR of a region containing a GT base sequence to be specified As a primer, an oligonucleotide having the base sequence shown in SEQ ID NO: 11 instead of the oligonucleotide of SEQ ID NO: 1 and the oligonucleotide of SEQ ID NO: 2 Example 1 except that the oligonucleotide having the base sequence shown in SEQ ID NO: 12 was used as a primer and that HL-60 (peripheral blood premyelocytes) was also used in addition to HEK293 cells as a human genome sample. The same was done.

Procedure (6) Specific dNTP, ddNTP uptake reaction The PCR product prepared in procedure (5) was added to the oligonucleotide of SEQ ID NO: 13 or SEQ ID NO: 15, Cy3 or Cy5 labeled dNTP (Cy3-dNTP or Cy5-dNTP), Unlabeled ddNTP, DNA Polymerase, and the like were added to incorporate dNTP and ddNTP. Since the GT base sequence was analyzed this time, Cy3-dATP, Cy5-dCTP, ddGTP, and ddTTP were used. As DNA Polymerase, modified T7 DNA Polymerase “Thermo Sequenase DNA Polymerase” (GE Healthcare Bioscience) widely used in the Sanger method was used.

  Here, for dNTP and ddNTP incorporation reaction, in the case of HEK293 cells, 0.3 μM HO-1 GT base sequence analysis primer (SEQ ID NO: 13) 2 μl, Reaction Buffer 2 μl, PCR reaction solution (after HEK293 column purification) 800 ng, 10 μMCy3-dATP 1 μl, 10 μMCy3-dCTP 1 μl, 10 μMddGTP 1 μl, 10 μMddTTP 1 μl, and Thermo Sequenase DNA Polymerase 1.0 units were used in a pure up to 20 μl. In the case of HL-60, 0.3 μMHO-1 GT base sequence analysis primer (SEQ ID NO: 15) 2 μl, Reaction Buffer 2 μl, PCR reaction solution (HL-60, after column purification) 800 ng, 10 μMCy3-dATP 1 μl, 10 μMCy3-dCTP 1 μl, 10 μM ddGTP 1 μl, 10 μM ddTTP 1 μl, and Thermo Sequenase DNA Polymerase 1.0 units were purely diluted to 20 μl. The dNTP and ddNTPs uptake reaction was performed in the same manner as in Example 1.

  Procedure (7) Hybridization was carried out in the same manner as in Example 1 except that 4.65 μl of each of the two types of uptake reaction solutions performed in Procedure (6) was used.

  Procedure (8) The fluorescence intensity was measured in the same manner as in Example 1.

  The results are shown in FIG. The way of viewing the figure is the same as that of FIG. 11 of the first embodiment.

  The tag capture probe Cy3 and Cy5 signal / noise of SEQ ID NO: 14 and the signal / noise of tag capture probe SEQ ID NO: 16 were calculated, and prepared using a sample having a known number of repetitions of FIG. It was found from the calibration curve that the number of repetitions of the repeated nucleotide sequence was calibrated, and the number of repeated nucleotide sequences in the case of HEK293 cells was 28 times, and the number of repeated nucleotide sequences in the case of HL-60 cells was 20 times. .

It is a schematic diagram which illustrates the hybridization of the primer for base sequence analysis to the target nucleic acid of the primer for base sequence analysis. It is a schematic diagram which illustrates base extension reaction which adds a labeled nucleotide complementary to the single base sequence of a specific position to the primer for base sequence analysis. It is a schematic diagram which illustrates base extension reaction which adds the labeled nucleotide complementary to the repetition base sequence of a specific position to the primer for base sequence analysis. It is a schematic diagram which illustrates the process of capturing specifically the labeled product containing the primer for a base sequence analysis using the tag capture probe fixed to the support body. It is a schematic diagram which illustrates the process of specifically capture | acquiring the label | marker product containing the primer for base sequence analysis using the some tag capture probe fixed to the support body. It is a graph which shows the relationship between the repetition frequency of a repetitive base sequence, and signal intensity. This is a chemical reaction scheme in which a carboxy group is generated on the surface of the support by the surface treatment of the PMMA support. It is a condensation reaction scheme between a carboxy group on the surface of a PMMA support and an aminated probe DNA. It is the perspective view and sectional drawing which illustrate the outline of the biochip of this invention by which the tag capture probe containing a support body and a cover was fix | immobilized. It is sectional drawing of an example of the biochip by which the tag capture probe of this invention was fix | immobilized. 4 is a graph showing the measurement results of fluorescence intensity in Example 1. 6 is a graph showing the measurement results of fluorescence intensity of Comparative Example 1. 6 is a graph showing the results of measurement of fluorescence intensity in Example 2. 6 is a graph showing the measurement results of fluorescence intensity of Example 3.

Explanation of symbols

1 Support 2 Cover 3 Adhesive Layer (PDMS)
4 Through-hole 5 Tag capture probe immobilized on support 6 Bead

Claims (9)

A method for analyzing a base sequence at a specific position in a target nucleic acid,
Using a base sequence analysis primer having a base sequence complementary to the base sequence adjacent to the 3 ′ side of the base sequence at the specific position and a tag sequence that is a base sequence that does not bind to a natural nucleic acid,
A first step of hybridizing the target nucleic acid and the base sequence analysis primer to form a complex of the base sequence analysis primer and the target nucleic acid;
A second step of obtaining a labeled product by performing a base extension reaction in which a labeled nucleotide complementary to the base sequence at the specific position is added to the base sequence analysis primer in the complex obtained in the first step;
A third step of specifically capturing the labeled product obtained in the second step via the tag sequence;
A fourth step of measuring the presence or signal intensity of the label contained in the labeled product and analyzing the base sequence at a specific position using the presence or signal intensity of the label as an index;
A method for analyzing a base sequence.   The base sequence analysis method according to claim 1, wherein the tag sequence in the base sequence analysis primer is a base sequence having a non-natural nucleotide.   The method for analyzing a base sequence according to claim 2, wherein the non-natural nucleotide has a non-natural sugar.   The method for analyzing a base sequence according to claim 3, wherein the unnatural sugar is either L-type ribose or L-type deoxyribose.   The base sequence at a specific position in the target nucleic acid is a single base sequence, and the second step is a label complementary to the single base sequence as a labeled nucleotide complementary to the base sequence at the specific position The base sequence analysis method according to any one of claims 1 to 4, which is a step of performing a base extension reaction using the dideoxynucleotide thus obtained to obtain a labeled product.   The base sequence at a specific position in the target nucleic acid is a repetitive base sequence consisting of 1 to 3 types of bases selected from the group consisting of adenine, guanine, cytosine and thymine or uracil, wherein the second step is specified A base extension reaction is performed using a labeled deoxynucleotide complementary to the repetitive base sequence and an unlabeled dideoxynucleotide that is not complementary to the repetitive base sequence as a labeled nucleotide complementary to the base sequence at the position. The method for analyzing a base sequence according to any one of claims 1 to 4, wherein the deoxynucleotide has a label different from each other.   The method for analyzing a base sequence according to any one of claims 1 to 5, wherein the labeling substance of the labeled nucleotide complementary to the base sequence at the specific position used in the second step is a cyanine fluorescent dye or a semiconductor fine particle.   The base sequence complementary to the base sequence adjacent to the 3 ′ side of the base sequence at a specific position in the target nucleic acid and the natural nucleic acid used in the base sequence analysis method according to claim 1 A primer for base sequence analysis having a tag sequence that does not bind. A biochip on which a tag capture probe having a base sequence capable of selective binding to the tag sequence in the primer for base sequence analysis according to claim 8 is immobilized.

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