JP2004191253A - Probe solid phase reaction array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a probe solid phase reaction array excellent in reaction efficiency and reproducibility and enabling visually easy confirmation. <P>SOLUTION: In the probe solid phase reaction array wherein probes are converted to a solid phase on a support, the support is internally equipped with the first - n-th reaction parts (wherein n is an integer of 2 or more) and the probes are grouped corresponding to the first - n-th reaction parts to be arranged. The fixing positions of the probes in the respective reaction parts are the same positions of the respective reaction parts so that the probes are positioned on one or more lines present at the same distance from the terminals of the respective reaction parts. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

プローブの長さを75merに設定し、計算を行った結果を表2に示す。
【００６４】
【表２】

【００６５】
計算結果から、45遺伝子において、Tm値が71℃±1℃とほぼ同一となったが、自己構造化の指標であるΔＧ値は、-2〜-30とばらつきがあった。プローブにより反応条件を揃えるための指標としてTmが挙げられるが、長鎖になるとTm値よりもより自己構造化がより強い影響を起こすものと思われる。
【００６６】
今回の計算結果から、自己構造化数値を指標として、自己構造化の高いプローブ、低いプローブ、またはその中間のプローブと、グループ分けを行うことで、これら特性の異なるプローブにおいても、至適反応条件にてハイブリダイゼーション反応を行うことが可能となる。
【００６７】
たとえば、図2に示すようなキャピラリ構造のアレイ基板を使用した場合、自己構造化の予測結果をもとにしてハイブリダイゼーション条件を変更することが可能である。図2ａにおいて、デバイス1には複数のキャピラリ2、3、4、5、6、7および8が形成されている。このキャピラリ内部に、自己構造化ΔＧ値の違いに基づいてグループ化されたDNAプローブが固定される。自己構造化ΔＧ値を段階的にグループ分けし、キャピラリ2には、ΔＧ値が最も高いＴＡＣＩ遺伝子プローブ（図では1種類）を固定化し、キャピラリ3には、ΔG値が-12〜-16であるプローブ（図では3種類）、キャピラリ4には、ΔG値が-16〜-18であるプローブ（図では12種類）という順に独立のキャピラリに搭載した。また、各キャピラリにおけるプローブの１つ以上がなるべく多く隣り合うようにプローブ群の一端を、基板の端縁から同一距離上にそろえるとともに、他のプローブ同士もキャピラリをまたいで基板の端縁から同一距離において隣り合うように配置した。今回は7グループに分けたが、これ以上でもこれ以下でも制限されることはなく、またΔＧ値のグループ化閾値もこれに限定されない。
【００６８】
ここではグループ分けされた複数のDNAプローブの1つをそれぞれDNAプローブ9、10、11、12、13、14および15とした。また、キャピラリアレイには、それぞれ試料注入口16、17、18、19、20、21および22、並びに試料排出口23、24、25、26、27、28および29が形成されている。キャピラリ4における断面図が図2Bに示されており、デバイス1の蓋基板30と溝形成基板31、また、デバイスの下には温度制御用のヒータ33が設置されている。このように形成したデバイス1はヒータ33で一定温度、たとえば45℃に保持されそこに試料注入口から試料が注入する。
【００６９】
ターゲット試料の調製は、通常組織または細胞より抽出したmRNAもしくはtotal RNAからcDNAを合成する際に、Cy3-dUTPまたはCy5-dUTPを用いてラベルする。オリゴdTプライマーまたはランダムプライマーおよび逆転写酵素を用いて第一鎖cDNAを合成する際にラベリングする。ハイブリダイゼーション反応は、一般的に45℃から65℃前後の恒温状態で一定の湿度を保ちターゲットDNAの蒸発を防ぎながら、一時間から一晩の間、ターゲットDNAとの反応が行われる。
【００７０】
このデバイス構造を利用することにより、ハイブリ反応時の条件設定や洗浄の条件設定を個別にまたは併用しても使い分けることが出来るので、再現性の良い、高精度の測定が可能である。
【００７１】
実施の様態2
（遺伝子機能に基づいてグループ化されたTNFファミリー遺伝子解析用プローブ固相化アレイ）
TNF/TNFRファミリー遺伝子は、上述の通り45種類以上が知られており、レセプター/リガンドの関係が解明されている遺伝子が多い。表3にTNF/TNFRファミリー遺伝子間におけるレセプターとそのリガンドとの関係の一例を示す。
【００７２】
【表３】

【００７３】
これらレセプター／リガンドとの関係等、遺伝子の機能に着目して、プローブのグループ化を行うことが可能である。グループ化を行った例を図3に示す。キャピラリ34、35、36、37、38、39および40には、レセプターおよびそのリガンドに対する5グループのプローブ群を配置した。7本のキャピラリは、いずれも同じ種類のプローブからなる同じグループとし、キャピラリ間で同種のグループ同士が隣り合うように、それぞれ基板の端縁から同一距離に並ぶ配置にした。このプローブ固相化アレイを用いた解析例を示す。
【００７４】
従来、ある細胞または個体において抗体刺激等によって引き起こされる遺伝子発現量の経時的変化を調べる場合、独立のプローブアレイ基板を使用し実験を行っていた。しかし、異なる独立のアレイを使用するとアレイ作製時、ハイブリダイゼーションおよび洗浄操作時、検出時点などのばらつきが大きく、本来の遺伝子変化量を調べることが難しいという状況があった。図4は、図3に示したプローブアレイを用いた解析の結果を示した模式図である。キャピラリ42、43、44、45、46、47、48には、細胞に抗体刺激を加えた後の遺伝子変化の経時的変化の検出の例である。キャピラリ42〜48は、ヒト単球U937細胞に対してTNFα抗体によって抗体刺激を加えた後、0時間、1時間、3時間、6時間、12時間、24時間、48時間経過後の細胞から、実施の態様1に述べた要領で標識化ターゲットを用意する。そのターゲットを同一基板上でハイブリダイゼーションさせることによって、図4に示したような遺伝子変化パターンを得ることが出来るであろう。TNFαによる抗体刺激によって、そのレセプターであるTNFR1の発現が6時間後から上昇し始め、経時的にさらに発現頻度上昇が見られ、それに呼応するように、遅れて24時間後にTNFα遺伝子の発現頻度が上昇するであろう。これまでは、画像データ取得後、解析ソフトを用いて数値化を行った後に、はじめて遺伝子発現頻度差の結果を得ることが出来たが、この遺伝子特性によるグループ化をおこなったプローブ固相化アレイをキャピラリーアレイシステム（たとえば特開2001-136963を参照）に使用することによって、いずれのレセプター／リガンド対の発現が変化しているのか、視覚的に経時変化を捉えることが出来る。また、一枚の基板上でハイブリダイゼーションおよび洗浄を行うことで、より再現性の高い結果を得ることが可能となる。上記の通り、プローブ群毎に一つの至適反応条件を適用して各プローブ群がいずれも最良の結果を得ることが可能となる。プローブ特性の指標としてTm値が挙げられるが、プローブとして使用するDNAが長鎖になった場合には、Tm値よりもプローブ自身が自己構造を取ることによりハイブリダイゼーションの阻害可能性が問題となる。このため本発明で示したように、プローブの自己構造化を計算により算出し、プローブ特性指標としてグループ化を行えば最良の結果を得ることが可能である。また、プローブ特性だけではなく、標的物質（たとえば標的核酸）の特性、特に標的物質の機能的特性によりグループ分けを行うことにより、これまで行うことができなかった視覚的に被検試料中の標的物質量の変化を捉えることが可能となる。
【００７５】
本発明の態様に従うと、ハイブリダイゼーションにおける従来における問題点が解決される。そればかりか従来では問題とされているこのような特徴を利用してしまうという逆転の発想により、反応効率に優れ、かつ再現性に優れたプローブ固相化反応アレイと、これを用いた反応方法が提供される。プローブ特性や、標的物質の機能的特性に着目して、プローブのグループ化または配置レイアウトの工夫を行うことで、より再現性に優れた、視覚的に見易いプローブ反応アレイ実験を実現することが可能となる。
【００７６】
【発明の効果】
本発明のプローブ固相化アレイを使用することにより、プローブ特性や、標的物質の機能的特性に着目して、プローブのグループ化または配置レイアウトの工夫を行うことで、より再現性に優れた、視覚的に見易いプローブ反応アレイ実験を実現することが可能となる。
【図面の簡単な説明】
【図１】計算フィルターの例を示すフローチャート図。
【図２】TNF/TNFRファミリー遺伝子を自己構造化指標によってグループ化したレイアウト図。
【図３】TNF/TNFRファミリー遺伝子の遺伝子機能によってグループ化したレイアウト図。
【図４】TNF/TNFRファミリー遺伝子の遺伝子機能によってグループ化したアレイの解析例を示す図。
【符号の説明】
1 プローブ固相化反応アレイ
２，３，４，５，６，７，８，３５，３６，３７，３８，３９，４０，４１，４２，４３，４４，４５，４６，４７，４８ キャピラリ
９，１０，１１，１２，１３，１４，１５ 核酸プローブ群
１６，１７，１８，１９，２０，２１，２２ 試料注入口
２３，２４，２５，２６，２７，２８，２９ 試料排出口
３０，３１ 基板
３３ ヒータ
３４a、３４ｂ 連結部[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a probe-immobilized reaction array. Specifically, the present invention relates to a probe-immobilized reaction array for use in analyzing nucleic acids, proteins, and the like.
[0002]
[Prior art]
As one of the methods for detecting the gene expression frequency, analysis using a probe array has been performed. Conventionally, in this probe array analysis, a nylon membrane has been used as a substrate for performing detection by a nucleic acid hybridization reaction, and the probe DNA has been immobilized on the substrate by the negative charge of the DNA (see Patent Document 1). Detection after the hybridization reaction has been performed by chemiluminescence or radioisotope.
[0003]
In recent years, a method in which various types of probe DNAs are solid-phased onto a slide glass (approximately 7.5 cm x 2.5 cm) of a certain standard by microarray technology (see Patent Document 2), and DNA probes on a semiconductor substrate by photolithography technology Are known. By using these microarray technologies, a conventional nylon film has one spot of 3 mm to 5 mm, but a glass substrate can have an extremely small spot of 100 μm to 300 μm, making it possible to increase the density of spots It became. Therefore, thousands to tens of thousands of probes can be arranged on one microarray, which is a useful means for large-scale gene expression frequency analysis. In addition, microarray technology is expected to improve detection sensitivity, save samples by micronization, automate data acquisition, and simplify data processing.
[0004]
The current mainstream of gene expression frequency analysis is a system that uses a DNA microarray as described above in the dual fluorescent labeling method to view differential gene expression. The principle is to detect a gene expression frequency difference between two samples. In other words, mRNA or tRNA obtained from two samples are labeled with different fluorescent substances, these two samples are subjected to competitive hybridization on a DNA microarray, and both fluorescence obtained on the DNA microarray are measured. This is a method for comparing the gene expression of two samples. Generally, a sample to be analyzed by the DNA microarray as described above is an RNA such as mRNA or total RNA extracted from a tissue or a cell. Therefore, the above-mentioned fluorescence is derived from a labeling substance such as Cy3-dUTP or Cy5-dUTP, which is labeled when cDNA is synthesized from RNA. In addition, labeling is often performed when cDNA is synthesized using an oligo dT primer or a random primer and a reverse transcriptase.
[0005]
However, the method for analyzing a nucleic acid using such a DNA microarray has the following problems. DNA probes immobilized on the same support generally have various types of sequences different in base sequence from each other, and the hybridization reaction conditions for each DNA probe are not constant. Therefore, in the conventional DNA microarray, it is difficult to efficiently use all the probes immobilized on the solid phase, and it cannot be said that all the desired targets are efficiently detected. Therefore, conventionally, the detection results obtained using the DNA microarray have low reproducibility.
[0006]
Further, in the conventional microarray experiment, since a plurality of probes are randomly spotted on the array, the result analysis cannot be performed without acquiring a fluorescence observation image and digitizing the image data. In microarray experiments, expression frequency analysis is generally performed based on the numerical data after image data has been digitized.However, in order to easily and quickly confirm the reliability of the array experiment, visual analysis of the image data is also required. It is desirable to have good visibility.
[0007]
[Patent Document 1]
JP 2000-135081 A
[0008]
[Patent Document 2]
JP 2001-17166 A
[0009]
[Non-patent document 1]
N. Uchikoga, A. Suyama, “Genome Infomatics”, 1998, Volume 9, p.388-389
[0010]
[Problems to be solved by the invention]
In view of the above situation, an object of the present invention is to provide a probe-immobilized reaction array excellent in reaction efficiency and reproducibility, and a probe-immobilized reaction array that can be easily confirmed visually.
[0011]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have conceived a means for grouping probes, and have completed the present invention.
[0012]
That is, the present invention is a probe-immobilized reaction array in which a probe is immobilized on a support,
The support includes a plurality of first to n-th reaction units (where n is an integer of 2 or more) arranged in parallel therein,
The probes are grouped for each of the first to n-th reaction units, and the immobilization position of the probe in each reaction unit is on one or more lines at the same distance from the end of each reaction unit. A probe-immobilized reaction array is provided, wherein the probe is positioned at the same position in each reaction part.
[0013]
The present invention also provides the probe-immobilized reaction array, wherein the grouping is classified according to probe characteristics.
[0014]
The present invention provides the probe-immobilized reaction array, which is the probe-immobilized reaction array, wherein the probe characteristics are structural characteristics of the probe.
[0015]
The present invention provides the probe-immobilized reaction array, which is the probe-immobilized reaction array, wherein the grouping is grouped according to characteristics of a target substance.
[0016]
The present invention provides the probe-immobilized reaction array, which is the probe-immobilized reaction array, wherein the characteristics of the target substance are functional properties of the target substance.
[0017]
The present invention provides the probe-immobilized reaction array, wherein the probe is a nucleic acid.
[0018]
The present invention provides the above-described probe-immobilized reaction array, wherein the probe is selected from the group consisting of a peptide, a protein, an antigen, and an antibody.
[0019]
The present invention provides the probe-immobilized reaction array, wherein the reaction section has a capillary shape.
[0020]
According to this structure, one optimal reaction condition can be applied to each reaction part, that is, to each probe group, the reaction can proceed in a plurality of reaction parts in parallel, and the analysis result can be visually checked. Can be easily compared. Further, depending on the mode of grouping, it is possible to obtain the best reaction result for each probe group. Therefore, not only the reaction efficiency of the probe-immobilized reaction array but also the reproducibility are significantly improved.
[0021]
BEST MODE FOR CARRYING OUT THE INVENTION
The probe-immobilized reaction array of the present invention is a probe-immobilized reaction array in which a probe is immobilized on a support, and the probe-immobilized reaction array includes first to n-th reaction units. A plurality of probes are provided in parallel, the probes are grouped and arranged for each of the first to n-th reaction units, and the immobilization position of the probe in each reaction unit is the same from the end of each reaction unit. It is characterized in that it is located at the same position in each reaction part so that the probe is located on one or more lines at a distance. Therefore, the probes included in each of the first to n-th reaction sections of the support are a probe group grouped for each reaction section.
[0022]
Grouping is performed based on the characteristics of the probe, the characteristics of the target substance, the reaction conditions, and the like according to the purpose. The probe characteristics include, for example, the Tm value of the probe, the self-structuring index value (free energy ΔG), and other characteristics of the probe if the characteristics are nucleic acid probe characteristics, and grouping may be performed based on these. Grouping can also be performed using the tuple method (grouping is efficiently performed based on Tm or expression frequency).
[0023]
For example, if the Tm value is used as an index, the Tm value of a probe to be used is measured, and probes having the same Tm value are put into the same group. If grouping is performed based on the self-structuring index value, probes having the same ΔG value are assigned to the same group using the energy ΔG value as an index, as shown in the following embodiments. Further, these may be combined and grouped.
[0024]
As described above, the Tm value can also be used as an index of the probe characteristics.However, when the nucleic acid used as the probe has a long chain, the probe takes a self-structure rather than the influence of the Tm value. It is possible that hybridization may be hindered. In such a case, in order to group by the structural characteristics of the probe, it is preferable to calculate the self-structuring of the probe by calculation and use it as a probe characteristic index, and use the Tm value as an index for the probe characteristic. Is undesirable.
[0025]
In addition, if the grouping is performed based on the characteristics of the target substance, functional characteristics (for example, a receptor / ligand relationship) and other characteristics may be used as indices. For example, if the genes are grouped according to their functional characteristics, as shown in the examples below, probes for each ligand / receptor gene are provided in each reaction site as a probe-immobilized array for TNF family gene analysis. It is conceivable to arrange them in pairs.
[0026]
Such grouping based on gene characteristics is not limited to grouping based on interaction such as receptor / ligand relationship, but also grouping based on other gene functions. For example, in the TNF / TNFR family gene group, apoptosis functions essential for ontogeny and homeostasis are known, and among them, TNFα, Fas and TRAIL are typical cytokines capable of inducing apoptosis. TNFα induces apoptosis via TNFR1 and Fas ligand via Fas, and TRAIL induces apoptosis via TRAIL receptor. These receptors have a characteristic nucleotide sequence domain called a death domain in an intracellular region. By utilizing this feature, it is also conceivable to perform grouping using differences due to gene structure and gene function such as the presence or absence of a certain domain (such as a common domain).
[0027]
Also, the probe immobilization position in each reaction section is the same in each reaction section so that the probe is located on one or more lines at the same distance from the end of each reaction section as shown in FIG. The probe may be immobilized on a solid phase. It is not necessary that the same number of probes be immobilized in all reaction units, and it is sufficient that at least some of the probes in each reaction unit are immobilized on the same line between the reaction units. Furthermore, the probe may not be immobilized on all the reaction units on the same line, and there may be a reaction unit that is not immobilized. For example, the case shown in FIG. 2 may be adopted, in which the leftmost probe is immobilized on the same line in all reaction sections, while the rightmost probe is immobilized only in the reaction section of the capillary 4. Have been. As shown in FIG. 3, the same probes may be grouped by immobilizing the same probe on the same line in each reaction part (in the figure, various ligand / receptor pairs are immobilized on each reaction part in the same arrangement). did). In particular, by performing such grouping, the expression in different test substances can be simultaneously visualized and rapidly analyzed. For example, as shown in FIG. 4, changes in expression over time can be easily compared.
[0028]
In addition, if the grouping is performed based on the reaction conditions, the probes can be grouped using the temperature at which the probes are hybridized, the salt concentration, the reaction time, or a combination thereof as an index.
[0029]
Further, the above-described grouping may be performed by combining the respective groups, such as combining the grouping based on the functional characteristics such as the receptor / ligand relationship and the grouping based on the probe characteristics.
[0030]
As used herein, the term `` nucleic acid '' refers to various naturally occurring DNAs and RNAs, as well as artificially synthesized peptide nucleic acids, morpholino nucleic acids, methylphosphonate nucleic acids and S-oligonucleic acids. Refers to nucleic acid analogs and the like.
[0031]
The term "target substance" as used herein refers to a substance to be detected by a probe. The target nucleic acid means a case where the target substance is a nucleic acid. Generally, a nucleic acid probe is designed to have a base sequence complementary to a target nucleic acid. When the test nucleic acid contained in the test sample has the base sequence of the target sequence, hybridization occurs between the nucleic acid probe and the target sequence. Therefore, by detecting this hybridization, it is possible to analyze the nucleic acid contained in the test sample. Hybridization may be detected by a means known per se. The base sequence that becomes the target of the target nucleic acid is referred to as “target sequence”.
[0032]
As used herein, the term “test sample” refers to a sample prepared from a biological sample such as cells, tissues, organs, blood, serum, lymph, tissues, hair and earwax collected from an individual as desired. And a sample that is to be subjected to a test containing a substance that is artificially synthesized or manufactured. Further, the “test sample” may be a sample obtained by subjecting a biological sample to any necessary pretreatment such as homogenation and extraction, if necessary. Such a pretreatment could be selected by one skilled in the art depending on the biological sample of interest.
[0033]
The term "individual" as used herein refers to any mammal, including humans, dogs, cats, cows, goats, pigs, sheep, and monkeys, as well as non-mammalian organisms such as plants and insects.
[0034]
Hereinafter, an embodiment of a probe-immobilized reaction array in which probes are grouped as described above will be described with reference to a nucleic acid probe as an example (FIG. 2).
[0035]
FIG. 2 (A) is a plan view of the probe-immobilized reaction array 1 according to the present embodiment as viewed from above. The probe-immobilized reaction array 1 is an example of a probe-immobilized reaction array manufactured using a transparent glass substrate 30 and a silicon substrate 31.
[0036]
Inside the substrate of the probe-immobilized reaction array 1, a plurality of capillaries 2, 3, 4, 5, 6, 7, and 8 are formed in parallel on the same plane. Since the capillaries are formed independently of each other, they do not mix with the fluid contained in other capillaries. Openings are formed at both ends of each capillary. Specifically, the capillaries 2, 3, 4, 5, 6, 7 and 8 have sample inlets 16, 17, 18, 19, 20, 21 and 22, respectively, and sample outlets 23, 24, 25, 26, 27, 28 and 29 are formed. Inside the capillaries 2, 3, 4, 5, 6, 7 and 8, fixed nucleic acid probe groups 9 to 15 are fixed.
[0037]
FIG. 2 (B) is a cross-sectional view of the probe-immobilized reaction array 1 according to the present embodiment cut along the capillary 4.
[0038]
The above-described probe-immobilized reaction array 1 can be manufactured, for example, as follows. A glass substrate and a silicon substrate having the same size are prepared, and are referred to as a substrate 30 and a substrate 31. A groove is formed in the substrate 31 by etching. Desired probes are grouped in advance as described above. These probes may be fixed by spotting the bottom of the groove for each group. On the other hand, through holes are formed in the substrate 30 at portions corresponding to both ends of the groove of the substrate 31. Next, the substrate 30 and the substrate 31 are joined. By bonding a glass tube of a desired length to each opening, the connecting portions 34a and 34b are formed, and the capillaries 2 to 8 are formed. The size of the capillary-shaped space can be set to various sizes according to the application, but practically, the width is 10 μm to several mm, the depth is 1 μm to 500 μm, the length is several mm to 100 mm, DNA A capillary interval of about 10 μm to several mm is sufficient. However, considering the efficiency of the reaction, since the diffusion rate of mRNA to be measured, or nucleic acid such as cDNA converted from mRNA, is as low as several μm per second, the cross-sectional shape of the capillary-shaped space should be wide. By adopting a flat structure with a shallow depth, it can be expected to shorten the reaction time, reduce the amount of sample, increase the observation field of view, and the like.
[0039]
In the above example, the groove is formed in the substrate 31, but the groove may be formed in the substrate 30 or in both the substrate 30 and the substrate 31. Further, as a material of the support, a glass substrate was used as a substrate used as a lid, and a silicon substrate was used as a substrate for forming a groove. However, the present invention is not limited to this. A substrate may be used, and a glass substrate may be used as the substrate for forming the groove. Further, two substrates used may be made of the same material. Further, the member to be used may be determined so that the transparent member is arranged in the observation direction. Alternatively, a support formed of a plastic resin, rubber, or the like may be used. Further, a support formed of a material such as glass, silicon, plastic resin and rubber may be used in combination. In the above example, a plate-like substrate is used as the support, but the present invention is not limited to this.
[0040]
In a substrate such as glass or a silicon wafer, grooves and through holes can be formed by a technique such as photolithography. In the case of plastic resin or rubber, grooves and through holes can be formed by machining or molding. As a means for immobilizing the nucleic acid probe, any means known per se may be used, for example, a spotting method and a photo-immobilization method. In the above example, the nucleic acid probe is fixed to the bottom of the groove of the substrate 31, but it may be fixed to the substrate 30, or it may be fixed to the side surface of each reaction section.
[0041]
FIG. 2 shows an example in which one capillary has various numbers of spots. However, the number of spots immobilized on one capillary is not limited to this, and the number of spots may be any number as long as the number of spots is 1 or more, or may be a different number of spots for each capillary. . It is not necessary that all capillaries included in the probe-immobilized reaction array include a plurality of types of probes, and a control capillary having no immobilized probe may be provided.
[0042]
Further, the bonding of the two substrates may be performed before or after fixing the nucleic acid probe by a means known per se. When they are bonded before immobilization, it is preferable to immobilize the nucleic acid probe by applying a photo-solid phase method (for example, see Japanese Patent Application Laid-Open No. 6-504997). For example, in the case of silicon-quartz glass, an adhesive may be printed by a screen printer and bonded. For example, in the case of silicon-pyrex glass, bonding may be performed at high temperature and high voltage by an anodic bonding method used in a semiconductor process.
[0043]
In the probe-immobilized reaction array 1 formed as described above, hybridization is performed in parallel in a region where the densities are integrated under reaction conditions suitable for the probes provided in the respective reaction portions. For example, the temperature may be controlled for each of the capillaries, or the optimum reaction conditions may be obtained based on the ion concentration and the salt concentration while maintaining all the reaction sections at a constant temperature.
[0044]
For example, when only one heater 33 is used to maintain the respective optimum temperatures for all the reaction parts having different optimum reaction temperatures, the salt concentration contained in the reaction solution used at the time of the hybridization is adjusted. Just adjust it. That is, even when the optimum concentration in each reaction part is different, it can be achieved by adjusting the salt concentration of a buffer or the like used as a solution of the test sample containing the target nucleic acid, for example. . When the components of the solution used in the actual hybridization reaction contain a DNA denaturant or the like, it is necessary to further adjust the salt concentration as appropriate.
[0045]
Washing as a treatment after the hybridization reaction is also important for realizing a precise reaction. In a DNA hybridization reaction, it is necessary to minimize mismatches and increase the efficiency of hybridization with a target nucleic acid as much as possible. For that purpose, it is necessary to remove only the test nucleic acid that is bound to the nucleic acid probe due to the mismatch so as not to remove the target nucleic acid in which the hybridization reaction has normally occurred. In the case of a mismatched nucleic acid, the number of hydrogen bonds in the duplex formed by the nucleic acid probe and the test nucleic acid in question is smaller than that in the case of a match. Therefore, Tm in the case of a mismatch is lower. Therefore, it is expected that the mismatched test nucleic acid is specifically removed by controlling the temperature or the salt concentration of the washing solution. Since the Tm of the mismatched test nucleic acid changes depending on the location and the number of mismatches, it is difficult to define the Tm collectively in any case. However, under such conditions, if an experiment is performed for each group of capillaries, it is possible to set optimal conditions for desired analysis. A method for preventing such a mismatch is also included in the scope of the present invention.
[0046]
According to the embodiment of the present invention, the use of the probe-immobilized reaction array 1 makes it possible to set the conditions for the hybridization reaction and the conditions for washing individually for each capillary or for each unit composed of a plurality of capillaries. It is possible. Therefore, according to the aspect of the present invention, it is possible to detect a target sequence with excellent reproducibility and high accuracy. It is also possible to prevent mismatch in hybridization.
[0047]
In the present embodiment, the form of the reaction portion included in the probe-immobilized reaction array 1 is a capillary, but is not limited to this shape. That is, various structures of the device structure in which the reaction parts physically separated from each other are formed are conceivable. In the above example, the probe-immobilized reaction array 1 includes seven reaction sections, and each reaction section has one or more openings that can be used as a sample inlet and / or a sample outlet. The number is not limited, and it is sufficient if one or more reaction units are provided.
[0048]
The reaction section provided in the probe-immobilized reaction array according to the present invention refers to a region in the region where a reaction and processing intended by the user such as a chemical reaction or a biochemical reaction are performed. Therefore, in the above-described embodiment, the example in which the reaction section has a capillary shape has been described, but the shape of the reaction section is not limited to this. The reaction units may be independent of each other. For example, a lid or a bottom having an opening may be arranged in a region partitioned by a concave or a convex to form a reaction unit having an effective volume. The volume of each reaction section of the reaction array of the present invention may be from 0.01 μL to 1 mL.
[0049]
Further, according to the aspect of the present invention, not only a closed reaction array in which a reaction portion consisting of a lumen is formed inside a substrate, but also a plurality of reaction portions independent of each other and arranged in parallel in an arbitrary direction on a support. If it is provided, even for a container simply partitioned by concave or convex portions, or even in a region without a special partition consisting of a plane, the liquid is sufficiently separated or provided by a large number of vertical holes. Since diffusion is prevented, they can be used in embodiments of the present invention if they are independent of each other. Such an apparatus and a reaction method using the same are also included in the scope of the present invention.
[0050]
Further, the shape of the reaction portion may be a well shape in which the bottom surface is a square, a circle, or an ellipse. Further, the area of the bottom surface and the area of the ceiling surface of the reaction section may be equal or different. The “well shape” used herein does not mean, for example, a form in which the bottom surface and the ceiling surface of the reaction section are spread only in a specific direction as in a capillary shape, but a two-dimensional structure that forms the bottom surface and the ceiling surface. It refers to a shape having a shape that shows a certain degree of spread in any of the dimensional directions, and has a concave portion or a porous portion that is divided so that a plurality of probe fixing portions can be collectively processed as a group (for example, , See Japanese Patent Application Laid-Open No. 9-504864).
[0051]
Further, in this embodiment, an example is shown in which the heater is separate from the reaction array according to the present invention. However, a heater, a sensor, necessary wiring, and the like may be incorporated in the reaction array. The reaction array may not be used.
[0052]
The `` probe-immobilized reaction array '' according to the present invention is an apparatus in which a plurality of reaction units in which the grouped probes are immobilized as described above are arranged on the same support in a desired pattern. Good. Such a probe-immobilized array only needs to be able to react a probe with a target substance having high affinity specific to the probe. Further, it is preferable that the bond generated by the reaction can be observed while being contained in the support. For example, by labeling the test sample with a visually recognizable label (such as a fluorescent dye), the fluorescence of the target substance bound to the probe can be visually recognized as it is.
[0053]
Further, in the present embodiment, an example of a nucleic acid probe-immobilized reaction array in which a nucleic acid probe is immobilized on a support has been described, but instead of nucleic acid as a probe, a protein or peptide, an antigen or an antibody, or a combination thereof is It may be used as a probe. In such a case, a substance that specifically binds to the target substance to be detected is used as a probe and grouped according to the property of the substance, and each group is arranged so that the probes to be compared are adjacent to each other and fixed to a support. Just fine.
[0054]
In recent years, several protein chips in which a plurality of proteins and peptides are immobilized on a support have been disclosed. For example, a chip in which more than 10,000 proteins are immobilized using a spotter for DNA microarray is disclosed (Science (2000) Sep 8; 289 (5485): 1673). In addition, Cyphergen has proposed a protein structural analysis using a protein chip and a mass spectrometer, and a system for analyzing their interaction. The present invention can also be applied to such a conventional protein chip known per se.
[0055]
【Example】
Here, specific examples of the probe-immobilized array of the present invention will be described.
[0056]
Embodiment 1
(Microarray for TNF family gene analysis)
DNA probes used in array experiments include oligonucleotide probes and cDNA probes. Oligonucleotide probes are superior to cDNA probes in the following respects: (1) less errors due to cross-hybridization; (2) no previously required operations such as cloning and PCR reactions are required; (3 ) Hybridization time is short; (4) Alternative transcription and alternative splicing can be distinguished. The design of oligonucleotide probes requires that the base sequence of genomic DNA be sufficiently determined, and such conditions have been established in many organisms with the rapid progress of the genome project.
[0057]
A method for designing a base sequence of a nucleic acid probe (for example, an oligonucleotide probe or the like) used in the probe-immobilized array of the present invention will be described. For the probe-immobilized reaction array of the present invention, a tuple method (see Non-Patent Document 1), which is a method for efficiently designing probe sequences grouped by Tm or expression frequency, can also be used. The tuple method is a method of counting how many times a short base sequence called a tuple, which is about several bases, appears in all genes, and determining a base sequence to be used based on the result. This counting need only be performed once at the beginning.
[0058]
First, the tuples included in the base sequence of the candidate selected in advance are checked to see if there are any tuples that appear frequently. Then, the specificity of whether the candidate is a “common” sequence among all genes is calculated, and the specificity is evaluated. In practice, first, the tuple frequency is calculated, and then the candidate sequences are evaluated using tuples to select highly specific sequences. On the way, after applying the restrictions determined by the experimental conditions, the candidates are narrowed down by applying a filter that allows only the candidates having the thermal characteristics suitable for the experiment. Furthermore, since DNA has the property of binding complementarily, if single-stranded DNA alone is sufficiently long, it may bind within its own molecule, making it difficult for hybridization to occur. Therefore, whether or not such a structure can be adopted is also excluded by prediction in calculation.
[0059]
Next, as an index of probe self-structuring, a predicted free energy ΔG value of a double-stranded region in a DNA molecule is used for predicting a secondary structure. The structure that DNA is most likely to take is the structure that is most stable in terms of energy, and the structure that maximizes the ΔG value is predicted from the possible combinations of the specified regions. The more stable the structure, the smaller the value of ΔG. FIG. 1 shows an example of a calculation filter obtained by combining the above methods.
[0060]
By the above-described method, a probe for analyzing human TNF / TNFR family genes was designed.
[0061]
Many family members of TNF (tumor necrosis factor) have been identified, and more than 40 genes, including ligands and receptors, form a superfamily. Its functions are diverse, including induction and inhibition of apoptosis, promotion of cell proliferation and differentiation, and play an important role in various life phenomena, from development and differentiation of individuals to immune responses. By analyzing these TNF family genes, it can be applied to basic research such as disease diagnosis and functional analysis of TNF family genes, and can be a useful analysis tool.
[0062]
Table 1 shows currently identified TNF family genes. Probe sequences for these 45 genes were designed.
[0063]
[Table 1]

The length of the probe was set to 75mer, and the result of calculation is shown in Table 2.
[0064]
[Table 2]

[0065]
From the calculation results, the Tm value of the 45 genes was almost the same as 71 ° C. ± 1 ° C., but the ΔG value as an index of self-structuring varied from −2 to −30. Tm can be cited as an index for adjusting the reaction conditions with a probe, but it is considered that self-structuring has a stronger effect than Tm value when the chain length is longer.
[0066]
Based on the results of this calculation, using the self-structuring value as an index, by grouping probes with high self-structuring, low self-structuring, or intermediate probes, optimum reaction conditions can be obtained even for probes with different characteristics. To perform a hybridization reaction.
[0067]
For example, when an array substrate having a capillary structure as shown in FIG. 2 is used, it is possible to change the hybridization conditions based on the prediction result of self-structuring. In FIG. 2a, the device 1 has a plurality of capillaries 2, 3, 4, 5, 6, 7 and 8 formed therein. DNA probes grouped based on the difference in the self-structured ΔG value are fixed inside the capillary. The self-structured ΔG values are grouped stepwise, and the TACI gene probe having the highest ΔG value (one type in the figure) is immobilized on the capillary 2, and the ΔG value is -12 to -16 on the capillary 3. A certain probe (three types in the figure) and a capillary 4 were mounted on independent capillaries in the order of probes having a ΔG value of -16 to -18 (12 types in the figure). In addition, one end of the probe group is arranged at the same distance from the edge of the substrate so that one or more of the probes in each capillary are adjacent to each other as much as possible, and the other probes are also identical from the edge of the substrate across the capillaries. They were arranged adjacent to each other at a distance. This time, it is divided into seven groups, but there is no limitation even if it is more or less than this, and the grouping threshold of the ΔG value is not limited to this.
[0068]
Here, one of the grouped DNA probes was designated as DNA probe 9, 10, 11, 12, 13, 14, and 15, respectively. The capillary array is provided with sample inlets 16, 17, 18, 19, 20, 21 and 22, and sample outlets 23, 24, 25, 26, 27, 28 and 29, respectively. A cross-sectional view of the capillary 4 is shown in FIG. 2B, and a lid substrate 30 and a groove forming substrate 31 of the device 1 and a heater 33 for temperature control are provided below the device. The device 1 thus formed is maintained at a constant temperature, for example, 45 ° C. by the heater 33, and a sample is injected into the device 1 from a sample injection port.
[0069]
In preparing a target sample, usually, when synthesizing cDNA from mRNA or total RNA extracted from tissues or cells, labeling is performed using Cy3-dUTP or Cy5-dUTP. Labeling when synthesizing first strand cDNA using oligo dT primer or random primer and reverse transcriptase. In the hybridization reaction, the reaction with the target DNA is generally performed for 1 hour to overnight while maintaining a constant humidity at a constant temperature of about 45 ° C. to 65 ° C. to prevent evaporation of the target DNA.
[0070]
By using this device structure, the setting of conditions for the hybridization reaction and the setting of the washing conditions can be used individually or in combination, so that measurement with good reproducibility and high accuracy can be performed.
[0071]
Implementation mode 2
(Probe-immobilized array for TNF family gene analysis grouped based on gene function)
As described above, more than 45 types of TNF / TNFR family genes are known, and many of them have a receptor / ligand relationship elucidated. Table 3 shows an example of the relationship between receptors and their ligands between TNF / TNFR family genes.
[0072]
[Table 3]

[0073]
Probes can be grouped by focusing on the function of the gene such as the relationship with these receptors / ligands. FIG. 3 shows an example of grouping. In capillaries 34, 35, 36, 37, 38, 39 and 40, five groups of probes for the receptor and its ligand were arranged. The seven capillaries were all in the same group of probes of the same type, and were arranged at the same distance from the edge of the substrate so that groups of the same type were adjacent to each other between the capillaries. An analysis example using this probe-immobilized array will be described.
[0074]
Conventionally, when examining the time-dependent change in the gene expression level caused by antibody stimulation or the like in a certain cell or individual, an experiment was performed using an independent probe array substrate. However, when different independent arrays are used, there are large variations in the time of array preparation, hybridization and washing operations, detection times, and the like, and it has been difficult to examine the original gene change amount. FIG. 4 is a schematic diagram showing the results of analysis using the probe array shown in FIG. Capillaries 42, 43, 44, 45, 46, 47, and 48 are examples of detecting changes over time in gene changes after antibody stimulation to cells. Capillary 42-48, after antibody stimulation with TNFα antibody to human monocyte U937 cells, 0 hours, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, from cells after 48 hours, A labeled target is prepared in the manner described in the first embodiment. By hybridizing the target on the same substrate, a gene change pattern as shown in FIG. 4 could be obtained. Due to the antibody stimulation by TNFα, the expression of its receptor, TNFR1, starts to increase after 6 hours, and the frequency of expression further increases over time.In response to this, the frequency of expression of the TNFα gene is increased 24 hours later. Will rise. Until now, it was possible to obtain the result of the gene expression frequency difference for the first time after obtaining the image data and digitizing it using analysis software. Is used in a capillary array system (see, for example, Japanese Patent Application Laid-Open No. 2001-136963), and it is possible to visually detect the change in the expression of any of the receptor / ligand pairs with time. Further, by performing hybridization and washing on one substrate, a result with higher reproducibility can be obtained. As described above, each probe group can obtain the best result by applying one optimal reaction condition to each probe group. Although the Tm value is an index of the probe characteristics, when the DNA used as the probe has a long chain, the probe itself has a self-structure rather than the Tm value. . Therefore, as shown in the present invention, the best result can be obtained by calculating the self-structuring of the probe by calculation and performing grouping as a probe characteristic index. In addition, by performing grouping based on not only the probe characteristics but also the characteristics of the target substance (eg, target nucleic acid), particularly the functional properties of the target substance, the target in the test sample, which could not be performed so far, has It becomes possible to capture changes in the amount of substances.
[0075]
According to the embodiments of the present invention, the conventional problems in hybridization are solved. In addition, based on the reversal idea of utilizing such characteristics that have been a problem in the past, a probe-immobilized reaction array with excellent reaction efficiency and excellent reproducibility, and a reaction method using the same Is provided. Focusing on the probe characteristics and the functional characteristics of the target substance, by devising probe grouping or arrangement layout, it is possible to realize more reproducible and visually recognizable probe reaction array experiments It becomes.
[0076]
【The invention's effect】
By using the probe-immobilized array of the present invention, by focusing on the probe characteristics and the functional characteristics of the target substance, by devising the grouping or arrangement layout of the probes, more excellent reproducibility, This makes it possible to implement a probe reaction array experiment that is visually easy to see.
[Brief description of the drawings]
FIG. 1 is a flowchart illustrating an example of a calculation filter.
FIG. 2 is a layout diagram in which TNF / TNFR family genes are grouped by a self-structuring index.
FIG. 3 is a layout diagram grouped by gene function of TNF / TNFR family genes.
FIG. 4 is a diagram showing an analysis example of an array grouped by gene functions of TNF / TNFR family genes.
[Explanation of symbols]
1 Probe-immobilized reaction array
2,3,4,5,6,7,8,35,36,37,38,39,40,41,42,43,44,45,46,47,48 capillaries
9,10,11,12,13,14,15 Nucleic acid probe group
16, 17, 18, 19, 20, 21, 22 Sample inlet
23, 24, 25, 26, 27, 28, 29 Sample outlet
30,31 substrate
33 heater
34a, 34b connecting part

Claims (8)

A probe-immobilized reaction array in which the probes are immobilized on a support,
The support includes a plurality of first to n-th reaction units (where n is an integer of 2 or more) arranged in parallel therein,
The probes are grouped and arranged for each of the first to n-th reaction units, and the immobilization position of the probe in each reaction unit is at least one position at the same distance from the end of each reaction unit. A probe-immobilized reaction array, wherein the probe is located at the same position in each reaction part so as to be located on a line. 2. The probe-immobilized reaction array according to claim 1, wherein the grouping is classified according to probe characteristics. 3. The probe-immobilized reaction array according to claim 2, wherein the probe characteristics are structural characteristics of the probe. 2. The probe immobilization reaction array according to claim 1, wherein the grouping is grouped according to characteristics of a target substance. 5. The probe-immobilized reaction array according to claim 4, wherein the characteristic of the target substance is a functional property of the target substance. The probe-immobilized reaction array according to any one of claims 1 to 5, wherein the probe is a nucleic acid. The probe-immobilized reaction array according to any one of claims 1 to 6, wherein the probes are selected from the group consisting of peptides, proteins, antigens, and antibodies. Reaction array. The probe-immobilized reaction array according to any one of claims 1 to 7, wherein the reaction section has a capillary shape.

Images