JP2004069489A - Probe carrier and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To immobilize a sequence for forming a hybrid with the target nucleic acid of a nucleic acid probe with a specific distance from the surface of a carrier via 5' end, and to provide a probe carrier which excludes influence on the sequence for forming the hybrid due to a reaction, when manufacturing the probe carrier and to provide a manufacturing method of the probe carrier. <P>SOLUTION: The probe is formed as a one-stranded nucleic acid having 5' end having an amino group for combining with the surface of the carrier, a part made of the continuous sequence of a thymine base, and a part for forming a hybrid made of a base sequence for forming a hybrid with a target nucleic acid, in this order from 5' side to 3' side. An epoxy group, provided on the carrier surface and an amino group at 5' end are commonly combined and immobilized on the carrier. In that case, the part for forming the hybrid is protected by the single-stranded nucleic acid for protection, thus excluding the influence to the part for forming the hybrid due to immobilization reaction. <P>COPYRIGHT: (C)2004,JPO

Description

【００３２】
３．ＦＩＴＣが結合した配列１の相補鎖（配列２）の合成
以下の配列の標識オリゴヌクレオチドを常法により合成した。
配列２（配列番号：２）：
５’−Ｆ−ＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ−３’
（Ｆ＝ＦＩＴＣ）
４．配列１の相補鎖（配列番号：３）の合成
以下の配列の標識オリゴヌクレオチドを常法により合成した。
配列３（配列番号：３）：
５’−ＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ−３’
５．ハイブリッド形成によるプローブ配列の保護
配列１からなるアミノリンクオリゴヌクレオチド２００μＭ、配列２からなるＦＩＴＣ標識相補鎖２０μＭを含む５０ｍＭＮａＣｌ／５０ｍＭりん酸ナトリウム緩衝液（ｐＨ８．０）溶液を用意し、６５℃にて１０分加温する。その後３５℃まで徐冷する。次に配列３からなるＦＩＴＣの結合していない相補鎖を１８０μＭになるように加え、ＮａＣｌ、及びりん酸ナトリウム緩衝液をそれぞれ最終濃度５０ｍＭになるように調製し、そのまま室温以下になるまで冷却しアニールする（一晩）。この反応により、二本鎖オリゴヌクレオチドが形成される。このとき、その後の反応のモニターのために、蛍光標識されたオリゴヌクレオチドを混入させている。
６．プローブの粒子への結合反応
上記１項で合成したエポキシ基導入粒子溶液１μｌ（約１０^８個）に上記５項で作製したＤＮＡ溶液を４０μＭになるように加え、回転攪拌装置を用いて室温で４時間反応させた。それに５倍量の１０ｍＭりん酸緩衝液を加え、遠心により粒子を回収した。上清の蛍光を日立蛍光分光光度計で測定し（励起波長４９８ｎｍ、蛍光５２２ｎｍ），反応前に比べ、蛍光強度が十分減少していることを確認した。再度りん酸緩衝液で、次に蒸留水にて洗浄を行った後、１Ｍエタノールアミンを加え室温にて一晩反応させることにより未反応のエポキシ基のブロッキングを行った。
【００３３】
反応後の粒子を１００μｌのシース液に懸濁し、そのうちの２０μｌを１ｍｌのシース液で希釈して、フローサイトメーターにより評価した。８５％以上の粒子に蛍光が導入されており、二本鎖オリゴヌクレオチドが粒子表面に結合したことが確認できた。
７．プローブ保護用相補鎖の除去（熱変性による方法）
ハイブリダイゼーション反応を行うためには、直接粒子に結合しているオリゴヌクレオチド以外の相補鎖の部分をはずす作業が必要である。
【００３４】
反応させた粒子を１００μｌの純水に懸濁し、８０℃で２分間加熱した。その後遠心操作を行って粒子を回収し、相補鎖が外れ一本鎖のプローブのみが結合したプローブ結合担体を得た。
８．プローブ保護用相補鎖の除去（アルカリ変性による方法）
熱変性以外の方法として、上記反応溶液２０μｌに１Ｍエタノールアミン５０μｌ加え、室温で５時間攪拌した。遠心により粒子を回収し、純水で数回遠心洗浄して、相補鎖を外した。フローサイトメーターで測定した結果、熱変性の場合も、アルカリ変性の場合も蛍光は完全に消失し、相補鎖が外れていることが確認された。
９．ハイブリダイゼーション反応
配列２のＦＩＴＣ標識オリゴヌクレオチド０．２ナノモルを０．５Ｍ　ＮａＣｌを含む０．５Ｍ　リン酸バッファーに溶解し、その中に７或いは８で作製したプローブ結合粒子を懸濁して３７℃一晩反応させた。反応後の懸濁液を遠心処理し、粒子を回収してその蛍光量をフローサイトメーターにより測定した。結果を以下の表１に示す。
【００３５】
【表１】

【００３６】
表１に示すとおり、Ｔの長さによりハイブリダイゼーションの効率が異なり、Ｔの長さが３の時に最大のハイブリッド効率を示した。
【００３７】
【発明の効果】
ビーズ等、粒子表面にオリゴヌクレオチドを固定する方法を提案した。通常のＤＮＡ合成の際に導入されるリンカー長に加えて、Ｔの三量体程度の長さのリンカーを連結し、リンカー長を長くすることが、ハイブリダイゼーション反応の効率をあげる上で重要であることを示した。
【００３８】
【配列表】

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a nucleic acid probe-bound fine particle carrier and a method for producing the same. More specifically, the present invention relates to a method effective for protecting a reactive functional group contained in a probe sequence when immobilizing a probe in a gene detection method based on a hybridization method or the like. The present invention also relates to a method for producing a carrier for binding a nucleic acid probe, which does not require protection of a functional group of a linker portion and is easy to operate. In addition, the present invention relates to a carrier for binding a nucleic acid probe to which a base sequence for hybrid formation with less nonspecific adsorption is bound.
[0002]
[Prior art]
Hybridization of nucleic acids is used as a means for extracting, separating or detecting a nucleic acid having a specific base sequence of interest in a test sample. In the reaction in this hybridization method, an oligonucleotide or a polynucleotide (ie, a probe) that is labeled or immobilized on a solid support surface forms a base pair with a target nucleic acid.
[0003]
In this hybridization method, a single-stranded DNA or RNA nucleic acid forms a hybrid under appropriate conditions with another single-stranded nucleic acid containing a complementary base sequence through hydrogen bonding between bases. Is used.
[0004]
Conventionally, in these hybridization reactions, a probe was bound using a nitrocellulose or nylon membrane filter as a support, and a hybridization reaction was generally performed. Is bonded to the surface of insoluble solid fine particles such as latex, and after the hybridization reaction, a method of detecting by fluorescence of a labeling agent or the like bound to the probe has been used.
[0005]
In Japanese Patent Application Laid-Open No. 63-27000, which discloses a method for binding a single-stranded nucleic acid to a solid surface, the fixation of the single-stranded nucleic acid to the particle surface cannot be controlled in advance, and There is a problem that can not be selected. Therefore, there was a problem that the hybridization efficiency was extremely low and was not suitable for practical use.
[0006]
In order to solve the above-mentioned drawbacks of the nucleic acid immobilization method, various nucleic acid immobilization supports and methods for producing the same have been proposed. For example, a method of indirectly immobilizing a nucleic acid by interposing a carbon chain having 4 to 20 carbon atoms between a sequence portion of nucleotides constituting the nucleic acid and an immobilization support is known.
[0007]
In addition, when immobilizing on a support, these amino groups are protected to prevent the reactive primary amino group of the nucleotide portion important as a probe from being changed by a chemical reaction accompanying the immobilization. It is necessary to do.
[0008]
To solve these problems, JP-A-6-335380 discloses that a nucleotide portion having a primary amino group having high reactivity is protected by forming a double strand with a complementary strand. . Then, a continuous sequence of deoxyadenylic acid or deoxycytidylic acid having a primary amino group is arranged in the bond between the hybridizable base sequence and the immobilized support, and the primary amino group of the base is A method of binding to an immobilized carrier using reactivity is disclosed. That is, in this method, the carboxyl group on the solid surface and the amino group of deoxyadenylic acid or deoxycytidylic acid are fixed by an amide bond.
[0009]
However, in this method, since it is not possible to specify which portion of continuous deoxyadenylic acid or deoxycytidylic acid is bound to the immobilization support, the distance between the hybridizable base sequence and the immobilization support is not constant. However, there is a problem that a difference may occur in the reaction efficiency in the hybridization reaction. Further, there is a problem that the carboxyl group on the solid surface may react with a primary amino group in the target DNA during the hybridization reaction or induce non-specific adsorption of the target DNA.
[0010]
[Problems to be solved by the invention]
In order to quantitatively evaluate the hybridization reaction in this way, it is necessary to control the distance between the immobilized support and the hybridization base sequence to be constant. Furthermore, when performing these reactions, it is necessary to protect functional groups for binding, such as highly reactive primary amino groups, in the hybridizable base sequence.
[0011]
[Means for Solving the Problems]
According to the present invention, there is provided a method for obtaining a simple probe-bound immobilized carrier by using an oligonucleotide (oligo T) comprising a thymidine base as a linker between the immobilized carrier and the hybridizable base sequence. Can be. According to a preferred embodiment of the present invention, non-specific adsorption is avoided by covering the surface of the solid support with polyglycidyl methacrylate, and the epoxy group in the polyglycidyl methacrylate further reacts with the amino group at the terminal of the probe nucleic acid. By doing so, a stable probe-bound and immobilized carrier can be obtained.
[0012]
That is, the probe carrier according to the present invention is a probe carrier in which a nucleic acid probe is immobilized on the surface of a carrier, wherein the nucleic acid probe has a 5 ′ end bonded to the surface of the carrier and a continuous sequence of thymine bases. And a hybrid-forming portion comprising a base sequence capable of forming a hybrid with a target nucleic acid in this order from the carrier side.
[0013]
Further, the method for producing a probe carrier according to the present invention includes a carrier, and a nucleic acid probe bound to the surface of the carrier, wherein the nucleic acid probe has a 5 ′ end bound to the surface of the carrier, and thymine. A method for producing a probe carrier which is a single-stranded nucleic acid having a portion consisting of a continuous sequence of bases and a portion for hybrid formation consisting of a base sequence capable of forming a hybrid with a target nucleic acid in this order from the carrier side,
(1) A single-stranded nucleic acid probe in which a 5 'end having a functional group for binding, a continuous sequence of thymine bases, and a hybrid-forming portion comprising a base sequence capable of forming a hybrid with the nucleic acid probe are arranged in this order. Preparing a;
(2) The single-stranded nucleic acid probe is hybridized with a single-stranded nucleic acid for protection having a sequence having a portion complementary to the sequence for hybridization of the single-stranded nucleic acid probe to have a double-stranded portion. Forming a nucleic acid and protecting the binding functional group on the hybridization moiety;
(3) a step of preparing a carrier having on its surface a functional group that binds to the 5 ′ end;
(4) reacting a functional group on the single-stranded nucleic acid probe side of the nucleic acid having the double-stranded portion with a 5′-terminal functional group and a functional group on the surface of the carrier to bond them;
(5) eliminating the reactivity of unreacted functional groups on the surface of the carrier to which the nucleic acid having the double-stranded portion is bound;
(6) a step of obtaining a probe carrier by removing a single-stranded nucleic acid for protection from the nucleic acid having a double-stranded portion bound to the carrier to obtain a probe carrier.
[0014]
According to the present invention, the sequence for hybridization of a nucleic acid probe with a target nucleic acid is immobilized at a predetermined distance from the carrier surface via the 5 'end, and the sequence for hybridization by reaction during the production of a probe carrier is immobilized. It is possible to provide a probe carrier and a method for producing the probe carrier, the influence of which is eliminated.
[0015]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present specification, a probe immobilized on a carrier is one that can specifically bind to a specific target substance. In the present invention, the probe is composed of a nucleic acid, that is, an oligonucleotide or a polynucleotide. The term "probe" refers to both a molecule having a probe function, such as an individual polynucleotide molecule, and a population of molecules having the same probe function, such as polynucleotides of the same sequence surface-immobilized at dispersed locations, often a ligand. Also included are molecules called. Also, probes and targets are often used interchangeably, where the probe is capable of binding to or becoming capable of binding to the target as part of a ligand-antiligand (sometimes referred to as a receptor) pair. is there. Probes and targets in the present invention can include bases as found in nature, or analogs thereof.
[0016]
The probe used in the present invention is appropriately selected according to the purpose of use, but in order to suitably carry out the method of the present invention, the probe may be DNA, RNA, cDNA (complementary DNA). , PNA, oligonucleotides, polynucleotides, and other nucleic acids.
[0017]
In the present invention, a probe carrier in which a plurality of types of these probes are fixed on the carrier surface as independent regions, for example, as dot spots, is called a probe carrier, and those arranged at predetermined intervals are called a probe array. In addition, there is a microarray in which spots are arranged at high density.
[0018]
The probe according to the present invention comprises a single-stranded nucleic acid, a 5′-terminal having a functional group used for binding to a carrier, a 5′-terminal, a linker portion composed of a thymine continuous sequence, A hybrid-forming portion comprising a base sequence capable of forming a hybrid with a nucleic acid.
[0019]
Each of these portions can be connected via a plurality of, for example, several bases within a range where the effects of the present invention can be obtained, but a configuration in which these portions are directly bonded is more preferable. . The length of the entire probe is preferably in the range of 10 to 70 bases.
[0020]
The portion consisting of a continuous thymine sequence has a function as a linker for keeping the distance between the surface of the carrier and the portion for forming a hybrid constant. The number of thymine bases is preferably 3 to 10, and more preferably 3.
[0021]
The binding functional group at the 5 'end has a function of binding the nucleic acid probe to the carrier accurately from the 5' end by binding to the binding functional group provided on the carrier surface. As such a combination of functional groups for binding, various combinations of functional groups for immobilization that form a bond for immobilization, preferably a covalent bond can be used. For example, an amino group (primary group) ) Is particularly preferred in combination with an epoxy group on the carrier side. Such a structure having an amino group at the 5 ′ end can be formed using a nucleotide having an amino group.
[0022]
For the introduction of the epoxy group to the surface of the carrier, a method of forming the entire carrier or a predetermined portion of the carrier with polyglycidyl methacrylate is preferable.
[0023]
Further, for example, a probe carrier for analyzing a plurality of target nucleic acids by arranging immobilized regions of a plurality of nucleic acid probes having different sequences of hybridization portions separately from each other on the surface of a plate-shaped carrier, for example. Can be obtained, and a plurality of immobilized regions can be arranged in a predetermined sequence to prepare a probe array. Furthermore, a microarray can be manufactured by arranging the immobilization regions at a high density.
[0024]
One preferred embodiment of the probe carrier according to the present invention comprises the following steps:
(1) A single-stranded nucleic acid probe is prepared in which a 5 ′ end having an amino group, a continuous sequence of thymine bases, and a portion for forming a hybrid comprising a base sequence capable of forming a hybrid with the nucleic acid probe are arranged in this order. Process and
(2) The single-stranded nucleic acid probe is hybridized with a single-stranded nucleic acid for protection having a sequence having a portion complementary to the sequence for hybridization of the single-stranded nucleic acid probe to have a double-stranded portion. Forming a nucleic acid and protecting the amino group in the hybridization moiety;
(3) providing a carrier having an epoxy group bonded to the 5 ′ end on the surface;
(4) reacting an amino group at the 5 ′ end of the nucleic acid having a double-stranded portion on the single-stranded nucleic acid probe side with an epoxy group on the surface of the carrier to covalently bond them;
(5) eliminating the reactivity of unreacted epoxy groups on the surface of the carrier to which the nucleic acid having the double-stranded portion is bound;
(6) a step of removing a single-stranded nucleic acid for protection from a nucleic acid having a double-stranded portion bound to the carrier to obtain a probe carrier;
[0025]
A known method can be used for a specific operation in performing each step. For example, heating or treatment by a change in the ionic strength of the reaction medium can be used to convert the double-stranded portion into a single strand.
[0026]
The single-stranded nucleic acid for protection may be any as long as it forms a double-stranded structure with a portion of the nucleic acid probe for hybridization with the target nucleic acid and protects it. By protecting the hybridizing portion of the nucleic acid probe, the amino group (primary) in the hybridizing portion is prevented from reacting with the epoxy group on the carrier side, and the amino group and the epoxy group are strongly bonded. Further, the probe nucleic acid can be immobilized on the carrier while maintaining a necessary and favorable state in the reaction of the portion for forming the probe nucleic acid with the target nucleic acid while utilizing the selective binding.
[0027]
As the carrier, those having various shapes such as a solid flat plate and a solid particle can be used. When a particulate material is used, it is preferable that the average particle size is 0.8 μm.
[0028]
When a solid particulate carrier is used, the probe carrier can be used for easy handling and use peculiar to the particulate form. When probe molecules are densely arranged on a solid particulate carrier, there is a problem that a sample such as a sample gene, which is tested for the presence of a sequence complementary to the probe, may not be able to enter between probe molecules. In the present invention, the occurrence of such a problem is prevented by arranging the probe-side hybridization portion at a good distance from the carrier surface by a linker composed of a continuous sequence of thymine bases, thereby achieving more efficient hybridization. The effect of making it possible can be obtained. Further, in the present invention, a double-strand with a nucleic acid probe is formed in advance using a single-stranded nucleic acid for protection (the double-strand formation has a function of blocking a functional group such as an amino group) to form a double strand with the specimen. By controlling the arrangement and density of the nucleic acid probes on the carrier so that effective probe binding is performed, it is possible to obtain an effect of eliminating or reducing the possibility of inhibiting the sample reaction due to tight probe binding.
[0029]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples.
1. Preparation of Epoxy Group-Introduced Immobilized Carrier Emulgit 49 (Daiichi Kogyo Seiyaku Co., Ltd.) (2 g, 20% by weight) was added to 10 g of ultra-low carboxylic acid-modified polystyrene particles Immutex G-1701 (0.789 μm in particle system). Next, glycidyl methacrylate was added in an amount 9 times the weight of the styrene resin (weight ratio), and a 2% aqueous solution of Potassium Persulfate was added as a polymerization initiator. After heating at 72 ° C. for 1 hour, 0.1 g of glycidyl methacrylate was added again every 30 minutes six times. Thereafter, it was heated at 75 ° C. for 2 hours, cooled, and washed. After washing with a 200 mesh, centrifugal washing using pure was performed twice. The obtained styrene resin was confirmed to have a diameter of 1.096 μm by flow cytometer analysis.
2. Synthesis of Amino Group Linked DNA n thymidine oligonucleotides were linked to the 5 ′ end of the 18-mer oligonucleotide, and 5′-Amino-Modifier C6 (Glen Research) was linked to the 5 ′ end of the oligonucleotide. The probe having the above configuration was synthesized by a phosphoramidite method using an ABI 381A DNA automatic synthesizer. The 5 'dimethoxytrityl group was removed on an automatic synthesizer (sequence 1).
[0030]
Sequence 1 (SEQ ID NO: 1):
5'-X- (T) n-ACTGGCCGTCGTTTTTACA-3 '
(N = 0, 3, 7, or 20)
[0031]
Embedded image

[0032]
3. Synthesis of Complementary Strand of Sequence 1 (Sequence 2) to which FITC was Bound A labeled oligonucleotide having the following sequence was synthesized by a conventional method.
Sequence 2 (SEQ ID NO: 2):
5'-F-TGTAAAACGACGGCCAGT-3 '
(F = FITC)
4. Synthesis of complementary strand of sequence 1 (SEQ ID NO: 3) A labeled oligonucleotide having the following sequence was synthesized by a conventional method.
Sequence 3 (SEQ ID NO: 3):
5'-TGTAAAACGACGGCCAGT-3 '
5. Protection of Probe Sequence by Hybridization A 50 mM NaCl / 50 mM sodium phosphate buffer (pH 8.0) solution containing 200 μM of amino link oligonucleotide consisting of sequence 1 and 20 μM of FITC-labeled complementary strand consisting of sequence 2 was prepared, and was heated at 65 ° C. Heat for 10 minutes. Thereafter, it is gradually cooled to 35 ° C. Next, a complementary strand of sequence 3 to which no FITC is bound is added to a concentration of 180 μM, and NaCl and a sodium phosphate buffer are each adjusted to a final concentration of 50 mM, and cooled to room temperature or lower. Anneal (overnight). This reaction forms a double-stranded oligonucleotide. At this time, a fluorescently labeled oligonucleotide is mixed in to monitor the subsequent reaction.
6. Probe Binding Reaction to Particles The DNA solution prepared in the above item 5 was added to 1 μl (about 10 ⁸ ) of the epoxy group-introduced particle solution synthesized in the above item 1 to a concentration of 40 μM, and the mixture was stirred at room temperature using a rotary stirring device. The reaction was performed for 4 hours. A 5-fold volume of 10 mM phosphate buffer was added thereto, and the particles were collected by centrifugation. The fluorescence of the supernatant was measured with a Hitachi fluorescence spectrophotometer (excitation wavelength 498 nm, fluorescence 522 nm), and it was confirmed that the fluorescence intensity was sufficiently reduced as compared with before the reaction. After washing again with a phosphate buffer and then with distilled water, unreacted epoxy groups were blocked by adding 1 M ethanolamine and reacting at room temperature overnight.
[0033]
The particles after the reaction were suspended in 100 μl of the sheath liquid, 20 μl of which was diluted with 1 ml of the sheath liquid, and evaluated by a flow cytometer. Fluorescence was introduced into 85% or more of the particles, confirming that the double-stranded oligonucleotide had bound to the particle surface.
7. Removal of complementary strand for probe protection (method by thermal denaturation)
In order to carry out the hybridization reaction, it is necessary to remove the complementary strand other than the oligonucleotide directly bound to the particles.
[0034]
The reacted particles were suspended in 100 μl of pure water and heated at 80 ° C. for 2 minutes. Thereafter, the particles were recovered by centrifugation to obtain a probe-bound carrier to which the complementary strand was removed and only a single-stranded probe was bound.
8. Removal of complementary strand for protecting probe (method by alkali denaturation)
As a method other than heat denaturation, 50 μl of 1 M ethanolamine was added to 20 μl of the above reaction solution, and the mixture was stirred at room temperature for 5 hours. The particles were collected by centrifugation and washed several times with pure water to remove the complementary strand. As a result of measurement with a flow cytometer, it was confirmed that the fluorescence completely disappeared in both the case of heat denaturation and the case of alkali denaturation, and that the complementary strand was removed.
9. 0.2 nmol of the FITC-labeled oligonucleotide of the hybridization reaction sequence 2 is dissolved in 0.5 M phosphate buffer containing 0.5 M NaCl, and the probe-bound particles prepared in 7 or 8 are suspended therein, and The reaction was performed overnight. The suspension after the reaction was centrifuged to collect particles, and the amount of fluorescence was measured by a flow cytometer. The results are shown in Table 1 below.
[0035]
[Table 1]

[0036]
As shown in Table 1, the hybridization efficiency was different depending on the length of T, and the maximum hybrid efficiency was exhibited when the length of T was 3.
[0037]
【The invention's effect】
A method for immobilizing oligonucleotides on the surface of particles such as beads was proposed. In order to increase the efficiency of the hybridization reaction, it is important to increase the linker length by linking a linker having a length of about T trimer in addition to the linker length introduced during normal DNA synthesis. Showed that there is.
[0038]
[Sequence list]

Claims (18)

A probe carrier having a nucleic acid probe immobilized on the surface of the carrier,
The nucleic acid probe has a 5 ′ end bonded to the surface of the carrier, a portion consisting of a continuous sequence of thymine bases, and a portion for forming a hybrid consisting of a base sequence capable of forming a hybrid with a target nucleic acid, A probe carrier, which is a single-stranded nucleic acid having this order from the side. The probe carrier according to claim 1, wherein the nucleic acid probe and the carrier surface are bonded by a covalent bond formed by a reaction between an amino group at the 5 'end of the linker and an epoxy group on the surface of the carrier. 3. The probe carrier according to claim 2, wherein the epoxy group has polyglycidyl methacrylate present on the surface of the carrier. The probe carrier according to any one of claims 1 to 3, wherein the length of the continuous sequence of the thymine base is 3 to 10 bases. The probe carrier according to claim 4, wherein the length of the continuous sequence of thymine bases is 3 bases. The probe carrier according to any one of claims 1 to 5, wherein the carrier is insoluble solid fine particles. The probe carrier according to claim 6, wherein the insoluble solid fine particles have an average diameter of 0.8 µm. The probe carrier according to any one of claims 1 to 5, wherein the carrier has a flat surface for immobilizing a nucleic acid probe. A carrier and a nucleic acid probe bound to the surface of the carrier, wherein the nucleic acid probe is hybridized with a target nucleic acid, a 5 ′ end bound to the surface of the carrier, a portion consisting of a continuous sequence of thymine bases, and a target nucleic acid. And a hybrid-forming portion consisting of a base sequence capable of forming a probe carrier, which is a single-stranded nucleic acid having in this order from the carrier side,
(1) A single-stranded nucleic acid probe in which a 5 'end having a functional group for binding, a continuous sequence of thymine bases, and a hybrid-forming portion comprising a base sequence capable of forming a hybrid with the nucleic acid probe are arranged in this order. Preparing a;
(2) The single-stranded nucleic acid probe is hybridized with a single-stranded nucleic acid for protection having a sequence having a portion complementary to the sequence for hybridization of the single-stranded nucleic acid probe to have a double-stranded portion. Forming a nucleic acid and protecting the binding functional group on the hybridization moiety;
(3) a step of preparing a carrier having on its surface a functional group that binds to the 5 ′ end;
(4) reacting a 5′-terminal functional group on the single-stranded nucleic acid probe side of the nucleic acid having the double-stranded portion with a functional group on the surface of the carrier to bond them; A step of eliminating the reactivity of unreacted functional groups on the surface of the carrier to which the nucleic acid having a double-stranded portion is bound,
(6) a step of obtaining a probe carrier by removing a single-stranded nucleic acid for protection from the nucleic acid having a double-stranded portion bound to the carrier, to obtain a probe carrier. The method according to claim 9, wherein the functional group for binding on the nucleic acid probe side is an amino group, and the functional group on the carrier side is an epoxy group. The method according to claim 10, wherein the epoxy group is an epoxy group of polyglycidyl methacrylate. The method according to any one of claims 9 to 11, wherein the length of the continuous sequence of the thymine base is 3 to 10 bases. The method according to claim 12, wherein the length of the continuous sequence of thymine bases is 3 bases. The method according to any one of claims 9 to 13, wherein the carrier is insoluble solid fine particles. The method according to claim 16, wherein the insoluble solid fine particles have an average diameter of 0.8 µm. The method according to any one of claims 9 to 13, wherein the carrier has a flat surface for immobilizing a nucleic acid probe. The production method according to claim 10, wherein the treatment for eliminating the reactivity of the epoxy group is performed by hydrolysis of the epoxy group. 18. The production method according to claim 17, wherein the hydrolysis of the epoxy group is performed with 0.1 M ethanolamine.