JP3568197B2 - Reactive solid support and DNA fragment detection tool - Google Patents

Description

【００４５】
【化２】

【００４６】
「−Ｘ」は、上記具体例中、（Ｘ１）、（Ｘ２）、（Ｘ３）、（Ｘ４）、（Ｘ７）、（Ｘ８）、（Ｘ１３）あるいは（Ｘ１４）であることが好ましく、（Ｘ１）あるいは（Ｘ２）であることがさらに好ましい。特に好ましいのは、（Ｘ１）で表わされるビニル基である。
【００４７】
Ｌの好ましい具体例を以下に示す。但し、ａは、１乃至６の整数であり、１もしくは２であることが好ましく、１であることが特に好ましい。ｂは、０乃至６の整数であり、２もしくは３であることが好ましい。
【００４８】
【化３】

【００４９】
Ｌとしては、上記記載の二価の連結基の他に、上記式のアルキレン基の水素原子が−ＳＯ_２ＣＨ＝ＣＨ_２基によって置換されてなる基も好ましい。
【００５０】
前記の式（１）で表わされるビニルスルホニル基もしくは反応性前駆体基が共有結合により固定された固相担体を得るために利用される二官能反応性化合物としては、下記の式（２）で表わされるジスルホン化合物が有利に利用できる。
【００５１】
Ｘ^１−ＳＯ_２−Ｌ^２−ＳＯ_２−Ｘ^２ （２）
【００５２】
［上記の式において、Ｘ^１およびＸ^２は互いに独立に、−ＣＲ^１＝ＣＲ^２Ｒ^３、または−ＣＨＲ^１−ＣＲ^２Ｒ^３Ｙ（反応性前駆体基）を表わし；Ｒ^１、Ｒ^２及びＲ^３は、互いに独立に、水素原子、炭素原子数が１乃至６のアルキル基、炭素原子数が６乃至２０のアリール基、及び炭素原子数が１乃至６のアルキル鎖を有する合計炭素原子数が７乃至２６のアラルキル基からなる群より選ばれる原子もしくは基を表わし；Ｙは、ハロゲン原子、−ＯＳＯ_２Ｒ^１１、−ＯＣＯＲ^１２、−ＯＳＯ_３Ｍ、及び四級ピリジニウム基からなる群より選ばれる原子もしくは基を表わし；Ｒ^１１は、炭素原子数が１乃至６のアルキル基、炭素原子数が６乃至２０のアリール基、及び炭素原子数が１乃至６のアルキル鎖を有する合計炭素原子数が７乃至２６のアラルキル基からなる群より選ばれる基を表わし；Ｒ^１２は、炭素原子数が１乃至６のアルキル基および炭素原子数が１乃至６のハロゲン化アルキル基からなる群より選ばれる基を表わし；Ｍは、水素原子、アルカリ金属原子およびアンモニウム基からなる群より選ばれる原子もしくは基を表わし；そして、Ｌ^２は連結基を表わす］。
【００５３】
すなわち、上記の式（２）で表わされるジスルホン化合物を、前記の固相担体と、例えば水性雰囲気にて、接触させることによって、本発明の反応性固相担体を容易に製造することができる。
【００５４】
本発明で好ましく用いるジスルホン化合物の代表例を下記に示す。なお、ジスルホン化合物は、二種類以上を混合して用いてもよい。
【００５５】
【化４】

【００５６】
【化５】

【００５７】
上記の式（２）で表わされるジスルホン化合物の代表的な例としては、１，２−ビス（ビニルスルホニルアセトアミド）エタン［上記のＳ１に相当する］を挙げることができる。
【００５８】
本発明で用いるジスルホン化合物の合成法については、たとえば、特公昭４７−２４２９号、同５０−３５８０７号、特開昭４９−２４４３５号、同５３−４１５５１号、同５９−１８９４４号等の各種公報に詳細が記載されている。
【００５９】
上記のようにして得られた反応性固相担体を利用して、ＤＮＡ、ＲＮＡ、ＤＮＡ断片、あるいはＲＮＡ断片などの天然起源のポリヌクレオチドあるいはオリゴヌクレオチドの検出固定のための検出具（一般にＤＮＡチップと呼ばれているもの）を作製するためには、上記の反応性固相担体を、その担体表面上のビニルスルホニル基もしくはその反応性前駆体基と反応して共有結合を形成するアミノ基などの反応性基を備えたヌクレオチド誘導体もしくはその類縁体と接触させる方法が利用される。すなわち、このようにして所望のヌクレオチド誘導体もしくはその類縁体からなるプローブ分子を備えた検出具（いわゆるＤＮＡチップ）を作製することができる。
【００６０】
本発明の固相基板表面に共有結合を介して結合されたビニルスルホニル基もしくはその反応性前駆体基は、加水分解に対して高い抵抗性を有しているため、容易に安定に保存することができ、また、アミノ基を予め備えているか、あるいはアミノ基などの反応性基が導入されているかヌクレオチド誘導体もしくはその類縁体の反応性基と迅速に反応して、安定な共有結合を形成することができる。
【００６１】
プローブ分子として用いるヌクレオチド誘導体もしくはその類縁体の代表例としては、オリゴヌクレオチド、ポリヌクレオチド、そしてペプチド核酸を挙げることができる。これらのヌクレオチド誘導体もしくはその類縁体としては、天然起源のもの（ＤＮＡ、ＤＮＡ断片、ＲＮＡ、あるいはＲＮＡ断片など）であってもよく、あるいは合成化合物であってもよい。また、ヌクレオチド誘導体もしくはその類縁体としては、その糖単位部分に架橋基を有するＬＮＡと呼ばれる化合物（Ｊ． Ａｍ． Ｃｈｅｍ． Ｓｏｃ． １９９８， １２０， １３２５２−１３２５３に記載）などの各種の類縁化合物が含まれる。
【００６２】
プローブ分子としてＤＮＡ断片を用いる場合は、目的によって二通りに分けることができる。遺伝子の発現を調べるためには、ｃＤＮＡ、ｃＤＮＡの一部、ＥＳＴ等のポリヌクレオチドを使用することが好ましい。これらのポリヌクレオチドは、その機能が未知であってもよいが、一般的にはデータベースに登録された配列を基にしてｃＤＮＡのライブラリー、ゲノムのライブラリーあるいは全ゲノムをテンプレートとしてＰＣＲ法によって増幅して調製する（以下、「ＰＣＲ産物」という）。ＰＣＲ法によって増幅しないものも、好ましく使用することができる。また、遺伝子の変異や多型を調べるには、標準となる既知の配列をもとにして、変異や多型に対応する種々のオリゴヌクレオチドを合成し、これを使用することが好ましい。さらに、塩基配列の分析を目的とする場合、４^ｎ（ｎは、塩基の長さ）種のオリゴヌクレオチドを合成して、それらを使用することが好ましい。ＤＮＡ断片の塩基配列は、一般的な塩基配列決定法によって予めその配列が決定されていることが好ましい。ＤＮＡ断片は、２乃至５０量体であることが好ましく、１０乃至２５量体であることが特に好ましい。
【００６３】
オリゴヌクレオチドやＤＮＡ断片などのヌクレオチド誘導体もしくはその類縁体の一方の末端には、前記のビニルスルホニル基もしくはその反応性前駆体基と反応して共有結合を形成する反応性基を導入する。このような反応性基は、アミノ基、イミノ基、ヒドラジノ基、カルバモイル基、ヒドラジノカルボニル基、もしくはカルボキシイミド基であることが好ましく、アミノ基であることが特に好ましい。オリゴヌクレオチドやＤＮＡ断片には、通常、クロスリンカーを介してこれらの反応性基が結合される。クロスリンカーとしては、たとえば、アルキレン基あるいはＮ−アルキルアミノ−アルキレン基が利用されるが、ヘキシレン基あるいはＮ−メチルアミノ−ヘキシレン基であることが好ましく、ヘキシレン基であることが特に好ましい。なお、ペプチド核酸（ＰＮＡ）はアミノ基を有しているため、通常は、改めて別に反応性基を導入する必要はない。
【００６４】
反応性基を備えたヌクレオチド誘導体もしくはその類縁体と反応性固相担体との接触は、通常、該ヌクレオチド誘導体もしくはその類縁体の水溶液を反応性固相担体の表面に点着することにより実施される。具体的には、反応性基を備えたヌクレオチド誘導体もしくはその類縁体を水性媒体に溶解あるいは分散して水性液としたのち、その水性液を、９６穴もしくは３８４穴プラスチックプレートに分注し、分注した水性液をスポッター装置等を用いて固相担体表面上に滴下して行うことが好ましい。
【００６５】
点着後のヌクレオチド誘導体もしくはその類縁体の乾燥を防ぐために、ヌクレオチド誘導体もしくはその類縁体が溶解あるいは分散してなる水性液中に、高沸点の物質を添加してもよい。高沸点の物質としては、点着後のヌクレオチド誘導体もしくはその類縁体が溶解あるいは分散してなる水性液に溶解し得るものであって、検出対象の核酸断片試料（標的核酸断片）などの試料とのハイブリダイゼーションを妨げることがなく、かつ粘性があまり大きくない物質であることが好ましい。このような物質としては、グリセリン、エチレングリコール、ジメチルスルホキシドおよび低分子の親水性ポリマーを挙げることができる。親水性ポリマーとしては、ポリアクリルアミド、ポリエチレングリコール、そしてポリアクリル酸ナトリウム等を挙げることができる。このポリマーの分子量は、１０^３乃至１０^６の範囲にあることが好ましい。高沸点の物質としては、グリセリンあるいはエチレングリコールを用いることがさらに好ましく、グリセリンを用いることが特に好ましい。高沸点の物質の濃度は、ヌクレオチド誘導体もしくはその類縁体の水性液中、０．１乃至２容量％の範囲にあることが好ましく、０．５乃至１容量％の範囲にあることが特に好ましい。
【００６６】
また、同じ目的のために、ヌクレオチド誘導体もしくはその類縁体を点着した後の固相担体を、９０％以上の湿度および２５乃至５０℃の温度範囲の環境に置くことも好ましい。
【００６７】
反応性基を有するヌクレオチド誘導体もしくはその類縁体を点着後、紫外線、水素化ホウ素ナトリウムあるいはシッフ試薬による後処理を施してもよい。これらの後処理は、複数の種類を組み合わせて行ってもよく、特に加熱処理と紫外線処理を組み合わせて行うことが好ましい。これらの後処理は、ポリ陽イオン化合物のみによって固相担体表面を処理した場合には特に有効である。点着後は、インキュベーションを行うことも好ましい。インキュベート後は、未反応のヌクレオチド誘導体もしくはその類縁体を洗浄して除去することが好ましい。
【００６８】
ヌクレオチド誘導体もしくはその類縁体の固定量（数）は、固相担体表面に対して、１０^２乃至１０^５種類／ｃｍ^２の範囲にあることが好ましい。ヌクレオチド誘導体もしくはその類縁体の量は、１乃至１０^−１５モルの範囲にあり、重量としては数ｎｇ以下であることが好ましい。点着によって、ヌクレオチド誘導体もしくはその類縁体の水性液は、固相担体表面にドットの形状で固定される。ドットの形状は、ほとんど円形である。形状に変動がないことは、遺伝子発現の定量的解析や一塩基変異を解析するために重要である。それぞれのドット間の距離は、０乃至１．５ｍｍの範囲にあることが好ましく、１００乃至３００μｍの範囲にあることが特に好ましい。１つのドットの大きさは、直径が５０乃至３００μｍの範囲にあることが好ましい。固相担体表面に点着するヌクレオチド誘導体もしくはその類縁体の量は、１００ｐＬ乃至１μＬの範囲にあることが好ましく、１乃至１００ｎＬの範囲にあることが特に好ましい。
【００６９】
図１に、本発明の代表的な態様であるオリゴヌクレオチド固定固相担体の製造方法および代表的なオリゴヌクレオチド固定固相担体の構成を模式的に示す。
本発明のオリゴヌクレオチドが固定された凹凸を有する固相担体の製造方法としては、前記式（２）で表されるジスルホン化合物を用いた場合、そのＸ^１およびＸ^２によって四種類の製造方法が利用できる。
【００７０】
図１には、式（２）のジスルホン化合物のＸ^１とＸ^２とが何れも−ＣＨＲ^１−ＣＲ^２Ｒ^３Ｙ（反応性前駆体基）である場合のオリゴヌクレオチドが固定された固相担体（Ｃ１）の製造方法（ａ）、およびＸ^１が−ＣＨＲ^１−ＣＲ^２Ｒ^３Ｙであって、Ｘ^２が−ＣＲ^１＝ＣＲ^２Ｒ^３である場合のオリゴヌクレオチド固定固相担体（Ｃ２）の製造方法（ｂ）を示す。ただし、Ｘ^１を、固相担体１の表面に導入された反応性基（Ｒ）と最初に反応する基と仮定して、そのＸ^１を−ＣＲ^１＝ＣＲ^２Ｒ^３とする製造方法であってもよい。以下、「Ｘ^１」を、固相担体１の表面に導入された反応性基（Ｒ）と最初に反応する基として説明を行なう。
【００７１】
製造方法（ａ）および（ｂ）について説明する。
工程（１）：担体表面に反応活性基（Ｒ）が導入された固相担体１［固相担体（Ａ）］に、式（１）で表わされるジスルホン化合物を接触させ、Ｘ１の−Ｙ部分に反応性基（Ｒ）を置換させることによって、−（ＣＲ^１Ｒ^２）_ｎ−ＳＯ_２−Ｌ−ＳＯ_２−Ｘ^２基を固相担体表面に導入する。
【００７２】
工程（２）：工程（１）で導入された−（ＣＲ^１Ｒ^２）_ｎ−ＳＯ_２−Ｌ−ＳＯ_２−Ｘ^２基のＸ^２に、一方の末端に反応性基（Ｚ）を有するオリゴヌクレオチド２を接触させることによって反応性基（Ｚ）を付加させる、または該Ｘ^２の−Ｙに該オリゴヌクレオチド２を接触させることによって反応性基（Ｚ）を置換させる。
【００７３】
本発明のプローブ分子固定用担体は、Ｘ^１を固相担体１の表面に導入された反応性基（Ｒ）と最初に反応する基とする場合に、そのＸ^１を−ＣＲ^１＝ＣＲ^２Ｒ^３とする下記の方法によって製造されるものであってもよい。
【００７４】
工程（１）：凹凸を有する固相担体１の表面に活性基（Ｒ）が導入された固相担体（Ａ）に、式（Ｉ）で表されるジスルホン化合物を接触させ、Ｘ^１の−ＣＲ^１＝ＣＲ^２Ｒ^３に、反応性基（Ｒ）を付加させることによって、−Ｒ^３Ｒ^２Ｃ−Ｒ^１ＨＣ−ＳＯ_２−Ｌ−ＳＯ_２−Ｘ^２基を固相担体表面に導入する。
【００７５】
工程（２）：工程（１）において導入された−Ｒ^３Ｒ^２Ｃ−Ｒ^１ＨＣ−ＳＯ_２−Ｌ−ＳＯ_２−Ｘ^２基のＸ^２に、一方の末端に反応性基（Ｚ）を有するオリゴヌクレオチドを接触させることによって反応性基（Ｚ）を付加させるか、あるいは該Ｘ^２の−Ｙの部分に、該オリゴヌクレオチドを接触させることによって、この反応性基（Ｚ）を置換させる。
【００７６】
一方の末端に反応性基（Ｚ）を有するオリゴヌクレオチドは、図１において、２によって示される化合物である。クロスリンカー（Ｑ）は、必須ではないが、反応性のオリゴヌクレオチド２の調製の都合上、反応性基（Ｚ）とリン酸エステル基との間に存在するのが一般的である。−リン酸エステル基−ＮＮＮＮ・・・ＮＮは、オリゴヌクレオチドを表わす。Ｒ^４は、反応性基（Ｒ）とＸ^１との反応によって、Ｚ^１は、Ｘ^２と反応性基（Ｚ）との反応によってそれぞれ決定される基である。
【００７７】
図１の凹凸を有する固相担体（Ｂ）の表面に反応性基（Ｚ）を有するオリゴヌクレオチド２を点着させると、Ｘ^２またはＸ^２の−Ｙと該反応性オリゴヌクレオチド片２との反応が起こるが、固相担体（Ｂ）の表面には該オリゴヌクレオチド２が結合していない未反応のＸ^２も存在する。このようなＸ^２は、後に行なわれる標識された核酸断片試料とのハイブリダイゼーションにおいて非特異的な反応を生じる可能性があり、非特異的な結合を測定してしまうおそれがあるため、予め該Ｘ^２（即ち、例えば、Ｘ^２のハロゲン原子）をマスク処理しておくことが好ましい。マスク処理は、固相担体（Ｃ１）（もしくは（Ｃ２））の表面に、アミノ基もしくはメルカプト基を有するアニオン性化合物を接触させることによって行うことが好ましい。該オリゴヌクレオチド２は、負の電荷を有するため、固相担体（Ｃ）表面にも負の電荷を発生させることによって、オリゴヌクレオチド２が未反応のＸ^２と反応するのを防ぐことができる。このようなアニオン性化合物としては、Ｘ^２のハロゲン原子と反応し、かつ負の電荷（ＣＯＯ⁻、ＳＯ_３ ⁻、ＯＳＯ_３ ⁻、ＰＯ_３ ⁻、もしくはＰＯ_２ ⁻）を有するものであれば何れのものも用いることができるが、アミノ酸であることが好ましく、グリシンもしくはシステインであることが特に好ましい。また、タウリンも好ましく用いることができる。
【００７８】
本発明の方法によって製造されたヌクレオチド誘導体もしくはその類縁体が固定された固相担体の寿命は、ｃＤＮＡが固定されてなるｃＤＮＡ固定固相担体では通常、数週間であり、合成オリゴヌクレオチドが固定されてなる固相担体ではさらに長期間である。本発明のヌクレオチド誘導体もしくはその類縁体が固定された固相担体は、遺伝子発現のモニタリング、塩基配列の決定、変異解析、多型解析等に利用される。検出原理は、後述する標識した試料核酸断片とのハイブリダイゼーションである。
【００７９】
標識方法としては、ＲＩ法と非ＲＩ法（蛍光法、ビオチン法、化学発光法等）とが知られているが、本発明では蛍光法を用いることが好ましい。蛍光標識に利用される蛍光物質としては、核酸の塩基部分と結合できるものであれば何れも用いることができるが、シアニン色素（例えば、市販のＣｙ Ｄｙｅ^ＴＭシリーズのＣｙ３、Ｃｙ５等）、ローダミン６Ｇ試薬、Ｎ−アセトキシ−Ｎ^２−アセチルアミノフルオレン（ＡＡＦ）あるいはＡＡＩＦ（ＡＡＦのヨウ素誘導体）を使用することができる。
【００８０】
試料核酸断片としては、通常、その配列や機能が未知であるＤＮＡ断片試料あるいはＲＮＡ断片試料などの核酸断片試料が用いられる。
【００８１】
核酸断片試料は、遺伝子発現を調べる目的では、真核生物の細胞や組織サンプルから単離することが好ましい。試料がゲノムならば、赤血球を除く任意の組織サンプルから単離することが好ましい。赤血球を除く任意の組織は、抹消血液リンパ球、皮膚、毛髪、精液等であることが好ましい。試料がｍＲＮＡならば、ｍＲＮＡが発現される組織サンプルから抽出することが好ましい。ｍＲＮＡは、逆転写反応により標識ｄＮＴＰ（「ｄＮＴＰ」は、塩基がアデニン（Ａ）、シトシン（Ｃ）、グアニン（Ｇ）もしくはチミン（Ｔ）であるデオキシリボヌクレオチドを意味する。）を取り込ませて標識ｃＤＮＡとすることが好ましい。ｄＮＴＰとしては、化学的な安定性のため、ｄＣＴＰを用いることが好ましい。一回のハイブリダイゼーションに必要なｍＲＮＡ量は、点着する液量や標識方法によって異なるが、数μｇ以下である。なお、ヌクレオチド誘導体もしくはその類縁体固定固相担体上のＤＮＡ断片がオリゴＤＮＡである場合には、核酸断片試料は低分子化しておくことが望ましい。原核生物の細胞では、ｍＲＮＡの選択的な抽出が困難なため、全ＲＮＡを標識することが好ましい。
【００８２】
核酸断片試料は、遺伝子の変異や多型を調べる目的では、標識プライマーもしくは標識ｄＮＴＰを含む反応系において標的領域のＰＣＲを行なって得ることが好ましい。
【００８３】
ハイブリダイゼーションは、９６穴もしくは３８４穴プラスチックプレートに分注しておいた、標識した核酸断片試料が溶解あるいは分散してなる水性液を、本発明のヌクレオチド誘導体もしくはその類縁体を固定した固相担体上に点着することによって実施することが好ましい。点着の量は、１乃至１００ｎＬの範囲にあることが好ましい。ハイブリダイゼーションは、室温乃至７０℃の温度範囲で、そして６乃至２０時間の範囲で実施することが好ましい。ハイブリダイゼーションの終了後、界面活性剤と緩衝液との混合溶液を用いて洗浄を行い、未反応の核酸断片試料を除去することが好ましい。界面活性剤としては、ドデシル硫酸ナトリウム（ＳＤＳ）を用いることが好ましい。緩衝液としては、クエン酸緩衝液、リン酸緩衝液、ホウ酸緩衝液、トリス緩衝液、グッド緩衝液等を用いることができるが、クエン酸緩衝液を用いることが特に好ましい。
【００８４】
ヌクレオチド誘導体もしくはその類縁体を固定した固相担体を用いるハイブリダイゼーションの特徴は、標識した核酸断片試料の使用量を非常に少なくできることである。そのため、固相担体に固定するヌクレオチド誘導体もしくはその類縁体の鎖長や標識した核酸断片試料の種類により、ハイブリダイゼーションの最適条件を設定する必要がある。遺伝子発現の解析には、低発現の遺伝子も十分に検出できるように、長時間のハイブリダイゼーションを行うことが好ましい。一塩基変異の検出には、短時間のハイブリダイゼーションを行うことが好ましい。また、互いに異なる蛍光物質によって標識した核酸断片試料を二種類用意し、これらを同時にハイブリダイゼーションに用いることにより、同一のＤＮＡ断片固定固相担体上で発現量の比較や定量ができる特徴もある。
以下の実施例により本発明をより具体的に説明するが、本発明は実施例によって限定されることはない。
【００８５】
【実施例】
実施例１：ＤＮＡ断片固定スライドの作製、およびＤＮＡ断片の固定量の測定
凹凸性の反応性固相担体の作成とその性能、比較対照として凹凸性の無い反応性固相担体との比較を下記に示す。
【００８６】
（１）ビニルスルホニル基が導入された凹凸性固相担体（Ａ）の作成
ポリビニールアルコール７５ｍｇを１０００ｍｌの超純水に溶かし、溶解後、シランカップリング剤（信越科学製ＫＢＥ９０３）を５００μｌ添加し、マグネットスターラーで１０分間攪拌する。この液にガラス基板（７６ｍｍ×２６ｍｍ）を３０秒間浸してから引き上げ、余分な液を切った後に４５℃の乾燥機で乾燥させる。次にこのガラス基板を１１０℃のオーブンで１０分間加熱してシランをガラス基板に固着させる。さらに前述ガラス基板を冷却後、３％質量１，２−ビス（ビニルスルホニルアセトアミド）エタン／ｐＨ８．０ホウ酸緩衝溶液にスライドウオッシャーを使い、１２０分間反応させる。反応終了後、超純水で２０秒間×３回水洗する。乾燥は２５℃にセットした乾燥機で３０分間乾燥する。
【００８７】
（２）ＤＮＡ断片の点着と蛍光強度の測定
３’末端および５’末端がそれぞれアミノ基、蛍光標識試薬（ＦｌｕｏｒｏＬｉｎｋ Ｃｙ５−ｄＣＴＰ、アマシャム・ファルマシア・バイオテック社製）で修飾されたＤＮＡ断片（３’−ＴＣＣＴＣＣＡＴＧＴＣＣＧＧＧＧＡＧＧＡＴＣＴＧＡＣＡＣＴＴＣＡＡＧＧＴＣＴＡＧ−５’）を０．１Ｍ炭酸緩衝液（ｐＨ９．３）に分散してなる水性液（１×１０^−６Ｍ、１μＬ）を、上記（１）で得たスライド（Ａ）に点着した。直ちに、点着後の固相担体を２５℃、湿度９０％にて１時間放置した後、この固相担体を０．１重量％ＳＤＳ（ドデシル硫酸ナトリウム）と２×ＳＳＣ（２×ＳＳＣ：ＳＳＣの原液を２倍に希釈した溶液、ＳＳＣ：標準食塩−クエン酸緩衝液）との混合溶液で２回、０．２×ＳＳＣ水溶液で１回順次洗浄した。次いで、上記の洗浄後のスライドを０．１Ｍグリシン水溶液（ｐＨ１０）中に１時間３０分浸積した後、蒸留水で洗浄し、室温で乾燥させ、ＤＮＡ断片が固定された固相担体（Ｂ）を得た。この固相担体（Ｂ）表面の蛍光強度を蛍光スキャニング装置で測定したところ、２２２５０であった。
【００８８】
（３）比較対照スライドの作成と性能
ガラススライドを信越シリコーンＫＢＥ９０３（信越化学工業）の２重量％溶液２００ｍｌに市販のスライドウオッシャーを使い３分間反応させる。反応終了後２００ｍｌの超純水で１分間（スライドウオッシャー使用）水洗する。超純水を交換しながら、さらに前述水洗条件で２回繰り返す。水洗終了後、４５℃の乾燥機で１０分間乾燥させた後に、１１０℃にセットしたオーブンに入れ１０分間熱処理する。冷却後、３重量％の１，２−ビス（ビニルスルホニルアセトアミド）エタン／ｐ Ｈ ８．０ホウ酸緩衝溶液にスライドウオッシャーを使い、１２０分間反応させる。反応終了後、超純水で２０秒間×３回水洗する。乾燥は２５℃にセットした乾燥機で３０分間乾燥する。上記（２）と同様の評価を行い蛍光強度１７５０を得た。
上記結果より、本発明の固定化方法により、ＤＮＡ断片が高密度に効率よくスライドガラスに固定されたことが分かる。
【００８９】
実施例２：試料ＤＮＡ断片の検出
（１）凹凸性ＤＮＡ断片固定固相担体の作製
３’末端がアミノ基で修飾された４０ｍｅｒのＤＮＡ断片（３’−ＴＣＣＴＣＣＡＴＧＴＣＣＧＧＧＧＡＧＧＡＴＣＴＧＡＣＡＣＴＴＣＡＡＧＧＴＣＴＡＧ−５’）を０．１Ｍ炭酸緩衝液（ｐＨ９．３）に分散してなる水性液（１×１０^−６Ｍ、１μＬ）を、実施例１の（１）で得た凹凸性固相担体（Ａ）に点着した。直ちに、点着後の固相担体を２５℃、湿度９０％にて１時間放置した後、この固相担体を０．１重量％ＳＤＳ（ドデシル硫酸ナトリウム）と２×ＳＳＣ（２×ＳＳＣ：ＳＳＣの原液を２倍に希釈した溶液、ＳＳＣ：標準食塩−クエン酸緩衝液）との混合溶液で２回、０．２×ＳＳＣ水溶液で１回順次洗浄した。次いで、上記の洗浄後のスライドを０．１Ｍグリシン水溶液（ｐＨ１０）中に１時間３０分浸積した後、蒸留水で洗浄し、室温で乾燥させ、ＤＮＡ断片が固定された固相担体（Ｃ）を得た。
【００９０】
（２）試料ＤＮＡ断片の検出
５’末端にＣｙ５が結合した２２ｍｅｒの試料オリゴヌクレオチド（ＣＴＡＧＴＣＴＧＴＧＡＡＧＴＴＣＣＡＧＡＴＣ−５’）をハイブリダイゼーション用溶液（４×ＳＳＣおよび１０重量％のＳＤＳの混合溶液）（２０μＬ）に分散させたものを、上記（１）で得た固相担体（Ｃ）に点着し、表面を顕微鏡用カバーガラスで保護した後、モイスチャンバー内にて６０℃で２０時間インキュベートした。次いで、このものを０．１重量％ＳＤＳと２×ＳＳＣとの混合溶液、０．１重量％ＳＤＳと０．２×ＳＳＣとの混合溶液、および０．２×ＳＳＣ水溶液で順次洗浄した後、６００ｒｐｍで２０秒間遠心し、室温で乾燥した。スライドガラス表面の蛍光強度を蛍光スキャニング装置で測定したところ、１８４００であった。
【００９１】
また比較対照スライドを用いて、試料ＤＮＡ断片の検出を行ったところ、蛍光強度は１５００であった。
本発明の固定化方法によって作製された凹凸性ＤＮＡ断片固定固相担体を用いることによって、固定されているＤＮＡ断片と相補性を有する試料ＤＮＡ断片を高感度に検出できることが分かる。
【００９２】
参考例１：ＤＮＡ断片固定スライドの作製、およびＤＮＡ断片の固定量の測定
（１）ビニルスルホニル基が導入された凹凸性固相担体の作成
ＤＮＡマイクロアレイ用コートスライドグラス（Ｔｙｐｅ２高密度化アミノ基導入タイプ［松浪硝子工業］）を、３重量％１，２−ビス（ビニルスルホニルアセトアミド）エタン／ｐＨ８．０ほう酸緩衝溶液にスライドウォッシャーを使い、１２０分間反応させる。反応終了後、超純水で２０秒間×３回水洗する。乾燥は２５℃にセットした乾燥機で３０分間乾燥する。実施例１の（２）と同様の評価を行い蛍光強度３６００を得た。
【００９３】
参考例２：試料ＤＮＡ断片の検出
（１）松浪ガラス製ＤＮＡ断片固定化担体の作製
３’末端がアミノ基で修飾された４０ｍｅｒのＤＮＡ断片（３’−ＴＣＣＴＣＣＡＴＧＴＣＣＧＧＧＧＡＧＧＡＴＣＴＧＡＣＡＣＴＴＣＡＡＧＧＴＣＴＡＧ−５’）を０．１Ｍ炭酸緩衝液（ｐＨ９．３）に分散してなる水性液（１×１０^−６Ｍ、１μＬ）を、実施例３の（１）で得た凹凸性固相担体に点着した。直ちに、点着後の固相担体を２５℃、湿度９０％にて１時間放置した後、この固相担体を０．１重量％ＳＤＳ（ドデシル硫酸ナトリウム）と２×ＳＳＣ（２×ＳＳＣ：ＳＳＣの原液を２倍に希釈した溶液、ＳＳＣ：標準食塩−クエン酸緩衝液）との混合溶液で２回、０．２×ＳＳＣ水溶液で１回順次洗浄した。次いで、上記の洗浄後のスライドを０．１Ｍグリシン水溶液（ｐＨ１０）中に１時間３０分浸積した後、蒸留水で洗浄し、室温で乾燥させ、ＤＮＡ断片が固定された固相担体（Ｄ）を得た。
【００９４】
（２）試料ＤＮＡ断片の検出
５’末端にＣｙ５が結合した２２ｍｅｒの試料オリゴヌクレオチド（ＣＴＡＧＴＣＴＧＴＧＡＡＧＴＴＣＣＡＧＡＴＣ−５’）をハイブリダイゼーション用溶液（４×ＳＳＣおよび１０重量％のＳＤＳの混合溶液）（２０μＬ）に分散させたものを、上記（１）で得た固相担体（Ｄ）に点着し、表面を顕微鏡用カバーガラスで保護した後、モイスチャンバー内にて６０℃で２０時間インキュベートした。次いで、このものを０．１重量％ＳＤＳと２×ＳＳＣとの混合溶液、０．１重量％ＳＤＳと０．２×ＳＳＣとの混合溶液、および０．２×ＳＳＣ水溶液で順次洗浄した後、６００ｒｐｍで２０秒間遠心し、室温で乾燥した。スライドガラス表面の蛍光強度を蛍光スキャニング装置で測定したところ、１５３６０であった。
【００９５】
【発明の効果】
本発明の固定方法を利用することによって、凹凸を有する固相担体の表面に、オリゴヌクレオチド、ポリヌクレオチド、あるいはペプチド核酸などのヌクレオチド誘導体あるいはその類縁体ヌクレオチドのプローブを高密度かつ安定に固定することができる。従って、本発明の固定方法によって作製されたヌクレオチド誘導体もしくはその類縁体が固定された固相担体は、加水分解によるプローブの離脱が起こりにくい非常に安定な固相担体となる。特に、固相担体として、その表面にアミノ基等をシランカップリング剤を用いて導入した場合には、アミノ基等の固相担体表面への結合も、プローブの結合も共に共有結合であるため、固相担体上に強固にプローブを固定することができる。プローブの安定な固定により、遺伝子解析等に有効に利用することができる高い検出限界を有する検出用具を得ることができる。
【００９６】
その一つの例として、本発明によって作製されたオリゴヌクレオチド固定固相担体を用いて、試料核酸断片とのハイブリダイゼーションを行なうことにより、オリゴヌクレオチド固定固相担体に固定されているプローブに相補性を有する核酸断片試料を感度よく検出することができる。また、オリゴヌクレオチドなどのプローブ試料を反応性固相担体の表面に点着後、グリシン等のアニオン性化合物で固相担体表面を処理することによって、試料核酸断片の非特的吸着を防ぐことができ、このことは相補性を有する核酸断片試料の高感度の検出に大きな効果を発揮する。
【００９７】
【配列表】

【図面の簡単な説明】
【図１】図１は、本発明の代表的なオリゴヌクレオチド固定固相担体および本発明の代表的なオリゴヌクレオチドの固定方法を示す模式図である。
【図２】図２は、実施例１の固定方法を示す模式図である。[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a detection tool useful for analyzing the structure of a biopolymer, and particularly useful for efficient analysis of gene expression, mutation, polymorphism, etc., and a large number of biopolymers or their analogs. The present invention relates to a detection tool in which is aligned and fixed on the surface of a solid support having irregularities. In particular, the present invention is a high-density array-type detection tool (DNA detection) in which a large number of nucleotide derivatives or analogs thereof are aligned and fixed at high density on a surface of a solid support having irregularities, which is useful for analyzing the base sequence of a DNA fragment sample. Chip), and a reactive solid phase carrier that can be advantageously used for producing the high-density array-type detection device.
[0002]
[Prior art]
Technology development for efficiently analyzing the gene functions of various organisms is progressing rapidly, and a large number of DNA fragments or synthetic fragments called DNA chips are used to analyze the base sequences of those DNAs or DNA fragments. A detection tool in which a nucleotide derivative such as an oligonucleotide is immobilized on the surface of a solid substrate is used. Such a detection molecule such as DNA or a fragment thereof or a synthetic oligonucleotide, such as a nucleotide derivative bound and immobilized on the surface of a solid substrate, is also called a probe molecule. A typical DNA chip is a microarray in which a large number of probe molecules are aligned and fixed on a solid support such as a slide glass. The technology relating to the production and use of DNA chips is considered to be applicable to the detection of biomolecules other than DNA, and therefore, is useful for drug discovery research, disease diagnosis and development of preventive methods. It is expected to provide a means.
[0003]
The DNA chip-related technology has been embodied since the invention of a method for determining the nucleotide sequence of DNA by hybridization with an oligonucleotide. Although this method could overcome the limitations of the base sequencing method using gel electrophoresis, it was not practical at first.
[0004]
Thereafter, a DNA chip having the above-described configuration and a technique for producing the same were developed, and it became possible to efficiently examine gene expression, mutation, polymorphism, and the like in a short time. That is, a DNA fragment sample (also referred to as a target DNA fragment) showing complementarity with a DNA fragment or oligonucleotide on a prepared DNA chip is generally prepared by combining a DNA fragment or oligonucleotide on a DNA chip with labeled DNA. Detection is performed using hybridization with the fragment sample.
[0005]
In order to put the DNA chip production technology into practical use, a technology for stably arranging a large number of DNA fragments and oligonucleotides on the surface of a solid support at high density is required.
[0006]
As a method for preparing a DNA chip, a method of directly synthesizing an oligonucleotide on the surface of a solid support (referred to as an “on-chip method”), or a method in which a DNA fragment or oligonucleotide prepared and prepared in advance is fixed to the surface of a solid support Methods and are known. The on-chip method combines the use of protecting groups that are selectively removed by light irradiation with the photolithography and solid-phase synthesis techniques used in semiconductor manufacturing to produce oligonucleotides in a predetermined small matrix area. A typical method is to selectively combine (hereinafter, referred to as "masking technique").
[0007]
The following methods are known as methods for binding and immobilizing a DNA fragment or oligonucleotide prepared and prepared in advance on the surface of a solid support, depending on the type of the DNA fragment or the type of the solid support.
(1) When the DNA fragment to be fixed is a cDNA (complementary DNA synthesized using mRNA as a template) or a PCR product (a DNA fragment obtained by amplifying cDNA by PCR), the cDNA or PCR product is transferred to a DNA chip. The spotter device provided in the production device spots the surface of the solid support that has been surface-treated with a polycationic compound (polylysine, polyethyleneimine, etc.), and uses the charge of the DNA fragment to electrostatically charge the solid support. It is common to combine. As a method for treating the surface of the solid support, a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group, or the like is also used. In the surface treatment using the silane coupling agent, amino groups, aldehyde groups, and the like are fixed to the surface of the solid support by covalent bonds, so that the surface treatment is more stable than in the case of the surface treatment using a polycationic compound. Immobilized on the phase carrier surface.
[0008]
As a modification of the above-described method utilizing the charge of the DNA fragment, a PCR product modified with an amino group is suspended in SSC (standard salt-citrate buffer), and this is spotted on the surface of a silylated slide glass. After the incubation, a method of sequentially performing a treatment with sodium borohydride and a heat treatment is reported. However, this fixing method has a problem that it is not always possible to obtain sufficient fixing stability of a DNA fragment. In DNA chip technology, the detection limit is important. Therefore, a sufficient amount (ie, high density) and stable fixation of the DNA fragment on the surface of the solid support can improve the detection limit of hybridization between the DNA fragment probe and the labeled sample nucleic acid fragment. Affects directly.
[0009]
(2) When the oligonucleotide (probe molecule) to be fixed is a synthetic oligonucleotide, first, an oligonucleotide having a reactive group introduced therein is synthesized, and the surface of the solid support is treated in advance to form a reactive group. A method is also known in which the oligonucleotide is spotted on a solid support and covalently immobilized on the surface of the solid support. For example, a method in which an amino group-introduced oligonucleotide is reacted with a slide glass having an amino group introduced into the surface thereof in the presence of PDC (p-phenylenediisothiocyanate), and an aldehyde group-introduced oligonucleotide is reacted with the slide glass. Methods are known. These two methods have an advantage that the oligonucleotide is stably fixed to the surface of the solid-phase carrier as compared with the above-mentioned method (1) in which the charge of the DNA fragment is used to fix the DNA fragment by electrostatic bonding. However, in the method in which PDC is present, the reaction between PDC and an amino group-introduced oligonucleotide is slow, and in the method using an aldehyde group-introduced oligonucleotide, the stability of the reaction product, the Schiff base, is low (hence, hydrolysis). Tends to occur).
[0010]
In recent years, instead of oligonucleotides or polynucleotides (including synthesized oligonucleotides or polynucleotides and DNA molecules and DNA fragments, and also RNA molecules and RNA fragments) as probe molecules of a DNA chip, PNA (peptide A technique using an oligonucleotide analog called “nucleic acid” has also been proposed. As a method for immobilizing PNA to a solid phase substrate by covalent bonding, a method using a combination of avidin and biotin is also known (JP-A-11-332595). This publication also describes a technique of using a surface plasmon resonance (SPR) biosensor as a solid substrate. Using a DNA chip having probe molecules immobilized on a surface plasmon resonance biosensor, a DNA fragment bonded and immobilized on its surface via hybridization can be detected using the surface plasmon resonance phenomenon.
[0011]
It is also known to use a charge-coupled device (CCD) as a substrate for a DNA chip (Nucleic Acids Research, 1994, Vol. 22, No. 11, 2124-2125).
[0012]
JP-A-4-228076 (corresponding to U.S. Pat. No. 5,387,505) discloses a technique for separating a target DNA by binding a target DNA with a biotin molecule to a substrate having an avidin molecule immobilized on the surface. Has been described.
[0013]
Japanese Patent Publication No. 7-43380 (corresponding to U.S. Pat. No. 5,094,962) discloses a detection tool for use in a ligand-receptor assay, in which a surface of a microporous polymer particle having a reactive group on the surface is received. Analytical devices having bound body molecules are described.
[0014]
[Problems to be solved by the invention]
The present invention is particularly advantageous in that a previously prepared oligonucleotide derivative such as an oligonucleotide, a polynucleotide, or a peptide nucleic acid or an analog thereof can be stably bonded and fixed at a high density on a solid support surface. It is intended to provide a solid phase carrier, and a tool for detecting DNA, RNA, or a fragment thereof having a specific base sequence portion, in which a nucleotide derivative or an analog thereof is fixed and bound to the reactive solid phase carrier. , The subject.
[0015]
[Means for Solving the Problems]
According to the present invention, a group of vinylsulfonyl groups (sometimes referred to as a vinylsulfone group or a sulfonylvinyl group) or a reactive precursor group thereof is provided on the surface of a solid support having irregularities formed by an organic substance. It is a reactive solid support that is covalently bonded and fixed via a linking group.
The organic substance is preferably a polymer or an aggregate of the polymer.
It is desirable that the linked body of the vinyl sulfonyl group or its reactive precursor group and the linking group is represented by the following formula (1).
[0016]
-L-SO₂-X (1)
[0017]
[In the above formula, X is -CR¹= CR²R³Or -CHR¹-CR²R³Represents Y; R¹, R²And R³Are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a total of 7 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. Y represents a halogen atom, -OSO, or an aralkyl group selected from the group consisting of₂R¹¹, -OCOR¹², -OSO₃M and an atom or group selected from the group consisting of a quaternary pyridinium group;¹¹Is composed of an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. R represents a group selected from the group¹²Represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; and M represents a group consisting of a hydrogen atom, an alkali metal atom and an ammonium group. And L represents a linking group].
[0018]
In the above formula (1), X is -CH = CH₂Is preferably a vinyl group represented by
[0019]
L is preferably a linking group containing a divalent or higher valent atom other than a carbon atom. L is preferably a linking group having a linking site such as -NH-, -S-, or -O-. As an example of L,-(L¹)_n-NH- (CR¹R²)₂-Or-(L¹)_n-S- (CR¹R²)₂-[However, R¹And R²Represents the same meaning as described above, and L¹Represents a linking group, and n is 0 or 1.]. In particular, if L is-(L¹)_n-NHCH₂CH₂− [However, L¹Represents a linking group, and n is 0 or 1.]. L above¹Is preferably a linking group containing a group represented by -OSi-.
[0020]
The reactive solid support of the present invention is obtained by adding the following formula (2) to a solid support having irregularities formed by an organic substance having a reactive group introduced on its surface.
[0021]
X¹-SO₂-L²-SO₂-X²
[0022]
[In the above formula, X¹And X²Are independently of each other,¹= CR²R³Or -CHR¹-CR²R³Y (reactive precursor group); R¹, R²And R³Are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a total of 7 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. Y represents a halogen atom, -OSO, or an aralkyl group selected from the group consisting of₂R¹¹, -OCOR¹², -OSO₃M and an atom or group selected from the group consisting of a quaternary pyridinium group;¹¹Is composed of an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. R represents a group selected from the group¹²Represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; and M represents a group consisting of a hydrogen atom, an alkali metal atom and an ammonium group. Represents an atom or group selected from²Represents a linking group]
Can be easily produced by contacting the disulfone compound represented by
[0023]
A typical example of the disulfone compound represented by the above formula (2) is 1,2-bis (vinylsulfonylacetamido) ethane.
[0024]
The reactive group introduced on the surface of the solid support is preferably an amino group, a mercapto group or a hydroxyl group.
[0025]
The present invention also provides a reactive solid support having a group of vinylsulfonyl groups or reactive precursor groups thereof, each of which is immobilized by a covalent bond. Contacting a nucleotide derivative having a group or an analog thereof, and a method for producing a solid support in which the nucleotide derivative or an analog thereof is bound and fixed to the support surface via a sulfonyl group-containing linking group. is there.
[0026]
Representative examples of nucleotide derivatives or their analogs immobilized on a solid support having irregularities include oligonucleotides, polynucleotides, and peptide nucleic acids. These nucleotide derivatives or analogs thereof include a vinylsulfonyl group such as an amino group, an imino group, a hydrazino group, a carbamoyl group, a hydrazinocarbonyl group, or a carboxyimide group at or near one end of the molecule. Those having a reactive group that forms a covalent bond that reacts with the group or its reactive precursor group are utilized.
[0027]
The above-mentioned solid support having irregularities on which the nucleotide derivative or its analog is immobilized may be an oligonucleotide or polynucleotide (DNA or its DNA) exhibiting complementarity to the immobilized nucleotide derivative or its analog in the presence of an aqueous medium. The complementary oligonucleotide or polynucleotide can be immobilized by contacting the fragment or RNA or a fragment thereof to cause hybridization. The complementary oligonucleotide or polynucleotide to be immobilized is desirably bound to a detectable label (eg, a fluorescent label) so that the immobilization can be detected from the outside.
The present invention also utilizes a solid support having irregularities in which the above-described nucleotide derivative or analog thereof is bonded and fixed to the surface of the support, or a solid support to which the above-described complementary oligonucleotide or polynucleotide is fixed and bonded. The present invention also relates to a method for identifying or screening a gene, characterized in that:
[0028]
BEST MODE FOR CARRYING OUT THE INVENTION
The reactive solid phase carrier of the present invention has a group of vinylsulfonyl groups or its reactive precursor groups bonded and immobilized on the surface of a solid phase carrier having irregularities formed by an organic substance via a linking group, respectively. It has the structure which was done. The reactive solid support of the present invention is prepared by preparing a solid support having irregularities in which a reactive group is introduced into the surface in advance, and reacting with the solid support to react with the reactive group provided on the support surface. Contacting a compound having a reactive group forming a bond at or near one end and having a vinylsulfonyl group or a reactive precursor group of a vinylsulfonyl group near or at the other end It can be manufactured by the following.
[0029]
The solid support is preferably a substrate having a particularly smooth surface with low hydrophobicity or low hydrophilicity. In addition, a substrate having a surface with unevenness and low flatness can be used. Examples of the material of the solid support include glass, cement, ceramics such as ceramics or new ceramics, polyethylene terephthalate, cellulose acetate, polycarbonate such as bisphenol A, polymers such as polystyrene and polymethyl methacrylate, silicon, activated carbon, porous glass, and porous. Examples include various porous substances such as ceramics, porous silicon, porous activated carbon, woven / knitted fabric, nonwoven fabric, filter paper, short fiber, and membrane filter. The pore size of the porous material is preferably in the range of 2 to 1000 nm, particularly preferably in the range of 2 to 500 nm. The material of the solid support is particularly preferably glass or silicon. This is due to the ease of surface treatment and the ease of analysis by electrochemical methods. The thickness of the solid support is preferably in the range of 100 to 2000 μm.
[0030]
Examples of the solid support having concavities and convexities used for the production of the reactive solid support of the present invention include various solid supports conventionally used for the production of DNA chips or proposed for the production of DNA chips. It can be preferably used. Examples of such solid carriers include a glass substrate, a resin substrate, a glass or resin substrate surface-treated with a silane coupling agent, or a glass or resin substrate having a coating layer on the surface. . The solid support is particularly preferably a silicate glass substrate, a silicate glass substrate surface-treated with a silane coupling agent, or a silicate glass substrate coated with an organic coating layer. An electrode substrate used as a substrate for a DNA chip used in an electrochemical analysis method may be used. Further, various functional substrates such as the above-described substrate for surface plasmon resonance (SPR) biosensor and charge-coupled device (CCD) may be used. Further, in addition to these substrates, a particulate solid support or the like can also be used.
Examples of the uneven organic substance include monosaccharides such as glucose and fructose or derivatives thereof, and polysaccharides such as dextran, amylose and starch or derivatives thereof, for example, carboxymethyl starch. Polymer polymers such as PVA, polyacrylic acid, cellulose, carboxymethyl cellulose, and polyacetal are exemplified. In particular, when an organic substance having a hydroxyl group is used in combination with a silane coupling agent, an aggregate can be formed, and unevenness can be easily formed. The size (height) of the unevenness is preferably 50 μm or less, and in particular, in the case of confocal laser fluorescence detection, it is preferable to use fine particles of 100 nm or less in order to suppress the background.
When an aminated silane coupling agent is used, an amino group can be introduced into the surface simultaneously with the unevenness. It is also possible to first form irregularities using TMOS and TEOS, and then react an aminated silane coupling agent to introduce an amino group into the surface.
[0031]
A polycationic compound (for example, poly-L-lysine, polyethyleneimine, polyalkylamine, etc.) is used for covalently bonding and fixing a bifunctional reactive compound such as a divinylsulfone compound on the surface of a solid support having irregularities. And the like, and more preferably poly-L-lysine). In this case, the reactive group introduced to the surface of the solid support is an amino group. Group). Alternatively, the surface of the solid support can be contact-treated with a surface treating agent having a reactive group such as a silane coupling agent that reacts with the surface of the solid support and another reactive group such as an amino group.
[0032]
In the case of a coating treatment with a polycation compound, an amino group or a mercapto group is introduced into the surface of the solid support having irregularities by electrostatic bonding between the polymer compound and the surface of the solid support. In the case of the surface treatment using a silane coupling agent, the amino group or the mercapto group is stably present on the surface of the solid support because the amino group or the mercapto group is covalently bonded and fixed to the surface of the solid support. Besides an amino group and a mercapto group, an aldehyde group, an epoxy group, a carboxyl group, or a hydroxyl group can be preferably introduced.
[0033]
Examples of the silane coupling agent having an amino group include γ-aminopropyltriethoxysilane, N-β (aminoethyl) -γ-aminopropyltrimethoxysilane or N-β (aminoethyl) -γ-aminopropylmethyldimethoxysilane. Is preferred, and γ-aminopropyltriethoxysilane is particularly preferred.
[0034]
The treatment using a polycation compound may be combined with the treatment using a silane coupling agent. By this method, electrostatic interaction between the DNA fragment and the solid support having low hydrophobicity or low hydrophilicity can be promoted. A layer made of a charged hydrophilic polymer or the like or a layer made of a crosslinking agent may be further provided on the surface of the solid support treated with the polycation compound. By providing such a layer, the unevenness of the solid support that has been treated with the polycationic compound can be reduced. Depending on the type of the solid support, a hydrophilic polymer or the like can be contained in the support, and a solid support subjected to such treatment can be preferably used.
[0035]
On the surface of a solid phase carrier for a normal DNA chip, a number of regions are set in advance or are assumed, and as described above, a bifunctional reactive compound such as a divinyl sulfone compound is provided for each region. A reactive group capable of reacting is introduced in advance. The surface of each region of the solid phase carrier to be used is provided with a reactive group such as the above-mentioned amino group, mercapto group, or hydroxyl group. As described above, the surface treatment with a silane coupling agent, or a method of applying a polymer having a reactive group such as an amino group in a side chain to the surface of the solid support by using a method of coating the reactive group, Introduction is performed.
[0036]
The solid support having a reactive group is contacted with a bifunctional reactive compound such as a divinylsulfone compound, whereby the reactive group and the bifunctional reactive compound react to form a covalent bond, The reactive group portion of the solid support is extended to form a reactive chain having a vinylsulfonyl group or a reactive precursor group at or near the tip thereof, thereby forming the reactive solid support of the present invention. .
[0037]
In the reactive solid support of the present invention, the conjugate of the vinyl sulfonyl group or its reactive precursor group and the linking group introduced on the surface of the solid support is represented by the following formula (1). Is desirable.
[0038]
-L-SO₂-X (1)
[0039]
In the above formula (1), X is -CR¹= CR²R³Or -CHR¹-CR²R³Represents Y (reactive precursor group). R¹, R²And R³Are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, or a total carbon atom having an alkyl chain having 1 to 6 carbon atoms. 7 to 26 aralkyl groups. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-hexyl group, and a methyl group is particularly preferable. . Examples of the aryl group include a phenyl group and a naphthyl group. R¹, R²And R³Are preferably both hydrogen atoms.
[0040]
Y is -OH, -OR⁰, -SH, NH₃, NH₂R⁰(However, R⁰Represents a group which is substituted by a nucleophile such as an alkyl group or the like except for a hydrogen atom) or a group which is eliminated as "HY" by a base, such as a halogen atom, -OSO₂R¹¹, -OCOR¹², -OSO₃M or a quaternary pyridinium group (R¹¹Represents an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, or an aralkyl group having 7 to 26 carbon atoms in total having an alkyl chain having 1 to 6 carbon atoms. R¹²Represents an alkyl group having 1 to 6 carbon atoms or a halogenated alkyl group having 1 to 6 carbon atoms; M represents a hydrogen atom, an alkali metal atom or an ammonium group.
[0041]
L is a solid support or a linking group bonded to the solid support, and -SO₂-Represents a divalent or higher linking group linking to the -X group. However, L may be a single bond. The divalent linking group includes an alkylene group having 1 to 6 carbon atoms, an aliphatic ring group having 3 to 16 carbon atoms, an arylene group having 6 to 20 carbon atoms, N, S, and P A heterocyclic group having 1 to 3 heteroatoms selected from the group and having 2 to 20 carbon atoms, -O-, -S-, -SO-, -SO₂-, -SO₃-, -NR¹¹It is preferable that the group is a group formed by combining one or more groups selected from the group consisting of-, -CO- and a combination thereof. R¹¹Is a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, an aryl group having 6 to 20 carbon atoms, or an aralkyl group having 7 to 21 carbon atoms having an alkyl group having 1 to 6 carbon atoms. It is more preferably a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and particularly preferably a hydrogen atom, a methyl group or an ethyl group.
L is -NR¹¹-, -SONR¹¹-, -CONR¹¹-, -NR¹¹COO- and -NR¹¹CONR¹¹When the group is a combination of two or more groups selected from the group consisting of¹¹They may combine with each other to form a ring.
[0042]
R¹¹An alkyl group of R¹¹An aryl group of¹¹May have a substituent. Examples of such a substituent include a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, a carbamoyl group having 2 to 7 carbon atoms, and a carbamoyl group having 1 to 6 carbon atoms. Alkyl groups, aralkyl groups having 7 to 16 carbon atoms, aryl groups having 6 to 20 carbon atoms, sulfamoyl groups (or Na salts, K salts, etc.), sulfo groups (or Na salts, K salts, etc.) ), A carboxylic acid group (or a Na salt or a K salt thereof), a halogen atom, an alkenylene group having 1 to 6 carbon atoms, an arylene group having 6 to 20 carbon atoms, a sulfonyl group, and a combination thereof. Examples include atoms or groups selected from the group.
[0043]
Preferred specific examples of the above-mentioned "-X" group are shown below. Also, "-L-SO₂Examples of groups that can be used as "-X" are also shown later.
[0044]
Embedded image

[0045]
Embedded image

[0046]
“−X” is preferably (X1), (X2), (X3), (X4), (X7), (X8), (X13) or (X14) in the above specific examples, and (X1) ) Or (X2). Particularly preferred is a vinyl group represented by (X1).
[0047]
Preferred specific examples of L are shown below. However, a is an integer of 1 to 6, preferably 1 or 2, and particularly preferably 1. b is an integer of 0 to 6, and is preferably 2 or 3.
[0048]
Embedded image

[0049]
As L, in addition to the divalent linking group described above, the hydrogen atom of the alkylene group of the above formula is -SO₂CH = CH₂A group substituted by a group is also preferable.
[0050]
The bifunctional reactive compound used to obtain a solid support to which a vinylsulfonyl group or a reactive precursor group represented by the above formula (1) is fixed by a covalent bond is represented by the following formula (2). The disulfone compounds represented can be advantageously used.
[0051]
X¹-SO₂-L²-SO₂-X²      (2)
[0052]
[In the above formula, X¹And X²Are independently of each other,¹= CR²R³Or -CHR¹-CR²R³Y (reactive precursor group); R¹, R²And R³Are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a total of 7 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. Y represents a halogen atom, -OSO, or an aralkyl group selected from the group consisting of₂R¹¹, -OCOR¹², -OSO₃M and an atom or group selected from the group consisting of a quaternary pyridinium group;¹¹Is composed of an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. R represents a group selected from the group¹²Represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; and M represents a group consisting of a hydrogen atom, an alkali metal atom and an ammonium group. Represents an atom or group selected from²Represents a linking group].
[0053]
That is, the reactive solid phase carrier of the present invention can be easily produced by bringing the disulfone compound represented by the above formula (2) into contact with the solid phase carrier, for example, in an aqueous atmosphere.
[0054]
Representative examples of the disulfone compound preferably used in the present invention are shown below. Note that two or more disulfone compounds may be used as a mixture.
[0055]
Embedded image

[0056]
Embedded image

[0057]
As a typical example of the disulfone compound represented by the above formula (2), 1,2-bis (vinylsulfonylacetamido) ethane [corresponding to S1 described above] can be given.
[0058]
Various methods for synthesizing the disulfone compound used in the present invention are described in, for example, JP-B-47-2429, JP-B-50-35807, JP-A-49-24435, JP-A-53-41551, and JP-A-59-18944. The details are described in.
[0059]
Using the reactive solid phase carrier obtained as described above, a detection tool (generally a DNA chip) for detecting and fixing a naturally occurring polynucleotide or oligonucleotide such as DNA, RNA, DNA fragment or RNA fragment. The above-mentioned reactive solid-phase carrier is prepared by reacting the above-mentioned reactive solid-phase carrier with a vinylsulfonyl group on the surface of the carrier or an amino group which forms a covalent bond by reacting with the reactive precursor group. A method of contacting with a nucleotide derivative having a reactive group or a derivative thereof is used. That is, a detection tool (so-called DNA chip) provided with a probe molecule comprising a desired nucleotide derivative or an analog thereof can be prepared in this manner.
[0060]
The vinylsulfonyl group or its reactive precursor group bonded via a covalent bond to the surface of the solid-phase substrate of the present invention has high resistance to hydrolysis, so that it can be easily and stably stored. And has an amino group in advance, or a reactive group such as an amino group is introduced, or reacts quickly with the reactive group of the nucleotide derivative or its analog to form a stable covalent bond. be able to.
[0061]
Representative examples of nucleotide derivatives or analogs thereof used as probe molecules include oligonucleotides, polynucleotides, and peptide nucleic acids. These nucleotide derivatives or analogs thereof may be of natural origin (DNA, DNA fragment, RNA, or RNA fragment, etc.) or may be synthetic compounds. Examples of the nucleotide derivative or its analog include various analogous compounds such as a compound called LNA having a cross-linking group in its sugar unit (described in J. Am. Chem. Soc. 1998, 120, 13252-13253). included.
[0062]
When a DNA fragment is used as a probe molecule, it can be divided into two types depending on the purpose. In order to examine gene expression, it is preferable to use a polynucleotide such as cDNA, a part of cDNA, or EST. These polynucleotides may be of unknown function, but are generally amplified by PCR using a cDNA library, a genomic library or a whole genome as a template based on sequences registered in a database. (Hereinafter referred to as “PCR product”). Those not amplified by the PCR method can also be preferably used. In order to examine gene mutations and polymorphisms, it is preferable to synthesize various oligonucleotides corresponding to the mutations and polymorphisms based on a known sequence as a standard, and use these. Furthermore, when the purpose is to analyze the base sequence, 4ⁿIt is preferable to synthesize (n is the length of a base) kinds of oligonucleotides and use them. The base sequence of the DNA fragment is preferably determined in advance by a general base sequence determination method. The DNA fragment is preferably a 2- to 50-mer, and particularly preferably a 10- to 25-mer.
[0063]
A reactive group that forms a covalent bond by reacting with the above-mentioned vinylsulfonyl group or its reactive precursor group is introduced into one end of a nucleotide derivative such as an oligonucleotide or a DNA fragment or an analog thereof. Such a reactive group is preferably an amino group, an imino group, a hydrazino group, a carbamoyl group, a hydrazinocarbonyl group, or a carboxyimide group, and particularly preferably an amino group. These reactive groups are usually bound to an oligonucleotide or a DNA fragment via a crosslinker. As the crosslinker, for example, an alkylene group or an N-alkylamino-alkylene group is used, but a hexylene group or an N-methylamino-hexylene group is preferable, and a hexylene group is particularly preferable. In addition, since the peptide nucleic acid (PNA) has an amino group, it is not usually necessary to separately introduce another reactive group.
[0064]
The contact between the nucleotide derivative having a reactive group or an analog thereof and the reactive solid support is usually carried out by spotting an aqueous solution of the nucleotide derivative or an analog thereof on the surface of the reactive solid support. You. Specifically, after dissolving or dispersing a nucleotide derivative having a reactive group or an analog thereof in an aqueous medium to form an aqueous liquid, the aqueous liquid is dispensed into a 96-well or 384-well plastic plate. It is preferable that the injection is performed by dropping the poured aqueous liquid onto the surface of the solid support using a spotter or the like.
[0065]
In order to prevent drying of the nucleotide derivative or its analog after spotting, a substance having a high boiling point may be added to an aqueous liquid in which the nucleotide derivative or its analog is dissolved or dispersed. The substance having a high boiling point is a substance which can be dissolved in an aqueous liquid in which a nucleotide derivative or an analog thereof is dissolved or dispersed after spotting, and is used as a sample such as a nucleic acid fragment sample (target nucleic acid fragment) to be detected. It is preferable that the substance does not hinder the hybridization and has a not so large viscosity. Such materials can include glycerin, ethylene glycol, dimethyl sulfoxide, and low molecular weight hydrophilic polymers. Examples of the hydrophilic polymer include polyacrylamide, polyethylene glycol, and sodium polyacrylate. The molecular weight of this polymer is 10³To 10⁶Is preferably within the range. As a substance having a high boiling point, glycerin or ethylene glycol is more preferably used, and glycerin is particularly preferably used. The concentration of the substance having a high boiling point is preferably in the range of 0.1 to 2% by volume, particularly preferably in the range of 0.5 to 1% by volume, in the aqueous liquid of the nucleotide derivative or its analog.
[0066]
For the same purpose, it is also preferable to place the solid support after spotting the nucleotide derivative or its analog in an environment having a humidity of 90% or more and a temperature range of 25 to 50 ° C.
[0067]
After spotting the nucleotide derivative having a reactive group or its analog, post-treatment with ultraviolet light, sodium borohydride or a Schiff reagent may be performed. These post-treatments may be performed in combination of a plurality of types, and particularly preferably performed in combination of the heat treatment and the ultraviolet treatment. These post-treatments are particularly effective when the surface of the solid support is treated only with the polycationic compound. After spotting, it is also preferable to carry out incubation. After incubation, it is preferable to wash and remove unreacted nucleotide derivatives or analogs thereof.
[0068]
The fixed amount (number) of the nucleotide derivative or its analog is 10²To 10⁵Kind / cm²Is preferably within the range. The amount of the nucleotide derivative or its analog is 1 to 10^-15It is preferably in the range of mol, and the weight is preferably several ng or less. By the spotting, the aqueous liquid of the nucleotide derivative or its analog is fixed in the form of dots on the surface of the solid support. The shape of the dot is almost circular. The absence of variation in shape is important for quantitative analysis of gene expression and analysis of single nucleotide mutation. The distance between each dot is preferably in the range of 0 to 1.5 mm, particularly preferably in the range of 100 to 300 μm. The size of one dot is preferably in the range of 50 to 300 μm in diameter. The amount of the nucleotide derivative or the analog thereof spotted on the surface of the solid support is preferably in the range of 100 pL to 1 μL, and particularly preferably in the range of 1 to 100 nL.
[0069]
FIG. 1 schematically shows a method for producing an oligonucleotide-immobilized solid phase carrier, which is a typical embodiment of the present invention, and a configuration of a typical oligonucleotide-immobilized solid phase carrier.
As a method for producing a solid support having irregularities on which the oligonucleotide of the present invention is immobilized, when a disulfone compound represented by the above formula (2) is used, X¹And X²Four types of manufacturing methods can be used.
[0070]
FIG. 1 shows the X of the disulfone compound of the formula (2).¹And X²Are both -CHR¹-CR²R³Method (a) for producing solid support (C1) on which oligonucleotide is immobilized in the case of Y (reactive precursor group), and X¹Is -CHR¹-CR²R³Y and X²Is -CR¹= CR²R³The production method (b) of the oligonucleotide-immobilized solid phase carrier (C2) in the case of is shown. Where X¹Is assumed to be a group that first reacts with the reactive group (R) introduced on the surface of the solid support 1, and¹To -CR¹= CR²R³Manufacturing method. Hereinafter, "X¹Is described as a group that first reacts with the reactive group (R) introduced on the surface of the solid support 1.
[0071]
The manufacturing methods (a) and (b) will be described.
Step (1): A disulfone compound represented by the formula (1) is brought into contact with a solid support 1 [solid support (A)] having a reactive group (R) introduced on the support surface, and a -Y portion of X1 By substituting a reactive group (R) for-(CR¹R²)_n-SO₂-L-SO₂-X²Groups are introduced on the surface of the solid support.
[0072]
Step (2):-(CR) introduced in step (1)¹R²)_n-SO₂-L-SO₂-X²Group X²To a reactive group (Z) by contacting the oligonucleotide 2 having a reactive group (Z) at one end, or²The reactive group (Z) is displaced by bringing the oligonucleotide 2 into contact with -Y.
[0073]
The carrier for immobilizing a probe molecule of the present invention comprises X¹Is a group that first reacts with the reactive group (R) introduced on the surface of the solid support 1,¹To -CR¹= CR²R³May be manufactured by the following method.
[0074]
Step (1): The disulfone compound represented by the formula (I) is brought into contact with the solid support (A) in which the active group (R) is introduced on the surface of the solid support 1 having irregularities, and X¹-CR¹= CR²R³By adding a reactive group (R) to³R²C-R¹HC-SO₂-L-SO₂-X²Groups are introduced on the surface of the solid support.
[0075]
Step (2): -R introduced in step (1)³R²C-R¹HC-SO₂-L-SO₂-X²Group X²To the reactive group (Z) by contacting an oligonucleotide having a reactive group (Z) at one end,²The reactive group (Z) is substituted by bringing the oligonucleotide into contact with the -Y portion of the above.
[0076]
The oligonucleotide having a reactive group (Z) at one end is a compound indicated by 2 in FIG. The crosslinker (Q) is not essential, but is generally present between the reactive group (Z) and the phosphate group for the purpose of preparing the reactive oligonucleotide 2. -Phosphate group -NNNN ... NN represents an oligonucleotide. R⁴Represents a reactive group (R) and X¹Reaction with Z¹Is X²And the reactive group (Z).
[0077]
When the oligonucleotide 2 having the reactive group (Z) is spotted on the surface of the solid support (B) having irregularities in FIG.²Or X²Of the reactive oligonucleotide fragment 2 occurs on the surface of the solid support (B), but unreacted X to which the oligonucleotide 2 is not bound²Also exists. X like this²May cause a non-specific reaction in the subsequent hybridization with a labeled nucleic acid fragment sample, which may result in measurement of non-specific binding.²(Ie, for example, X²Is preferably masked. The mask treatment is preferably performed by bringing an anionic compound having an amino group or a mercapto group into contact with the surface of the solid support (C1) (or (C2)). Since the oligonucleotide 2 has a negative charge, by generating a negative charge also on the surface of the solid support (C), the unreacted X²Can be prevented from reacting. Such anionic compounds include X²Reacts with a halogen atom of⁻, SO₃ ⁻, OSO₃ ⁻, PO₃ ⁻Or PO₂ ⁻) Can be used as long as it has (a), but is preferably an amino acid, and particularly preferably glycine or cysteine. Taurine can also be preferably used.
[0078]
The life span of the solid phase carrier to which the nucleotide derivative or its analog produced by the method of the present invention is immobilized is usually several weeks for the cDNA immobilized solid phase carrier in which cDNA is immobilized, and the synthetic oligonucleotide is immobilized. The longer the solid support, the longer it is. The solid-phase carrier on which the nucleotide derivative or its analog of the present invention is immobilized is used for gene expression monitoring, nucleotide sequence determination, mutation analysis, polymorphism analysis, and the like. The principle of detection is hybridization with a labeled sample nucleic acid fragment described below.
[0079]
As a labeling method, an RI method and a non-RI method (fluorescence method, biotin method, chemiluminescence method, etc.) are known, but it is preferable to use a fluorescence method in the present invention. As the fluorescent substance used for the fluorescent labeling, any substance can be used as long as it can bind to the base portion of the nucleic acid. Cyanine dyes (for example, commercially available Cy Dye)^TMSeries Cy3, Cy5, etc.), rhodamine 6G reagent, N-acetoxy-N²-Acetylaminofluorene (AAF) or AAIF (iodine derivative of AAF) can be used.
[0080]
As the sample nucleic acid fragment, a nucleic acid fragment sample such as a DNA fragment sample or an RNA fragment sample whose sequence or function is unknown is usually used.
[0081]
The nucleic acid fragment sample is preferably isolated from eukaryotic cell or tissue samples for the purpose of examining gene expression. If the sample is a genome, it is preferably isolated from any tissue sample except red blood cells. Preferably, any tissue other than red blood cells is peripheral blood lymphocytes, skin, hair, semen and the like. If the sample is mRNA, it is preferably extracted from a tissue sample in which mRNA is expressed. The mRNA is labeled by incorporating a labeled dNTP ("dNTP" means a deoxyribonucleotide whose base is adenine (A), cytosine (C), guanine (G) or thymine (T)) by a reverse transcription reaction. Preferably, it is cDNA. As dNTP, it is preferable to use dCTP for chemical stability. The amount of mRNA required for one hybridization varies depending on the amount of liquid to be spotted and the labeling method, but is several μg or less. When the DNA fragment on the solid support having immobilized nucleotide derivatives or analogs thereof is an oligo DNA, it is desirable that the nucleic acid fragment sample be reduced in molecular weight. In prokaryotic cells, since it is difficult to selectively extract mRNA, it is preferable to label total RNA.
[0082]
For the purpose of examining gene mutation or polymorphism, a nucleic acid fragment sample is preferably obtained by performing PCR of a target region in a reaction system containing a labeled primer or a labeled dNTP.
[0083]
Hybridization is performed by dissolving or dispersing a labeled nucleic acid fragment sample in a 96-well or 384-well plastic plate on a solid support on which the nucleotide derivative of the present invention or its analog is immobilized. It is preferably carried out by spotting on top. The amount of spotting is preferably in the range of 1 to 100 nL. Hybridization is preferably carried out in a temperature range from room temperature to 70 ° C. and for a time in the range from 6 to 20 hours. After the completion of the hybridization, it is preferable to wash with a mixed solution of a surfactant and a buffer to remove an unreacted nucleic acid fragment sample. It is preferable to use sodium dodecyl sulfate (SDS) as the surfactant. As the buffer, a citrate buffer, a phosphate buffer, a borate buffer, a Tris buffer, a Good buffer, and the like can be used, but a citrate buffer is particularly preferable.
[0084]
A feature of hybridization using a solid phase carrier on which a nucleotide derivative or an analog thereof is immobilized is that the amount of a labeled nucleic acid fragment sample to be used can be extremely reduced. Therefore, it is necessary to set the optimal conditions for hybridization depending on the chain length of the nucleotide derivative or its analog immobilized on the solid support and the type of the labeled nucleic acid fragment sample. For gene expression analysis, it is preferable to perform hybridization for a long time so that low-expressed genes can be sufficiently detected. For detection of a single nucleotide mutation, it is preferable to perform hybridization for a short time. Another feature is that by preparing two types of nucleic acid fragment samples labeled with different fluorescent substances and simultaneously using them for hybridization, the expression level can be compared and quantified on the same DNA fragment-immobilized solid phase carrier.
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
[0085]
【Example】
Example 1: Preparation of DNA fragment-immobilized slide and measurement of DNA fragment immobilization amount
The preparation of a reactive solid support having irregularities, its performance, and a comparison with a reactive solid support having no irregularities as a comparative control are shown below.
[0086]
(1) Preparation of uneven solid support (A) into which vinylsulfonyl group is introduced
75 mg of polyvinyl alcohol is dissolved in 1000 ml of ultrapure water, and after dissolution, 500 μl of a silane coupling agent (KBE903 manufactured by Shin-Etsu Kagaku) is added, and the mixture is stirred with a magnetic stirrer for 10 minutes. A glass substrate (76 mm × 26 mm) is immersed in this liquid for 30 seconds, then pulled up, cut off excess liquid, and dried with a dryer at 45 ° C. Next, the glass substrate is heated in an oven at 110 ° C. for 10 minutes to fix silane to the glass substrate. Further, after cooling the glass substrate, the slide substrate is reacted with a 3% by weight 1,2-bis (vinylsulfonylacetamide) ethane / pH 8.0 borate buffer solution for 120 minutes using a slide washer. After the completion of the reaction, the resultant is washed with ultrapure water for 20 seconds × 3 times. Drying is performed for 30 minutes using a dryer set at 25 ° C.
[0087]
(2) Spotting of DNA fragments and measurement of fluorescence intensity
The 3′-end and the 5′-end are each an amino group, and a DNA fragment (3′-TCCTCCATGTCCGGGGAGGATCTGACACTCAAGGCTCAG-5 ′) modified with a fluorescent labeling reagent (FluoroLink Cy5-dCTP, manufactured by Amersham Pharmacia Biotech) is used in 0.1M carbonate. Aqueous liquid (1 × 10 3) dispersed in a buffer (pH 9.3)^-6M, 1 μL) was spotted on the slide (A) obtained in the above (1). Immediately after leaving the solid support after spotting at 25 ° C. and 90% humidity for 1 hour, the solid support was combined with 0.1% by weight of SDS (sodium dodecyl sulfate) and 2 × SSC (2 × SSC: SSC). Was washed twice with a solution obtained by diluting the stock solution of 2-fold, SSC: standard salt-citrate buffer) twice, and once with a 0.2 × SSC aqueous solution. Next, the washed slide was immersed in a 0.1 M glycine aqueous solution (pH 10) for 1 hour and 30 minutes, washed with distilled water, dried at room temperature, and solid phase carrier (B) on which DNA fragments were immobilized. ) Got. The fluorescence intensity on the surface of the solid-phase support (B) was measured using a fluorescence scanning apparatus, and was found to be 22,250.
[0088]
(3) Preparation and performance of comparative slides
A glass slide is reacted with 200 ml of a 2% by weight solution of Shin-Etsu Silicone KBE903 (Shin-Etsu Chemical Co., Ltd.) using a commercially available slide washer for 3 minutes. After completion of the reaction, the plate is washed with 200 ml of ultrapure water for 1 minute (using a slide washer). While exchanging the ultrapure water, the process is further repeated twice under the aforementioned washing conditions. After completion of the water washing, the substrate is dried for 10 minutes in a dryer at 45 ° C., and then placed in an oven set at 110 ° C. and heat-treated for 10 minutes. After cooling, the mixture is reacted with 3% by weight of 1,2-bis (vinylsulfonylacetamide) ethane / pH 8.0 borate buffer solution using a slide washer for 120 minutes. After the completion of the reaction, the resultant is washed with ultrapure water for 20 seconds × 3 times. Drying is performed for 30 minutes using a dryer set at 25 ° C. The same evaluation as in the above (2) was performed to obtain a fluorescence intensity of 1750.
From the above results, it can be seen that the DNA fragment was efficiently and efficiently fixed to the slide glass by the immobilization method of the present invention.
[0089]
Example 2: Detection of sample DNA fragment
(1) Preparation of solid support on which uneven DNA fragment is fixed
An aqueous liquid (1 × 10 4) obtained by dispersing a 40-mer DNA fragment (3′-TCCTCCATGTCCGGGGAGGATCTGACACTTCAAGGTCTAG-5 ′) whose 3 ′ end is modified with an amino group in a 0.1 M carbonate buffer (pH 9.3).^-6M, 1 μL) was spotted on the uneven solid support (A) obtained in (1) of Example 1. Immediately after leaving the solid support after spotting at 25 ° C. and 90% humidity for 1 hour, the solid support was combined with 0.1% by weight of SDS (sodium dodecyl sulfate) and 2 × SSC (2 × SSC: SSC). Was washed twice with a solution obtained by diluting the stock solution of 2-fold, SSC: standard salt-citrate buffer) twice, and once with a 0.2 × SSC aqueous solution. Next, the washed slide was immersed in a 0.1 M glycine aqueous solution (pH 10) for 1 hour and 30 minutes, washed with distilled water, dried at room temperature, and solid phase carrier (C) on which DNA fragments were immobilized. ) Got.
[0090]
(2) Detection of sample DNA fragment
A 22-mer sample oligonucleotide (CTAGTCCTGTGAAGTCCAGATC-5 ′) having Cy5 bound to the 5 ′ end dispersed in a hybridization solution (a mixed solution of 4 × SSC and 10% by weight of SDS) (20 μL) was dispersed in the above (20 μL). After being spotted on the solid support (C) obtained in 1), the surface was protected with a cover glass for a microscope, and the mixture was incubated at 60 ° C. for 20 hours in a Moisture chamber. Next, this was sequentially washed with a mixed solution of 0.1% by weight SDS and 2 × SSC, a mixed solution of 0.1% by weight SDS and 0.2 × SSC, and a 0.2 × SSC aqueous solution. Centrifuged at 600 rpm for 20 seconds and dried at room temperature. The fluorescence intensity on the surface of the slide glass was measured by a fluorescence scanning apparatus and was found to be 18400.
[0091]
When a sample DNA fragment was detected using a comparative slide, the fluorescence intensity was 1500.
It can be seen that the use of the solid support on which the uneven DNA fragment is immobilized produced by the immobilization method of the present invention makes it possible to detect a sample DNA fragment complementary to the immobilized DNA fragment with high sensitivity.
[0092]
Reference Example 1: Preparation of DNA fragment-immobilized slide and measurement of DNA fragment immobilization amount
(1) Preparation of uneven solid support with vinylsulfonyl group introduced
Using a slide washer, a coated slide glass for DNA microarray (Type 2 densified amino group-introduced type [Matsunami Glass Industry]) was added to a 3 wt% 1,2-bis (vinylsulfonylacetamide) ethane / pH 8.0 borate buffer solution using a slide washer. Incubate for 120 minutes. After the completion of the reaction, the resultant is washed with ultrapure water for 20 seconds × 3 times. Drying is performed for 30 minutes using a dryer set at 25 ° C. The same evaluation as in (2) of Example 1 was performed to obtain a fluorescence intensity of 3600.
[0093]
Reference Example 2: Detection of sample DNA fragment
(1) Preparation of DNA fragment immobilization carrier made of Matsunami glass
An aqueous liquid (1 × 10 4) obtained by dispersing a 40-mer DNA fragment (3′-TCCTCCATGTCCGGGGAGGATCTGACACTTCAAGGTCTAG-5 ′) whose 3 ′ end is modified with an amino group in a 0.1 M carbonate buffer (pH 9.3).^-6M, 1 μL) was spotted on the uneven solid support obtained in Example 3 (1). Immediately after leaving the solid support after spotting at 25 ° C. and 90% humidity for 1 hour, the solid support was combined with 0.1% by weight of SDS (sodium dodecyl sulfate) and 2 × SSC (2 × SSC: SSC). Was washed twice with a solution obtained by diluting the stock solution of 2-fold, SSC: standard salt-citrate buffer) twice, and once with a 0.2 × SSC aqueous solution. Next, the washed slide was immersed in a 0.1 M glycine aqueous solution (pH 10) for 1 hour and 30 minutes, washed with distilled water, dried at room temperature, and solid phase carrier (D ) Got.
[0094]
(2) Detection of sample DNA fragment
A 22-mer sample oligonucleotide (CTAGTCCTGTGAAGTCCAGATC-5 ′) having Cy5 bound to the 5 ′ end dispersed in a hybridization solution (a mixed solution of 4 × SSC and 10% by weight of SDS) (20 μL) was dispersed in the above (20 μL). After spotting on the solid support (D) obtained in 1) and protecting the surface with a microscope cover glass, the mixture was incubated in a Mois chamber at 60 ° C. for 20 hours. Next, this was sequentially washed with a mixed solution of 0.1% by weight SDS and 2 × SSC, a mixed solution of 0.1% by weight SDS and 0.2 × SSC, and a 0.2 × SSC aqueous solution. Centrifuged at 600 rpm for 20 seconds and dried at room temperature. The fluorescence intensity of the surface of the slide glass was 15360 when measured with a fluorescence scanning device.
[0095]
【The invention's effect】
By using the immobilization method of the present invention, a probe of a nucleotide derivative such as an oligonucleotide, a polynucleotide, or a peptide nucleic acid or a nucleotide probe thereof can be stably fixed at a high density on the surface of a solid support having irregularities. Can be. Therefore, the solid phase carrier to which the nucleotide derivative or its analog produced by the immobilization method of the present invention is immobilized becomes a very stable solid phase carrier in which the detachment of the probe due to hydrolysis does not easily occur. In particular, when an amino group or the like is introduced to the surface of the solid support using a silane coupling agent, both the bond of the amino group to the surface of the solid support and the bond of the probe are covalent bonds. In addition, the probe can be firmly immobilized on the solid support. By stably fixing the probe, it is possible to obtain a detection tool having a high detection limit that can be effectively used for gene analysis and the like.
[0096]
As one example, by using the oligonucleotide-immobilized solid-phase support prepared according to the present invention and performing hybridization with a sample nucleic acid fragment, complementarity with the probe immobilized on the oligonucleotide-immobilized solid-phase support is obtained. Nucleic acid fragment sample can be detected with high sensitivity. In addition, after spotting a probe sample such as an oligonucleotide on the surface of the reactive solid support, and treating the solid support surface with an anionic compound such as glycine, nonspecific adsorption of sample nucleic acid fragments can be prevented. This has a great effect on highly sensitive detection of a nucleic acid fragment sample having complementarity.
[0097]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing a typical oligonucleotide-immobilized solid phase carrier of the present invention and a typical oligonucleotide-immobilizing method of the present invention.
FIG. 2 is a schematic diagram illustrating a fixing method according to the first embodiment.

Claims (21)

A glass substrate, a resin substrate, a glass substrate or a resin substrate surface-treated with a silane coupling agent, and a sheet substrate selected from the group consisting of a glass substrate or a resin substrate having a coating layer on the surface, and a smooth surface. A group of vinylsulfonyl groups or their reactive precursor groups are shared via a linking group on the surface of a solid support obtained by forming irregularities on a substrate by a polymer or an aggregate of a polymer. A reactive solid support immobilized by bonding. The reactive solid phase carrier according to claim 1, wherein the linked product of the vinylsulfonyl group or its reactive precursor group and the linking group is represented by the following formula:
-L-SO ₂ -X
[In the above formula, X represents -CR ¹ = CR ² R ³ or -CHR ¹ -CR ² R ³ Y; R ¹ , R ² and R ³ independently of one another are a hydrogen atom, a carbon atom number; Is an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an atom selected from the group consisting of an aralkyl group having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. or it represents a group; Y is a halogen ^{_{atom, -OSO 2 R 11, -OCOR 12}} , -OSO 3 represents M, and the atom or group selected from the group consisting of quaternary pyridinium group; R ¹¹ has a number of carbon atoms Is a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. the expressed; R ¹² is M represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M is selected from the group consisting of a hydrogen atom, an alkali metal atom and an ammonium group And L represents a linking group]. X is reactive solid support as claimed in claim 2, characterized in that a vinyl group represented by -CH = CH _2. The reactive solid support according to claim 2, wherein L is a linking group containing a divalent or higher valent atom other than a carbon atom. The reactive solid support according to claim 2, wherein L is a linking group having a linking site selected from the group consisting of -NH-, -S-, and -O-. L is-(L ¹ ) _n -NH- (CR ¹ R ² ) ₂ -or-(L ¹ ) _n -S- (CR ¹ R ² ) _2- wherein R ¹ and R ² are the same as above. Wherein L ¹ represents a linking group, and n represents 0 or 1.]. _{3. The method according to claim 2} , wherein L is a linking group represented by-(L ¹ ) _n -NHCH ₂ CH _2-wherein L ¹ represents a linking group and n is 0 or 1. 22. The reactive solid support according to any one of the preceding claims. The reactive solid support according to claim 6, wherein L ¹ is a linking group containing a group represented by —OSi—, and n is 1. 9. The solid support is a sheet-shaped substrate selected from the group consisting of a silicate glass substrate, a silicate glass substrate surface-treated with a silane coupling agent, and a silicate glass substrate coated with an organic coating layer. 9. The reactive solid support according to any one of 1 to 8. A solid support having a reactive group introduced on its surface has the following formula:
X ¹ -SO ₂ -L ² -SO ₂ -X ²
[In the above formula, X ¹ and X ² independently represent each other —CR ¹ ＣＲCR ² R ³ , or —CHR ¹ —CR ² R ³ Y; R ¹ , R ² and R ³ represent each other A hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. It represents an atom or group selected from the group consisting of groups; Y is a halogen ^{_{atom, -OSO 2 R 11, -OCOR 12}} , -OSO 3 M, and represents an atom or group selected from the group consisting of quaternary pyridinium group R ¹¹ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms; Select from group consisting of groups R ¹² represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M represents a hydrogen atom, an alkali metal Represents an atom or group selected from the group consisting of an atom and an ammonium group; and L ² represents a linking group]
The method for producing a reactive solid support according to claim 2, wherein a disulfone compound represented by the following formula: The method for producing a reactive solid support according to claim 10, wherein the reactive group introduced on the surface of the solid support is an amino group, a mercapto group or a hydroxyl group. A sulfonyl compound, comprising: contacting the surface of the reactive solid support according to claim 1 with a nucleotide derivative having a reactive group capable of reacting with the reactive group to form a covalent bond or an analog thereof. A method for producing a solid support in which a nucleotide derivative or an analog thereof is bound and immobilized on a support surface via a linking group having a group. The method according to claim 12, wherein the nucleotide derivative or its analog is selected from the group consisting of an oligonucleotide, a polynucleotide, and a peptide nucleic acid. As the reactive solid support having irregularities, a vinyl sulfonyl group represented by the following formula or a reactive solid support in which a conjugate of a reactive precursor group and a linking group is bonded and fixed is used. The production method according to claim 12 or 13.
-L-SO ₂ -X
[In the above formula, X represents -CR ¹ = CR ² R ³ or -CHR ¹ -CR ² R ³ Y; R ¹ , R ² and R ³ independently of one another are a hydrogen atom, a carbon atom number; Is an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an atom selected from the group consisting of an aralkyl group having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. or it represents a group; Y is a halogen ^{_{atom, -OSO 2 R 11, -OCOR 12}} , -OSO 3 represents M, and the atom or group selected from the group consisting of quaternary pyridinium group; R ¹¹ has a number of carbon atoms Is a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms having an alkyl chain having 1 to 6 carbon atoms. the expressed; R ¹² is M represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M is selected from the group consisting of a hydrogen atom, an alkali metal atom and an ammonium group And L represents a linking group or a single bond]. X ^{is, -CR 1 = CR 2 R 3} [R 1, R 2 and R ³ are the the same meaning as' manufacturing method according to claim 14, wherein it is a reactive group represented by . A solid support having irregularities, wherein the nucleotide derivative or its analog obtained by the production method according to any one of claims 12 to 15 is bonded and fixed to the surface of the support. An oligonucleotide or polynucleotide showing complementarity to the immobilized nucleotide derivative or its analog in the presence of an aqueous medium on a solid support having irregularities on which the nucleotide derivative or its analog according to claim 16 is immobilized. And a method for binding and immobilizing a complementary oligonucleotide or polynucleotide. 18. The method of claim 17, wherein a detectable label is attached to the complementary oligonucleotide or polynucleotide. An oligonucleotide or polynucleotide having complementarity to the immobilized nucleotide derivative or its analog is complementarily bound to the solid support having irregularities on which the nucleotide derivative or its analog according to claim 16 is immobilized. A solid support to which a complementary oligonucleotide or polynucleotide is bound and fixed. 20. The solid support according to claim 19, wherein a detectable label is bound to the complementary oligonucleotide or polynucleotide. A solid support having irregularities wherein the nucleotide derivative or the analog thereof according to claim 16 is bonded and fixed to the surface of the support, or a solid support to which the complementary oligonucleotide or polynucleotide according to claim 19 is bonded and fixed. A method for identifying or screening a gene, characterized in that it is used.

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