JP2004121087A - Method for detecting single nucleotide polymorphism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting a single nucleotide polymorphism accurately, simply and rapidly. <P>SOLUTION: The method for detecting the single nucleotide polymorphism comprises using two kinds of allele-specific primers designed so as to have substantially the same amplification amount of each allele in a hetero junction. <P>COPYRIGHT: (C)2004,JPO

Description

【００４６】
本発明に適したピロ燐酸の検出方法は式２または式３に示した方法である。式２または式３に示した方法は、ピロ燐酸（ＰＰｉ）をピロホスファターゼで無機燐（Ｐｉ）に変換し、プリンヌクレオシドホスホリラーゼ（ＰＮＰ）により無機燐（Ｐｉ）をキサントシンまたはイノシンと反応させ、生じたキサンチンまたはヒポキサンチンをキサンチンオキシダーゼ（ＸＯＤ）により酸化して尿酸を生成させ、この酸化過程で生じる過酸化水素（Ｈ_２Ｏ_２）を用いてペルオキシダーゼ（ＰＯＤ）により発色剤（色素前駆体）を発色させ、これを比色するものである。これら式２または式３に示した方法では結果を比色で検出できるため、目視または簡単な比色測定装置を用いてピロ燐酸（ＰＰｉ）の検出が可能である。
式２及び式３：
【００４７】
【化２】

【００４８】
【化３】

【００４９】
ピロホスファターゼ（ＥＣ３，６，１，１）プリンヌクレオシドホスホリラーゼ（ＰＮＰ，ＥＣ２．４．２．１）、キサンチンオキシダーゼ（ＸＯＤ，ＥＣ１．２．３．２）及びペルオキシダーゼ（ＰＯＤ，ＥＣ１．１１．１．７）は市販のものを使用することができる。発色剤（すなわち色素前駆体）は、過酸化水素とペルオキシダーゼ（ＰＯＤ）により色素を生成させるものであればよく、例えば、ロイコ色素の酸化によって色素を生成する組成物（例、米国特許４，０８９，７４７等に記載のトリアリールイミダゾールロイコ色素、特開昭５９−１９３３５２号公報（ＥＰ　０１２２６４１Ａ）等に記載のジアリールイミダゾーロイコ色素）；酸化されたときに他の化合物とカップリングにより色素を生成する化合物を含む組成物（例えば４−アミノアンチピリン類とフェノール類又はナフトール類）などを使用することができる。
【００５０】
（Ｆ）乾式分析素子：
本発明において使用することのできる乾式分析素子とは、一層または複数層の機能層からなる分析素子であって、その少なくとも一層（または複数の層に渡って）に検出試薬を含有させ、層内での反応により生じた生成色素を、分析素子の外から反射光あるいは透過光により比色定量するものである。
【００５１】
このような乾式分析素子を用いて定量分析するには、液体試料を展開層の表面に一定量点着する。展開層で展開された液体試料は試薬層に達し、ここで試薬と反応し、発色する。点着後、乾式分析素子を適当な時間、一定温度に保って（インクベーション）発色反応を充分に進行させた後、例えば透明支持体側から照明光を試薬層に照射し、特定波長域で反射光量を測定して反射光学濃度を求め、予め求めておいた検量線に基づいて定量分析を行う。
【００５２】
乾式分析素子においては、検出を行うまでは乾燥状態で貯蔵・保管されるため、試薬を用時調製する必要がなく、また一般に乾燥状態の方が試薬の安定性が高いことから、試薬溶液を用時調製しなければならないいわゆる湿式法より簡便性、迅速性に優れている。また、微量の液体試料で、精度の高い検査を迅速に行うことができる検査方法としても優れている。
【００５３】
（Ｇ）ピロ燐酸定量用乾式分析素子：
本発明で使用することのできるピロ燐酸定量用乾式分析素子は、公知の多種の乾式分析素子と同様の層構成とすることができる。乾式分析素子は、前記（Ｅ）項（ピロ燐酸（ＰＰｉ）の検出）における、式２または式３の反応を行うための試薬の他、支持体、展開層、検出層、光遮蔽層、接着層、吸水層、下塗り層その他の層を含む多重層としてもよい。このような乾式分析素子として、例えば特開昭４９−５３８８８号公報（対応米国特許３，９９２，１５８）、特開昭５１−４０１９１号公報（対応米国特許４，０４２，３３５）、及び特開昭５５−１６４３５６号公報（対応米国特許４，２９２，２７２）、特開昭６１−４９５９号公報（対応ＥＰＣ公開特許０１６６３６５Ａ）の各明細書に開示されたものがある。
【００５４】
本発明で用いることができる乾式分析素子としては、ピロ燐酸を無機燐に変換する試薬、および無機燐の量に応じた発色反応を行う試薬群を含有する試薬層を備えるピロ燐酸定量用乾式分析素子が挙げられる。
このピロ燐酸定量用乾式分析素子においては、ピロホスファターゼを用いて酵素的にピロ燐酸（ＰＰｉ）を無機燐（Ｐｉ）に変換するまでは本明細書中上記した通り行うことができ、それ以降は、生化学検査分野で既知の以下に述べる「無機燐の定量法」（及びそれらに用いられる各反応の組み合わせ）を用いることにより、無機燐（Ｐｉ）の量に応じた発色反応を行うことができる。
【００５５】
なお、「無機燐」を表記する場合、燐酸（燐酸イオン）として、「Ｐｉ」と表記する場合と「ＨＰＯ_４ ^２−、Ｈ_２ＰＯ_４ ^１−」と表記する両方の場合がある。以下に示す反応の例では、「Ｐｉ」として表記するが、同じ反応式に対して「ＨＰＯ_４ ^２−」と表記する場合もある。
【００５６】
無機燐の定量法としては酵素法と燐モリブテン酸塩法が知られている。以下、無機燐の定量法としての酵素法と燐モリブテン酸塩法について説明する。
【００５７】
Ａ．酵素法
Ｐｉを定量検出するための一連の反応における最後の「呈色反応」に用いる酵素に応じて、ペルオキシダーゼ（ＰＯＤ）を用いる方法とグルコース−６−燐酸デヒドロゲナーゼ（Ｇ６ＰＤＨ）を用いる方法がある。以下、これらの方法の具体例を説明する。
【００５８】
（１）ペルオキシダーゼ（ＰＯＤ）を用いる方法の例
（１−１）
無機燐（Ｐｉ）を、プリンヌクレオシドホスホリラーゼ（ＰＮＰ）により、イノシンと反応させ、生じたヒポキサンチンをキサンチンオキシダーゼ（ＸＯＤ）により酸化して尿酸を生成する。この酸化過程で生じる過酸化水素（Ｈ_２Ｏ_２）を用いて、ペルオキシダーゼ（ＰＯＤ）により、４−アミノアンチピリン（４−ＡＡ）とフェノールとを酸化縮合させてキノンイミン色素を形成し、これを比色する。
【００５９】
（１−２）
無機燐（Ｐｉ）、コカルボキシラーゼ（ＴＰＰ）、フラビンアデニンジヌクレオチド（ＦＡＤ）、Ｍｇ^２＋の存在下で、ピルビン酸をピルビン酸オキシダーゼ（ＰＯＰ）により酸化してアセチル酢酸を生成する。この酸化過程で生じる過酸化水素（Ｈ_２Ｏ_２）を用いて、上記（１−１）の場合と同様に、ペルオキシダーゼ（ＰＯＤ）により、４−アミノアンチピリン（４−ＡＡ）とフェノールとを酸化縮合させてキノンイミン色素を形成し、これを比色する。
【００６０】
なお、上記の（１−１）および（１−２）における最後の呈色反応は、過酸化水素の検出試薬として既知の「Ｔｒｉｎｄｅｒ試薬」を使用して行うことができる。この反応で、フェノールは「水素供与体」として働く。「水素供与体」として用いるフェノールは古典的で、現在は改良された様々な「水素供与体」が使用されている。このような水素供与体の具体例としては、Ｎ−エチル−Ｎ−スルホプロピル−ｍ−アニリジン、Ｎ−エチル−Ｎ−スルホプロピルアニリン、Ｎ−エチル−Ｎ−スルホプロピル−３，５−ジメトキシアニリン、Ｎ−スルホプロピル−３，５−ジメトキシアニリン、Ｎ−エチル−Ｎ−スルホプロピル−３，５−ジメチルアニリン、Ｎ−エチル−Ｎ−スルホプロピル−ｍ−トルイジン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−ｍ−アニリジン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）アニリン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−３，５−ジメトキシアニリン、Ｎ−（２−ヒドロキシ−３−スルホプロピル）−３，５−ジメトキシアニリン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−３，５−ジメチルアニリン、Ｎ−エチル−Ｎ−（２−ヒドロキシ−３−スルホプロピル）−ｍ−トルイジン、及びＮ−スルホプロピルアニリンなどが挙げられる。
【００６１】
（２）グルコース−６−燐酸デヒドロゲナーゼ（Ｇ６ＰＤＨ）を用いる方法
（２−１）
無機燐（Ｐｉ）とグリコーゲンとをホスホリラーゼを用いて反応させ、グルコース−１−燐酸（Ｇ−１−Ｐ）を生成させる。生じたグルコース−１−燐酸をホスホグルコムターゼ（ＰＧＭ）により、グルコース−６−燐酸（Ｇ−６−Ｐ）にする。グルコース−６−燐酸とニコチアミドアデニンジヌクレオチド（ＮＡＤ）との存在下、グルコース−６−燐酸デヒドロゲナーゼ（Ｇ６ＰＤＨ）により、ＮＡＤを還元してＮＡＤＨにし、これを比色する。
【００６２】
（２−２）
無機燐（Ｐｉ）とマルトースとをマルトースホスホリラーゼ（ＭＰ）を用いて反応させ、グルコース−１−燐酸（Ｇ−１−Ｐ）を反応させる。以下、上記（２−１）と同様に、生じたグルコース−１−燐酸をホスホグルコムターゼ（ＰＧＭ）により、グルコース−６−燐酸（Ｇ−６−Ｐ）にする。グルコース−６−燐酸とニコチアミドアデニンジヌクレオチド（ＮＡＤ）との存在下、グルコース−６−燐酸デヒドロゲナーゼ（Ｇ６ＰＤＨ）により、ＮＡＤを還元してＮＡＤＨにし、これを比色する。
【００６３】
Ｂ．燐モリブテン酸塩法
酸性下で無機燐（燐酸塩）と水溶性モリブテン酸イオンとを錯化させた「燐モリブテン酸塩（Ｈ_３［ＰＯ_４Ｍｏ_１２Ｏ_３６］）を直接定量する「直接法」と、上記直接法の反応に続いて、還元剤により、Ｍｏ（ＩＶ）からＭｏ（ＩＩＩ）として、モリブテン青（Ｍｏ（ＩＩＩ））を定量する「還元法」とがある。水溶性モリブテン酸イオンの例としては、モリブテン酸アルミニウム、モリブテン酸カドミウム、モリブテン酸カルシウム、モリブテン酸バリウム、モリブテン酸リチウム、モリブテン酸カリウム、モリブテン酸ナトリウム、モリブテン酸アンモニウムなどが挙げられる。還元法で使用される代表的な還元剤の例としては、１，２，４アミノナフトールスルホン酸、硫酸第一鉄アンモニウム、塩化第一鉄、塩化第一スズ−ヒドラジン、硫酸−ｐ−メチルアミノフェノール、Ｎ，Ｎ−ジメチル−フェニレンジアミン、アスコルピン酸、マラカイトグリーンなどが挙げられる。
【００６４】
光透過性水不透過性支持体を用いる場合の乾式分析素子は、実用的に次のような構成を取り得る。ただし、本発明の内容はこれに限定されない。
（１）　　支持体上に試薬層を有するもの。
（２）　　支持体上に検出層、試薬層をこの順に有するもの。
（３）　　支持体上に検出層、光反射層、試薬層をこの順に有するもの。
（４）　　支持体上に第２試薬層、光反射層、第１試薬層をこの順に有するもの。
（５）　　支持体上に検出層、第２試薬層、光反射層、第１試薬層をこの順に有するもの。
【００６５】
上記（１）ないし（３）において試薬層は異なる複数の層から成ってもよい。例えば第１試薬層には、式２または式３に示すピロホスファターゼ反応に必要な酵素ピロホスファターゼ、ＰＮＰ反応に必要な基質キサントシンまたは基質イノシンと酵素ＰＮＰを、第２試薬層には、式２または式３に示すＸＯＤ反応に必要な酵素ＸＯＤを、そして第３試薬層には、式２または式３に示すＰＯＤ反応に必要な酵素ＰＯＤと発色色素（色素前駆体）を、それぞれ含有させてもよい。あるいは試薬層を２層として、第１試薬層ではピロホスファターゼ反応とＰＮＰ反応を、第２試薬層ではＸＯＤ反応とＰＯＤ反応を進行させてもよい。又は、第１試薬層ではピロホスファターゼ反応とＰＮＰ反応とＸＯＤ反応を、第２試薬層でＰＯＤ反応を進行させてもよい。
【００６６】
なお支持体と試薬層又は検出層との間には吸水層を設けてもよい。また各層の間には濾過層を設けてもよい。また試薬層の上には展開層を設けてもよく、その間に接着層を設けてもよい。
【００６７】
支持体は光不透過性（不透明）、光半透過性（半透明）、光透過性（透明）のいずれのものも用いることができるが、一般的には光透過性で水不透過性の支持体が好ましい。光透過性水不透過性支持体の材料として好ましいものはポリエチレンテレフタレート、ポリスチレンである。親水性層を強固に接着させるため通常、下塗り層を設けるか、親水化処理を施す。
【００６８】
試薬層として多孔性層を用いる場合、その多孔性媒体は繊維質であってもよいし、非繊維質であってもよい。繊維質材料としては、例えば濾紙、不織布、織物布地（例えば平織り布地）、編物布地（例えばトリコット編物布地）、ガラス繊維濾紙等を用いることができる。非繊維質材料としては特開昭４９−５３８８８号公報等に記載の酢酸セルロースなどからなるメンブランフイルター、特開昭４９−５３８８８号公報、特開昭５５−９０８５９号公報（対応米国特許４，２５８，００１）特開昭５８−７０１６３号公報（対応米国特許４，４８６，５３７）等に記載の無機物又は有機物微粒子からなる連続空隙含有粒状構造物層等のいずれでもよい。特開昭６１−４９５９号公報（対応欧州公開ＥＰ　０１６６３６５Ａ）、特開昭６２−１１６２５８号公報、特開昭６２−１３８７５６号公報（対応欧州公開ＥＰ　０２２６４６５Ａ）、特開昭６２−１３８７５７号公報（対応欧州公開ＥＰ　０２２６４６５Ａ）、特開昭６２−１３８７５８号公報（対応欧州公開ＥＰ　０２２６４６５Ａ）等に記載の部分接着された複数の多孔性層の積層物も好適である。
【００６９】
多孔性層は、供給される液体の量にほぼ比例した面積に液体を展開する、いわゆる計量作用を有する展開層であってもよい。展開層としては、これらのうち織物布地、編物布地などが好ましい。織物布地などは特開昭５７−６６３５９号公報に記載されたようなグロー放電処理をしてもよい。展開層には、展開面積、展開速度等を調節するため特開昭６０−２２２７７０号公報（対応：ＥＰ　０１６２３０１Ａ）、特開昭６３−２１９３９７号公報（対応西独特許公開ＤＥ　３７１７９１３Ａ）、特開昭６３−１１２９９９号公報（対応：ＤＥ　３７１７９１３Ａ）、特開昭６２−１８２６５２号公報（対応：ＤＥ　３７１７９１３Ａ）に記載したような親水性高分子あるいは界面活性剤を含有させてもよい。
【００７０】
例えば紙、布、高分子からなる多孔質膜等に本発明の試薬を予め含浸又は塗布した後、支持体上に設けた他の水浸透性層、例えば検出層の上に、特開昭５５−１６４５３５６号公報のような方法で接着させるのも有用な方法である。
【００７１】
こうして作られる試薬層の厚さは特に制限されないが、塗布層として設ける場合には、１μｍ〜５０μｍ程度、好ましくは２μｍ〜３０μｍの範囲が適当である。ラミネートによる積層など、塗布以外の方法による場合、厚さは数十μｍから数百μｍの範囲で大きく変化し得る。
【００７２】
親水性ポリマーバインダーからなる水浸透性層で試薬層を構成する場合、使用できる親水性ポリマーとしては、例えば、以下のものがある。ゼラチン及びこれらの誘導体（例えばフタル化ゼラチン）、セルロース誘導体（例えばヒドロキシエチルセルロース）、アガロース、アルギン酸ナトリウム、アクリルアミド共重合体やメタアクリルアミド共重合体（例えば、アクリルアミド又はメタアクリルアミドと各種ビニル性モニマーとの共重合体）、ポリヒドロキシエチルメタクリレート、ポリビニルアルコール、ポリビニルピロリドン、ポリアクリル酸ナトリウム、アクリル酸と各種ビニル性モノマーとの共重合体などである。
【００７３】
親水性ポリマーバインダーで構成される試薬層は、特公昭５３−２１６７７号公報（対応米国特許３，９９２，１５８）、特開昭５５−１６４３５６号公報（対応米国特許４，２９２，２７２）、特開昭５４−１０１３９８号公報（対応米国特許４，１３２，５２８）等の明細書に記載の方法に従って本発明の試薬組成物と親水性ポリマーを含む水溶液又は水分散液を支持体又は検出層等の他の層の上に塗布し乾燥することにより設けることができる。親水性ポリマーをバインダーとする試薬層の乾燥時の厚さは約２μｍ〜約５０μｍ、好ましくは約４μｍ〜約３０μｍの範囲、被覆量では約２ｇ／ｍ^２〜約５０ｇ／ｍ^２、好ましくは約４ｇ／ｍ^２〜約３０ｇ／ｍ^２の範囲である。
【００７４】
試薬層には式２または式３の試薬組成物の他に、塗布特性、拡散性化合物の拡散性、反応性、保存性等の諸性能の向上を目的として、酵素の活性化剤、補酵素、界面活性剤、ｐＨ緩衝剤組成物、微粉末、酸化防止剤、その他、有機物あるいは無機物からなる各種添加剤を加える事ができる。試薬層に含有させることができる緩衝剤はの例としては、日本化学学会編「化学便覧　基礎」（丸善（株）、１９６６年発行）１３１２−１３２０頁、Ｒ．Ｍ．Ｃ．Ｄａｗｓｏｎ　ｅｔ　ａｌ編、「Ｄａｔａ　ｆｏｒ　Ｂｉｏｃｈｅｍｉｃａｌ　Ｒｅｓｅａｒｃｈ」第２版（Ｏｘｆｏｒｄ　ａｔ　ｔｈｅ　Ｃｌａｒｅｎｄｏｎ　Ｐｒｅｓｓ，１９６９年発行）４７６−５０８頁、「Ｂｉｏｃｈｅｍｉｓｔｒｙ」５，４６７−４７７頁（１９６６年）、「Ａｎａｌｙｔｉｃａｌ　Ｂｉｏｃｈｅｍｉｓｔｒｙ」１０４，３００−３１０頁（１９８０年）に記載のｐＨ緩衝剤系がある。ｐＨ緩衝剤系の具体例として硼酸塩を含む緩衝剤；クエン酸又はクエン酸塩を含む緩衝剤；グリシンを含む緩衝剤；ビシン（Ｂｉｃｉｎｅ）を含む緩衝剤；ＨＥＰＥＳを含む緩衝剤；ＭＥＳを含む緩衝剤などのグッド緩衝剤等がある。なお燐酸塩を含む緩衝剤は、ピロ燐酸検出用乾式分析素子に使用することはできない。
【００７５】
本発明で使用することのできる、ピロ燐酸定量用乾式分析素子は前述の諸特許明細書に記載の公知の方法により調製することができる。ピロ燐酸定量用乾式分析素子は一辺約５ｍｍから約３０ｍｍの正方形またはほぼ同サイズの円形等の小片に裁断し、特公昭５７−２８３３３１号公報（対応米国特許４，１６９，７５１）、実開昭５６−１４２４５４号公報（対応米国特許４，３８７，９９０）、特開昭５７−６３４５２号公報、実開昭５８−３２３５０号公報、特表昭５８−５０１１４４号公報（対応国際公：ＷＯ０８３／００３９１）等に記載のスライド枠に収めて化学分析スライドとして用いることが製造，包装，輸送，保存，測定操作等の観点で好ましい。使用目的によっては、長いテープ状でカセットまたはマガジンに収めて用いたり、又は小片を開口のある容器内に収めて用いたり、又は小片を開口カードに貼付または収めて用いたり、あるいは裁断した小片をそのまま用いることなどもできる。
【００７６】
本発明で使用することのできるピロ燐酸定量用乾式分析素子は前述の諸特許明細書等に記載の操作と同様の操作により液体試料中の被検物であるピロ燐酸の定量検出ができる。例えば約２μＬ〜約３０μＬ、好ましくは４μＬ〜１５μＬの範囲の水性液体試料液を試薬層に点着する。点着した分析素子を約２０℃〜約４５℃の範囲の一定温度で、好ましくは約３０℃〜約４０℃の範囲内の一定温度で１〜１０分間インキュベーションする。分析素子内の発色又は変色を光透過性支持体側から反射測光し、予め作成した検量線を用いて比色測定法の原理により検体中のピロ燐酸の量を求めることができる。点着する液体試料の量、インキュベーション時間及び温度を一定にすることにより定量分析を高精度に実施できる。
【００７７】
測定操作は特開昭６０−１２５５４３号公報、特開昭６０−２２０８６２号公報、特開昭６１−２９４３６７号公報、特開昭５８−１６１８６７号公報（対応米国特許４，４２４，１９１）などに記載の化学分析装置により極めて容易な操作で高精度の定量分析を実施できる。なお、目的や必要精度によっては目視により発色の度合いを判定して、半定量的な測定を行ってもよい。
【００７８】
本発明で使用することのできる、ピロ燐酸定量乾式分析素子においては、分析を行うまでは乾燥状態で貯蔵・保管されるため、試薬を用時調製する必要がなく、また一般に乾燥状態の方が試薬の安定性が高いことから、試薬溶液を用時調製しなければならないいわゆる湿式法より簡便性、迅速性に優れている。また、微量の液体試料で、精度の高い検査を迅速に行うことができる検査方法としても優れている。
【００７９】
本発明の第二の形態において使用することのできる無機燐定量用乾式分析素子は、前記のピロ燐酸定量乾式分析素子における試薬層からピロホスファターゼを除くことで調製することができる。また、特開平７−１９７号公報に記載の乾式分析素子を使用することも可能である。無機燐定量用乾式分析素子は、試薬層にピロホスファターゼを含有しない以外は、その層構成、製造方法、使用方法において、前記ピロ燐酸定量乾式分析素子と同様である。
以下、実施例にて本発明を詳細に説明する。しかしながら、本実施例により本発明の技術的範囲が限定されるものではない。
【００８０】
【実施例】
＜参考例１（比較例）＞
ピロ燐酸定量用乾式分析素子を用いたアルデヒド脱水素酵素遺伝子（ＡＬＤＨ２遺伝子）関連部位の１塩基多型（ＳＮＰｓ）検出　（１塩基多型に対応する部分をプライマーの３’末端付近に設定した例）
（１）ターゲット核酸断片を含む核酸試料液の調製
予め塩基配列のシーケンシングにより、ＡＬＤＨ２遺伝子関連部位の特定の１塩基種が異なることにより、ＡＬＤＨ２活性型、ＡＬＤＨ２低活性型、不活性型であることが既知である、各々１人から採取した血液試料のそれぞれから、市販の核酸抽出、精製キット（ＱＩＡＧＥＮ社製、ＱＩＡａｍｐ　ＤＮＡ　ＢｌｏｏｄＭｉｎｉ　Ｋｉｔ）を用いて抽出、精製したゲノミック核酸断片を１ｍＬの精製蒸留水中に回収することで、ターゲット核酸断片を含む核酸試料液を調製した。
【００８１】
（２）ピロ燐酸定量用乾式分析素子の作製
ゼラチン下塗層が設けられている厚さ１８０μｍの無色透明ポリエチレンテレフタレ−ト（ＰＥＴ）平滑フイルムシ−ト（支持体）上に表１記載の組成（ａ）の水溶液を、以下の被覆率となるように塗布し、乾燥して試薬層を設けた。
【００８２】
【表１】

【００８３】
この試薬層の上に下記の表２記載の組成（ｂ）の接着層水溶液を以下の被覆率となるように塗布し、乾燥して接着層を設けた。
【００８４】
【表２】

【００８５】
次いで接着層の上に３０ｇ／ｍ^２の割合で水を全面に供給してゼラチン層を膨潤させ、その上に純ポリエステル製のブロ−ド織物布地をほぼ一様に軽く圧力をかけてラミネ−トして多孔性展開層を設けた。
【００８６】
次にこの展開層の上から下記の表３記載の組成（ｃ）の水溶液を以下の被覆率となるようにほぼ均一塗布し、乾燥させ、１３ｍｍ×１４ｍｍに裁断し、プラスチック製マウント材内に収めることで、ピロ燐酸定量用乾式分析素子を作成した。
【００８７】
【表３】

【００８８】
（３）ＰＣＲ増幅
上記（１）で、ＡＬＤＨ２活性型またはＡＬＤＨ２不活性型それぞれのヒト全血試料から抽出・精製して得たタ−ゲット核酸断片を含む核酸試料液をそのまま用いて、以下の条件でＰＣＲ増幅を行った。
【００８９】
＜プライマー＞
プライマーは、１２番染色体上のＡＬＤＨ２遺伝子関連部位のなかに、共通のプライマー（ｕｐｐｅｒ）と、ＡＬＤＨ２の活性を決定する１塩基多型に対応する部分を３’末端付近に設定（ｌｏｗｅｒ−１とｌｏｗｅｒ−２に記載のプライマー塩基配列の下線部分）した、ＡＬＤＨ２活性型および不活性型に対応する、２種のプライマ−（ｌｏｗｅｒ−１）および、プライマ−（ｌｏｗｅｒ−２）のセットを使用した。なお。ｌｏｗｅｒプライマーの１塩基多型に対応する配列の１塩基分５‘上流の塩基を変え（Ｔ→Ａ）、人為的にミスマッチを作り出した．
【００９０】
活性型検出用プライマー
プライマー（ｕｐｐｅｒ）：
５’−ＡＡＣＧＡＡＧＣＣＣＡＧＣＡＡＡＴＧＡ−３’（配列番号１）
プライマー（ｌｏｗｅｒ−１）：
５’−ＧＧＧＣＴＧＣＡＧＧＣＡＴＡＣＡＣＡＧＡ−３’（配列番号２）
不活性型検出用プライマー
プライマ−（ｕｐｐｅｒ）：
５’−ＡＡＣＧＡＡＧＣＣＣＡＧＣＡＡＡＴＧＡ−３’（配列番号１）
プライマ−（ｌｏｗｅｒ−２）：
５’−ＧＧＧＣＴＧＣＡＧＧＣＡＴＡＣＡＣＡＡＡ−３’（配列番号３）
【００９１】
以下に示す反応液の組成で、［変性：９４℃・２０秒、アニーリング：６０℃・３０秒、ポリメラーゼ伸長反応：７２℃・１分３０秒］を３５サイクル繰り返すことでＰＣＲ増幅を実施した。
【００９２】
＜反応液の組成＞
１０×ＰＣＲバッファ−　　　　　　　　　５μＬ
２．５ｍＭ　ｄＮＴＰ　　　　　　　　　　５μＬ
５μＭプライマ−（ｕｐｐｅｒ）　　　　　　　　　　　　　　　　２μＬ
５μＭプライマ−（ｌｏｗｅｒ−１または−２）　　　　　　２μＬ
Ｔａｑ　　　　　　　　　　　　　　　０．５μＬ
（１）で得た核酸断片試料液　　　　　０．５μＬ
精製水　　　　　　　　　　　　　　　　３５μＬ
【００９３】
（４）ピロ燐酸定量用分析素子を用いた検出
前記（３）におけるＰＣＲ増幅反応後の溶液をそのまま、上記（２）で製作したピロ燐酸定量用乾式分析素子上に各々２０μＬ点着し、ピロ燐酸定量用乾式分析素子を３７℃にて５分間インキュベーション後、波長６５０ｎｍにて支持体側から反射光学濃度（ＯＤ_Ｒ）を測定した。得られた結果を以下の表４に示す。
【００９４】
【表４】

【００９５】
参考例１の結果より、試料中の既知であるＡＬＤＨ２の活性型とピロ燐酸定量用乾式分析素子を用いて測定した反射光学濃度（ＯＤｒ）との関係は一致している。すなわちアルデヒド脱水素酵素遺伝子（ＡＬＤＨ２遺伝子）関連部位の１塩基多型（ＳＮＰｓ）を検出できていることがわかる。しかしながら、低活性型の各対立遺伝子におけるピロリン酸の生成量が異なるため、活性型と低活性型の判定が難しいことがわかる。
【００９６】
＜実施例１＞
ピロ燐酸定量用乾式分析素子を用いたアルデヒド脱水素酵素遺伝子（ＡＬＤＨ２遺伝子）関連部位の１塩基多型（ＳＮＰｓ）検出　（プライマーの塩基を各対立遺伝子に応じて設定した例）
【００９７】
（１）ターゲット核酸断片を含む核酸試料液の調製
参考例１と同様に、予め塩基配列のシーケンシングにより、ＡＬＤＨ２活性型、ＡＬＤＨ２低活性型、不活性型であることが既知であるそれぞれ１人の血液試料から、ターゲット核酸断片を含む核酸試料液を調製した。
【００９８】
（２）ピロ燐酸定量用乾式分析素子の作製
参考例１と同様に行った．
【００９９】
（３）ＰＣＲ増幅
上記（１）で、ＡＬＤＨ２活性型またはＡＬＤＨ２不活性型それぞれのヒト全血試料から抽出・精製して得たタ−ゲット核酸断片を含む核酸試料液をそのまま用いて、以下の条件でＰＣＲ増幅を行った。
【０１００】
＜プライマー＞
プライマーは、１２番染色体上のＡＬＤＨ２遺伝子関連部位のなかに、共通のプライマー（ｕｐｐｅｒ）と、ＡＬＤＨ２の活性を決定する１塩基多型に対応する部分を３’末端付近に設定（ｌｏｗｅｒ−１とｌｏｗｅｒ−２に記載のプライマー塩基配列の下線部分）した、ＡＬＤＨ２活性型および不活性型に対応する、２種のプライマ−（ｌｏｗｅｒ−１）および、プライマ−（ｌｏｗｅｒ−２）のセットを使用した。なお、人為的にミスマッチを作り出すため、ｌｏｗｅｒプライマーの１塩基多型に対応する配列の１塩基分５‘上流の塩基を（Ｔ→Ａ）に変えたものと、（Ｃ→Ａ）に変えたもののそれぞれを用いた．
【０１０１】
○活性型検出用プライマー
プライマー（ｕｐｐｅｒ）：
５’−ＡＡＣＧＡＡＧＣＣＣＡＧＣＡＡＡＴＧＡ−３’（配列番号１）
プライマー（ｌｏｗｅｒ−１Ａ）：
５’−ＧＧＧＣＴＧＣＡＧＧＣＡＴＡＣＡＣＡＧＡ−３’（配列番号４）
プライマー（ｌｏｗｅｒ−１Ｃ）：
５’−ＧＧＧＣＴＧＣＡＧＧＣＡＴＡＣＡＣＣＧＡ−３（配列番号５）
○不活性型検出用プライマー
プライマ−（ｕｐｐｅｒ）：
５’−ＡＡＣＧＡＡＧＣＣＣＡＧＣＡＡＡＴＧＡ−３’（配列番号１）
プライマ−（ｌｏｗｅｒ−２Ａ）：
５’−ＧＧＧＣＴＧＣＡＧＧＣＡＴＡＣＡＣＡＡＡ−３’（配列番号６）
プライマ−（ｌｏｗｅｒ−２Ｃ）：
５’−ＧＧＧＣＴＧＣＡＧＧＣＡＴＡＣＡＣＣＡＡ−３’（配列番号７）
【０１０２】
以下に示す反応液の組成で、［変性：９４℃・２０秒、アニーリング：６０℃・３０秒、ポリメラーゼ伸長反応：７２℃・１分３０秒］を３５サイクル繰り返すことでＰＣＲ増幅を実施した。
【０１０３】
＜反応液の組成＞
１０×ＰＣＲバッファ−　　　　　　　　　５μＬ
２．５ｍＭ　ｄＮＴＰ　　　　　　　　　　５μＬ
５μＭプライマ−（ｕｐｐｅｒ）　　　　　　　　　　　　　　　　２μＬ
５μＭプライマ−（ｌｏｗｅｒ−１Ａ，Ｃ又は−２Ａ，Ｃ）　２μＬ
Ｔａｑ　　　　　　　　　　　　　　　０．５μＬ
（１）で得た核酸断片試料液　　　　　０．５μＬ
精製水　　　　　　　　　　　　　　　　３５μＬ
【０１０４】
（４）ピロ燐酸定量用分析素子を用いた検出
前記（３）におけるＰＣＲ増幅反応後の溶液をそのまま、上記（２）で製作したピロ燐酸定量用乾式分析素子上に各々２０μＬ点着し、ピロ燐酸定量用乾式分析素子を３７℃にて５分間インキュベーション後、波長６５０ｎｍにて支持体側から反射光学濃度（ＯＤ_Ｒ）を測定した。得られた結果を以下の表５に示す。
【０１０５】
【表５】

【０１０６】
実施例１の結果より、ピロ燐酸定量用乾式分析素子を用いて測定した反射光学濃度（ＯＤｒ）は、低活性型の試料において、活性型プライマーとして１Ａ、不活性型プライマーとして２Ｃを用いれば、等しくなっている。これにより、活性型／不活性型と低活性型との判別が明らかにできるようになった。
【０１０７】
【発明の効果】
本発明によれば、１塩基多型を正確、簡便かつ迅速に検出することが可能になった。特に、本発明によれば、対立遺伝子のヘテロ接合とホモ接合を明確に区別することが可能である。
【０１０８】
【配列表】

【０１０９】

【０１１０】

【０１１１】

【０１１２】

【０１１３】

【０１１４】

【０１１５】

【図面の簡単な説明】
【図１】図１は、本発明の実施形態を説明する概念図を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to detection of a specific sequence contained in DNA in a specimen, detection of gene polymorphism, single base substitution (SNPs) analysis, and the like.
[Prior art]
The results of genome analysis research are summarized in systematic gene polymorphism analysis and systematic gene expression analysis. In recent years, movements to apply these genomic information to the medical field have become active, and gene polymorphisms and expression analysis techniques have been dramatically advanced.
[0003]
Among gene polymorphisms, single nucleotide substitutions (SNPs) are said to be one in every 1000 bases, and these SNPs are considered to be the cause of individual differences, individual characteristics and innate constitutions. It has been. In addition, diseases that have been considered to have a relatively high rate of environmental factors (diabetes, hypertension, etc.) are also associated with factor genes as risk factors, many of which are defined by single nucleotide polymorphisms. It is becoming clear. Therefore, SNP analysis is considered to lead to medication and treatment (tailor-made medicine) tailored to the individual's constitution, and is attracting a great deal of attention.
[0004]
To date, various methods have been developed for SNP analysis, but broadly divided into a method for detecting an unknown base substitution and a method for detecting a known base substitution.
[0005]
Examples of known base substitution include analytical methods such as a method for directly analyzing a base sequence and a DNA chip using an oligonucleotide.
[0006]
From the viewpoint of genetic testing for tailor-made medicine, what is needed in the future is a technique for typing patient SNPs associated with each disease. In addition, more effective treatment can be expected by determining medication and treatment guidelines as quickly as possible. Therefore, it is desirable to develop a SNPs typing technology that allows anyone to easily perform inspections and obtain results quickly.
[0007]
Currently classified SNPs can be classified into two methods, that is, a method using a polymerase reaction and a method using hybridization.
[0008]
Methods utilizing hybridization include simple hybridization using a DNA chip (Sequence By Hybridization) (Drmanac R, et al: Genomics 4: 114-128 (1989)) and Dye-labeled oligonucleotide ligation (Chen X, et al. al .: Genome Res. 8: 549-556 (1998)) and the Invader method (Lyamichev, et al: Science 260: 778-783 (1993)). In any case, in principle, an oligonucleotide corresponding to each allele (allele) is prepared, and which allele is hybridized is detected in principle.
[0009]
These methods require a time since they require a hybrid operation, or have a problem that the apparatus is expensive because fluorescence is used in the detection system, and cannot be easily tested. .
[0010]
Primers are set near the SNP as in the SNaPShot method and Pyrosequence method (Alderborn, A. et. Al: Genome Res., 28: 1249-1258 (2000)), and the method using the polymerase reaction is performed at the SNP site. A method of determining which base has been incorporated, a method of designing a primer so as to include a SNP site corresponding to each allele in the vicinity of the 3 ′ end, and determining whether or not a polymerase reaction occurs (ARMS method (Amplification refractory) (mutation system), Newton CR, et al .: Nucl Acids Res.17: 2503-2516 (1989), PASA method (PCR-amplification of specific alleles), arker G et al: Anal Biochem 186: 64-68 (1990)) and to break up.
[0011]
The SNaPShot method is a method in which a primer is made up to just before the SNP site, an extension reaction is carried out with only dideoxynucleotide, and which base has been incorporated. Since this is an extension reaction of only one base, a sequencer must be used for analysis, and an expensive apparatus is required.
[0012]
The Pyrosequence method is a method in which a primer is designed several upstream or immediately before a SNP and a deoxynucleotide is added by one base at a time starting from this primer. At that time, pyrophosphate generated only when an extension reaction occurs is converted to ATP to cause chemiluminescence, and this luminescence is detected. Since pyrophosphoric acid is produced quantitatively with the amount of base incorporated, it is excellent in quantification. However, there remains a problem in terms of operability that it is necessary to add four types of dNTPs to the reaction part in order and a device for detecting luminescence.
[0013]
The ARMS method and PASA method make use of the fact that the extension reaction starting from the primer strongly depends on the matching between the primer 3 ′ end and the template (Kwok S. et al .: Nucleic Acids Res 18, 999-1005). (1990), Huang MM et al .: Nucleic Acids Res. 20, 4567-4573 (1992)). In other words, by preparing a primer complementary to each allele in advance and using the fact that an extension reaction occurs only when it matches the genotype of the sample, the genotype is determined based on whether an amplification reaction has occurred. is there. This method is excellent in that it can be examined quickly by a simple method such as electrophoresis.
[0014]
However, in reality, there is only one base difference between allele-specific primers, and non-specific amplification is often caused by mismatched primers depending on the template sequence (Huang MM et al .: Nucleic Acids Res. 20, 4567-4573 (1992)). In addition, since whether or not amplification occurs depends on delicate conditions such as the equipment used and the surrounding environment, it is difficult to suppress nonspecific amplification.
[0015]
In fact, in the ARMS method, another mismatch is artificially introduced one base upstream of the 3 ′ end of the primer in order to enhance the mismatch. This is because PCR amplification efficiency is greatly reduced by introducing a mismatch of 2 bases within 4 bases from the 3 ′ end. (Kwok S. et al .: Nucleic Acids Res 18, 999-1005 (1990)) Thereby, non-specific amplification can be suppressed to some extent without strictly controlling the conditions.
[0016]
[Problems to be solved by the invention]
Based on the above method, it seems that the nucleotide polymorphism can be clearly detected. However, if the reaction is carried out using different primers in an amplification reaction such as PCR, the efficiency for amplification derived from both primers is often different. If the amplification products corresponding to both alleles are not constant, there is a problem that when the SNP type is heterozygous, the amount of amplification of both alleles is different, making it difficult to distinguish from homozygotes.
[0017]
An object of this invention is to provide the method of detecting a single nucleotide polymorphism correctly, simply and rapidly. In particular, the present invention aims to provide a method of clearly distinguishing between heterozygotes and homozygotes of alleles.
[0018]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have designed primers specific to each allele so that the amount of amplification product derived from each allele in the heterozygote is substantially the same. By using such two types of allele-specific primers, it was found that an amplification product in heterozygote and an amplification product in homozygote can be distinguished.
[0019]
That is, primers specific to both alleles were set so as to include the SNP site, and in order to further enhance the mismatch, an artificial mismatch was designed near the SNP site. At this time, by selecting artificial mismatch bases so that the mismatches between each allele of the heterozygous sample and the corresponding primer are equal, the amount of amplification products derived from each allele becomes equal and homologous. Can now be distinguished from bonding.
[0020]
That is, according to the present invention, there is provided a method for detecting a single nucleotide polymorphism using two types of allele-specific primers designed so that the amount of amplification of each allele in a heterozygote is substantially the same. .
Preferably, an allele-specific primer designed so that the polymorphic site is present within 4 bases from the 3 ′ end of the allele-specific primer is used.
Preferably, an allele-specific primer having a mismatch base introduced into the adjacent base of the polymorphic site is used.
Preferably, an allele-specific primer is used in which mismatch bases adjacent to the polymorphic site are selected according to each allele.
[0021]
Preferably, a single nucleotide polymorphism is detected using a polymerase reaction.
Preferably, a single nucleotide polymorphism is detected using a polymerase reaction product.
Preferably, single nucleotide polymorphisms are detected using electrophoresis, chromatography or HPLC as a detection means.
[0022]
Preferably, a single nucleotide polymorphism is detected using a side reaction product during the polymerase reaction.
Preferably, the side reaction product is pyrophosphoric acid.
Preferably, pyrophosphoric acid is detected using a dry analytical element.
Preferably, the detection of the single nucleotide polymorphism includes discriminating the homozygous / heterozygous of the single nucleotide polymorphism.
[0023]
According to another aspect of the present invention, there is provided a primer set for carrying out the method of the present invention, comprising two allele-specific primers designed so that the amount of amplification of each allele is substantially the same. Is done.
[0024]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
The single nucleotide polymorphism detection method of the present invention is characterized by using two types of allele-specific primers designed so that the amount of amplification of each allele in a heterozygote is substantially the same. By utilizing the method of the present invention, it is possible to discriminate homo / heterozygotes of single nucleotide polymorphisms. In a preferred form of the method of the present invention, a polymerase reaction is performed using each allele-specific primer, and the presence or absence of the extension reaction is detected. Specific examples of the detection method include a method of directly measuring amplification products such as electrophoresis, mass spectrometry, and liquid chromatography, and a method of detecting pyrophosphoric acid produced during the polymerase extension reaction. FIG. 1 is a conceptual diagram illustrating an embodiment of the present invention.
[0025]
A first preferred embodiment of the method for discriminating homonucleotides / heterozygotes of a single nucleotide polymorphism according to the present invention is listed below.
An allele-specific primer is set to include the SNP site to be detected. At that time, a mismatch is artificially inserted in the vicinity of the SNP site in order to increase the mismatch to the allele. Artificial mismatches are selected so that the degree of mismatch of each primer for each allele is equal. These primers are used separately to cause the polymerase extension reaction.
[0026]
Whether or not an elongation reaction has actually occurred is detected by detecting pyrophosphoric acid. More preferably, it is carried out using a dry analytical element for quantifying pyrophosphate characterized by comprising a reagent layer containing xanthosine or inosine, pyrophosphatase, purine nucleoside phosphorylase, xanthine oxidase, peroxidase and a color former.
Hereinafter, embodiments of the present invention will be described in more detail.
[0027]
(A) Target nucleic acid fragment:
The target nucleic acid fragment to be analyzed in the present invention is a polynucleotide having at least a part of the known base sequence, and is a genomic DNA fragment isolated from all living organisms such as animals, microorganisms, bacteria and plants. obtain. In addition, RNA fragments or DNA fragments that can be isolated from viruses, and cDNA fragments synthesized using mRNA as a template can also be targeted. It is desirable that the target nucleic acid fragment is purified as much as possible, and extra components other than the nucleic acid fragment are removed. For example, when targeting a genomic DNA fragment isolated from the blood of an animal (eg, human) or when targeting a nucleic acid (DNA or RNA) fragment of an infected bacterium or virus present in the blood, The destroyed white blood cell membrane, hemoglobin eluted from the red blood cells, and other general chemicals present in the blood must be sufficiently removed. In particular, hemogrombin inhibits the subsequent polymerase extension reaction.
[0028]
(B) Target nucleic acid fragment and specific primer:
The primer specific to the target nucleic acid fragment used in the present invention is an oligonucleotide having a base sequence complementary to a target site where the base sequence of the target nucleic acid fragment is known. A primer complementary to the target nucleic acid fragment hybridizes to a target site of the target nucleic acid fragment, and a polymerase extension reaction proceeds using the target nucleic acid as a template from the 3 ′ end of the primer. That is, the point in the present invention is whether the primer recognizes the target site of the target nucleic acid fragment and specifically hybridizes. The preferable base number of the primer used in the present invention is 5 to 60 bases. Particularly preferred is 15 to 40 bases.
[0029]
Furthermore, it is necessary to set the primer in the present invention so as to include a polymorphic site for which at least one is to be detected. More preferably, it is set within 4 bases from the 3 ′ end. This is because the polymorphism detection in the present invention utilizes the fact that the extension reaction starting from the primer strongly depends on the matching between the 3 ′ end of the primer and the template, and there is a mismatch near the 3 ′ end. If present, the extension reaction will not proceed. However, in practice, if there is only a single base difference in the SNP site, which is the detection site, between each allele-specific primer, non-specific amplification can occur even with a mismatch primer depending on the template sequence. In addition, whether or not amplification occurs depends on delicate conditions such as the equipment used and the surrounding environment, so it is difficult to suppress non-specific amplification, and polymorphism cannot be determined. Therefore, it is necessary to artificially make a mismatch in addition to the SNP polymorphic site. The artificial mismatch is preferably in the vicinity of the SNP polymorphism site, and more preferably adjacent. Thereby, non-specific amplification can be suppressed to some extent.
[0030]
However, if the reaction is carried out using different primers in an amplification reaction such as PCR, the efficiency for amplification derived from both primers is often different.
In the case of heterozygosity, the amount of amplification derived from both alleles is not equal, and there is a possibility of misjudgment as homozygous. Therefore, an artificial mismatch is set so that the amounts of amplification products derived from both alleles are equal. For example, if a gene has a (G / A) SNP, the sequence of the primer for detecting the G type, ... AG ..., the sequence of the primer for detecting the A type, ... ... CA may be set so that.
[0031]
(C) Polymerase:
When the target nucleic acid is DNA, the polymerase used in the present invention is 5 ′ starting from a double-stranded portion formed by hybridization of a primer with a portion denatured into a single strand of the target nucleic acid fragment. → A DNA polymerase that catalyzes a complementary extension reaction in the 3 ′ direction using deoxynucleoside triphosphate (dNTP) as a material and a target nucleic acid fragment as a template. Specific examples of the DNA polymerase used include DNA polymerase I, Klenow fragment of DNA polymerase I, Bst DNA polymerase, and the like. DNA polymerases can be selected or combined depending on the purpose. For example, when a part of the target nucleic acid fragment is amplified (for example, PCR method), it is effective to use Taq DNA polymerase having excellent heat resistance. In addition, depending on the purpose, DNA polymerase α, T4 DNA polymerase, and T7 DNA polymerase having hexokinase activity in the 3 ′ → 5 ′ direction can be used in combination.
[0032]
Further, when the genomic nucleic acid or mRNA of the RNA virus is the target nucleic acid fragment, it is possible to use reverse transcriptase having reverse transcription activity. Further, reverse transcriptase and Taq DNA polymerase can be used in combination.
[0033]
(D) Polymerase extension reaction:
In the polymerase extension reaction of interest in the present invention, the above-mentioned (B), which specifically hybridizes to a part of the target nucleic acid fragment denatured to a single strand as described in (A) above. Starting from the 3 ′ end of a primer complementary to the target nucleic acid fragment as described in the above, using deoxynucleoside triphosphate (dNTP) as a material, and a polymerase as described in (C) above as a catalyst All of the complementary nucleic acid extension reactions that proceed using the target nucleic acid fragment as a template are included. This complementary nucleic acid extension reaction means that a continuous extension reaction occurs at least twice (for two bases).
[0034]
When the amount of the target nucleic acid is small, it is preferable to amplify the target portion of the target nucleic acid by some means using a polymerase extension reaction. Various methods developed and invented so far can be used for amplification of the target nucleic acid.
[0035]
Examples of nucleic acid amplification methods include PCR (Japanese Patent Publication No. 4-67960, Japanese Patent Publication No. 4-67957), LCR (Japanese Patent Laid-Open No. 5-2934), SDA (Strand Displacement Amplification: Japanese Patent Laid-Open No. 5-130870), RCA (Rolling). Circle Amplification: Proc. Natl. Acad. Sci, Vol. 92, 4641-4645 (1995)), ICAN (Isomalized and Chimerized Prime of Amplified Nucleic Acids). Vol.18, No.2 (2001)), NA BA (Nucleic acid Sequence-based Amplification method; Nature, 350, 91 (1991)), and TMA (Transcription mediated amplification method;. J.Clin Microbiol vol. 31, 3270 (1993)) and the like can be mentioned.
[0036]
The most common and widespread method for amplifying a target nucleic acid is a PCR (polymerase chain reaction) method. In the PCR method, the temperature of the reaction solution is periodically controlled to denature (step of denaturing nucleic acid fragments from double strands to single strands) → annealing (primers are applied to nucleic acid fragments denatured into single strands). This is a method of amplifying a target portion of a target nucleic acid fragment by repeating a periodic step of hybridization) → polymerase (Taq DNA polymerase) extension reaction → denature. Finally, the target site of the target nucleic acid fragment can be amplified up to 1 million times the initial amount.
[0037]
When the target nucleic acid fragment is an RNA fragment, a reverse transcriptase having reverse transcription activity can be used, and an extension reaction can be performed using the RNA strand as a template. Furthermore, RT-PCR method can be used in which reverse transcriptase and Taq DNA polymerase are used in combination and a PCR reaction is carried out following an RT (reverse transcription) reaction.
[0038]
LCR (Japanese Patent Laid-Open No. 5-2934) is a technique in which two complementary oligonucleotide probe strands are bonded to a single-stranded DNA in an end-to-tail manner, and a nick between the two oligonucleotide strands is formed with a heat-resistant ligase. To seal. In this method, the bound DNA strand is released by denaturation, becomes a template, and is amplified. The SNP can be determined by devising the probe sequence and determining whether amplification has occurred. In addition, a method for improving LCR by setting a gap between two primers and filling the gap with a polymerase (Gap-LCR: Nucleic Acids Research, Vol. 23, No. 4, No. 675 (1995)) has been developed. . When this method is used, SNP can also be determined by measuring the production of pyrophosphate by the polymerase extension reaction.
[0039]
SDA (Strand Displacement Amplification: JP-A-5-130870) is a cycling assay method using exonuclease, and is one of the methods for amplifying a target site of a target nucleic acid fragment using a polymerase extension reaction. This method is a method in which 5 ′ → 3 ′ exonuclease is allowed to act together with a polymerase extension reaction starting from a primer that specifically hybridizes to a target site of a target nucleic acid fragment, so that the primer is degraded in the reverse direction. A new primer hybridizes in place of the degraded primer, and the extension reaction by the DNA polymerase proceeds again. The elongation reaction by the polymerase and the decomposition reaction by exonuclease for removing the previously elongated strand are sequentially and periodically repeated. Here, the elongation reaction by polymerase and the degradation reaction by exonuclease can be carried out under isothermal conditions. By devising the primer sequence, it is possible to determine the SNP based on whether or not the polymerase reaction has occurred.
[0040]
The LAMP method is a method for amplifying a target site of a target nucleic acid fragment developed in recent years. In this method, at least four kinds of primers that complementarily recognize at least six specific sites of the target nucleic acid fragment, no nuclease activity in the 5 ′ → 3 ′ direction, and double-stranded DNA on the template are 1 This is a method of amplifying a target site of a target nucleic acid fragment as a special structure under isothermal conditions by using a strand displacement type Bst DNA polymerase that catalyzes an extension reaction while releasing it as a double-stranded DNA. By devising this primer sequence, it is possible to determine the SNP based on whether or not amplification has occurred. In addition, the amplification efficiency of this LAMP method is high, and the amount of pyrophosphate accumulated in the polymerase extension reaction is extremely large. Therefore, it is easy to detect SNP by detecting pyrophosphate.
[0041]
The ICAN method is also a method for amplifying a target site of a target nucleic acid fragment developed in recent years. This is an isothermal gene amplification method using RNA-DNA chimera primer, DNA polymerase having strand displacement activity and template exchange activity, and RNaseH. After the chimeric primer is bound to the template, a complementary strand is synthesized by DNA polymerase. Thereafter, RNase H cleaves the RNA portion derived from the chimeric primer, and the gene is amplified by repeating this reaction in which an elongation reaction accompanied by a strand displacement reaction and a template exchange reaction occurs from the cleaved portion. By devising this primer sequence, it is possible to determine the SNP based on whether or not amplification has occurred. In addition, the amplification efficiency of the ICAN method is high, and the amount of pyrophosphate accumulated in the polymerase extension reaction is extremely large. Therefore, it is easy to detect SNP by detecting pyrophosphate.
[0042]
(E) Detection:
Since the present invention relies on the purpose of making the amount of product after polymerase reaction such as PCR equal among products from alleles in heterozygotes, it is not limited as long as the amount of product can be quantified. Nor.
[0043]
Examples of the detection method include a method of directly measuring a product such as electrophoresis, liquid chromatography, and a mass spectrometer, or a method of detecting pyrophosphate and the like generated as a result of the polymerase reaction. In view of quantitativeness, it is preferably a detection method by quantifying pyrophosphate, and in view of simplicity, a method of quantifying pyrophosphate using a dry analytical element is more preferable.
[0044]
Conventionally, the method shown in Formula 1 is known as a method for detecting pyrophosphoric acid (PPi). In this method, pyrophosphate (PPi) is converted into adenosine triphosphate (ATP) by sulfurylase, and luminescence generated by adenosine triphosphate acting on luciferin by luciferase is detected. Therefore, an apparatus capable of measuring luminescence is required to detect pyrophosphoric acid (PPi) by this method.
[0045]
[Chemical 1]

[0046]
The pyrophosphoric acid detection method suitable for the present invention is the method shown in Formula 2 or Formula 3. The method shown in Formula 2 or Formula 3 is obtained by converting pyrophosphate (PPi) to inorganic phosphorus (Pi) with pyrophosphatase, and reacting purine nucleoside phosphorylase (PNP) with inorganic phosphorus (Pi) with xanthosine or inosine. Xanthine or hypoxanthine is oxidized with xanthine oxidase (XOD) to produce uric acid, and hydrogen peroxide (H ₂ O ₂ ) generated in this oxidation process is used to produce a color former (dye precursor) with peroxidase (POD). Color is developed, and this is colorimetric. Since the results shown in these formulas 2 and 3 can be detected by colorimetry, pyrophosphoric acid (PPi) can be detected visually or using a simple colorimetric measuring device.
Equation 2 and Equation 3:
[0047]
[Chemical 2]

[0048]
[Chemical 3]

[0049]
Pyrophosphatase (EC 3, 6, 1, 1) purine nucleoside phosphorylase (PNP, EC 2.4.2.1), xanthine oxidase (XOD, EC 1.2.3.2) and peroxidase (POD, EC 1.11.1. 7) can use a commercially available thing. The color former (that is, the dye precursor) may be any one that generates a dye by hydrogen peroxide and peroxidase (POD). For example, a composition that generates a dye by oxidation of a leuco dye (eg, US Pat. No. 4,089) Triarylimidazole leuco dyes described in JP, 747 et al., Diaryl imidazole leuco dyes described in JP-A-59-193352 (EP 0126241A), etc .; when oxidized, a dye is formed by coupling with other compounds And the like (for example, 4-aminoantipyrines and phenols or naphthols) can be used.
[0050]
(F) Dry analytical element:
The dry analytical element that can be used in the present invention is an analytical element composed of one or a plurality of functional layers, in which at least one layer (or across a plurality of layers) contains a detection reagent, The produced dye produced by the reaction in is colorimetrically determined from the outside of the analytical element by reflected light or transmitted light.
[0051]
In order to perform quantitative analysis using such a dry analytical element, a certain amount of liquid sample is spotted on the surface of the development layer. The liquid sample developed in the development layer reaches the reagent layer, where it reacts with the reagent and develops color. After spotting, the dry analytical element is kept at a certain temperature for an appropriate time (incubation), and the color development reaction proceeds sufficiently. For example, the reagent layer is irradiated with illumination light from the transparent support side and reflected in a specific wavelength range. The amount of reflected light is measured to determine the reflection optical density, and quantitative analysis is performed based on a calibration curve obtained in advance.
[0052]
Since dry analytical elements are stored and stored in a dry state until detection, there is no need to prepare the reagent at the time of use, and generally the reagent stability is higher in the dry state. It is easier and faster than the so-called wet method that must be prepared at the time of use. Moreover, it is also excellent as an inspection method capable of quickly performing a high-accuracy inspection with a small amount of liquid sample.
[0053]
(G) Dry analytical element for quantification of pyrophosphoric acid:
The dry analytical element for quantifying pyrophosphoric acid that can be used in the present invention can have the same layer structure as that of various known dry analytical elements. The dry analytical element includes a support, a developing layer, a detection layer, a light shielding layer, an adhesive, in addition to the reagent for performing the reaction of Formula 2 or Formula 3 in the item (E) (detection of pyrophosphoric acid (PPi)) A multilayer including a layer, a water absorbing layer, an undercoat layer and other layers may be used. As such dry analytical elements, for example, Japanese Patent Laid-Open No. 49-53888 (corresponding US Pat. No. 3,992,158), Japanese Patent Laid-Open No. 51-40191 (corresponding US Pat. No. 4,042,335), and Japanese Patent Laid-Open No. There are those disclosed in Japanese Patent Application Laid-Open No. 55-164356 (corresponding US Pat. No. 4,292,272) and Japanese Patent Application Laid-Open No. 61-4959 (corresponding EPC Publication No. 0166365A).
[0054]
The dry analytical element that can be used in the present invention includes a reagent for converting pyrophosphoric acid into inorganic phosphorus and a reagent layer containing a reagent group that performs a color reaction according to the amount of inorganic phosphorus. An element is mentioned.
In this dry analytical element for quantifying pyrophosphate, it can be carried out as described above in this specification until pyrophosphoric acid (PPi) is enzymatically converted into inorganic phosphorus (Pi) using pyrophosphatase. By using the following “quantitative method of inorganic phosphorus” (and combinations of reactions used in them) known in the field of biochemical testing, a color development reaction corresponding to the amount of inorganic phosphorus (Pi) can be performed. it can.
[0055]
Note that when “inorganic phosphorus” is represented, phosphoric acid (phosphate ion) may be represented as “Pi” or “HPO ₄ ²⁻ , H ₂ PO ₄ ¹⁻ ”. In the example of the reaction shown below, “Pi” is used, but “HPO ₄ ²⁻ ” is sometimes used for the same reaction formula.
[0056]
As a quantitative method for inorganic phosphorus, an enzyme method and a phosphorus molybdate method are known. Hereinafter, an enzyme method and a phosphomolybdate method as quantitative methods for inorganic phosphorus will be described.
[0057]
A. There are a method using peroxidase (POD) and a method using glucose-6-phosphate dehydrogenase (G6PDH) depending on the enzyme used in the last “color reaction” in a series of reactions for quantitative detection of the enzyme method Pi. Hereinafter, specific examples of these methods will be described.
[0058]
(1) Example of method using peroxidase (POD) (1-1)
Inorganic phosphorus (Pi) is reacted with inosine by purine nucleoside phosphorylase (PNP), and the resulting hypoxanthine is oxidized by xanthine oxidase (XOD) to generate uric acid. Using hydrogen peroxide (H ₂ O ₂ ) generated in this oxidation process, 4-aminoantipyrine (4-AA) and phenol are oxidized and condensed with peroxidase (POD) to form a quinoneimine dye, To color.
[0059]
(1-2)
Pyruvate is oxidized with pyruvate oxidase (POP) in the presence of inorganic phosphorus (Pi), cocarboxylase (TPP), flavin adenine dinucleotide (FAD), and Mg ²⁺ to produce acetylacetic acid. Using hydrogen peroxide (H ₂ O ₂ ) generated in this oxidation process, 4-aminoantipyrine (4-AA) and phenol are oxidized by peroxidase (POD) as in (1-1) above. Condensation forms a quinoneimine dye, which is colorimetric.
[0060]
The final color reaction in the above (1-1) and (1-2) can be performed using a known “Trinder reagent” as a hydrogen peroxide detection reagent. In this reaction, phenol acts as a “hydrogen donor”. Phenols used as “hydrogen donors” are classic, and various improved “hydrogen donors” are currently used. Specific examples of such hydrogen donors include N-ethyl-N-sulfopropyl-m-anilysine, N-ethyl-N-sulfopropylaniline, N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline. N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-sulfopropyl-3,5-dimethylaniline, N-ethyl-N-sulfopropyl-m-toluidine, N-ethyl-N- (2 -Hydroxy-3-sulfopropyl) -m-anilysine, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline, N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, N-ethyl-N- (2-hydroxy- - sulfopropyl) -3,5-dimethylaniline, N- ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine, and the like N- sulfopropyl aniline.
[0061]
(2) Method using glucose-6-phosphate dehydrogenase (G6PDH) (2-1)
Inorganic phosphorus (Pi) and glycogen are reacted with phosphorylase to produce glucose-1-phosphate (G-1-P). The resulting glucose-1-phosphate is converted to glucose-6-phosphate (G-6-P) by phosphoglucomutase (PGM). In the presence of glucose-6-phosphate and nicotiamide adenine dinucleotide (NAD), NAD is reduced to NADH with glucose-6-phosphate dehydrogenase (G6PDH), and this is colorimetric.
[0062]
(2-2)
Inorganic phosphorus (Pi) and maltose are reacted using maltose phosphorylase (MP), and glucose-1-phosphate (G-1-P) is reacted. Hereinafter, similarly to the above (2-1), the resulting glucose-1-phosphate is converted to glucose-6-phosphate (G-6-P) by phosphoglucomutase (PGM). In the presence of glucose-6-phosphate and nicotiamide adenine dinucleotide (NAD), NAD is reduced to NADH with glucose-6-phosphate dehydrogenase (G6PDH), and this is colorimetric.
[0063]
B. Under phosphomolybdate method <br/> acid was complexing with inorganic phosphate (phosphate) and a water-soluble molybdate ion "phosphomolybdate _{(H 3 [PO 4 Mo 12} O 36]) the direct determination There are “direct method” and “reduction method” in which molybden blue (Mo (III)) is quantified from Mo (IV) to Mo (III) with a reducing agent following the reaction of the direct method. Examples of water-soluble molybdate ions include aluminum molybdate, cadmium molybdate, calcium molybdate, barium molybdate, lithium molybdate, potassium molybdate, sodium molybdate, and ammonium molybdate. Examples of typical reducing agents used in the reduction method include 1,2,4 aminonaphtholsulfonic acid, ferrous ammonium sulfate, ferrous chloride, stannous chloride-hydrazine, sulfate-p-methylamino. Phenol, N, N-dimethyl-phenylenediamine, ascorbic acid, malachite green and the like can be mentioned.
[0064]
A dry analytical element in the case of using a light-transmissive water-impermeable support can practically have the following configuration. However, the content of the present invention is not limited to this.
(1) One having a reagent layer on a support.
(2) One having a detection layer and a reagent layer in this order on a support.
(3) One having a detection layer, a light reflection layer, and a reagent layer in this order on a support.
(4) What has a 2nd reagent layer, a light reflection layer, and a 1st reagent layer in this order on a support body.
(5) What has a detection layer, a 2nd reagent layer, a light reflection layer, and a 1st reagent layer in this order on a support body.
[0065]
In the above (1) to (3), the reagent layer may be composed of a plurality of different layers. For example, the first reagent layer contains the enzyme pyrophosphatase required for the pyrophosphatase reaction shown in Formula 2 or Formula 3, the substrate xanthosine or the substrate inosine and the enzyme PNP required for the PNP reaction, and the second reagent layer contains the formula 2 or The enzyme XOD required for the XOD reaction shown in Formula 3 and the enzyme POD required for the POD reaction shown in Formula 2 or Formula 3 and the coloring dye (dye precursor) may be contained in the third reagent layer, respectively. Good. Alternatively, two reagent layers may be used, and the pyrophosphatase reaction and the PNP reaction may proceed in the first reagent layer, and the XOD reaction and the POD reaction may proceed in the second reagent layer. Alternatively, the pyrophosphatase reaction, the PNP reaction, and the XOD reaction may proceed in the first reagent layer, and the POD reaction may proceed in the second reagent layer.
[0066]
A water absorption layer may be provided between the support and the reagent layer or the detection layer. Moreover, you may provide a filtration layer between each layer. Further, a spreading layer may be provided on the reagent layer, and an adhesive layer may be provided therebetween.
[0067]
The support can be any of light-opaque (opaque), light-translucent (translucent), and light-transmissive (transparent), but is generally light-transmissive and water-impermeable. A support is preferred. Preferable materials for the light transmissive water-impermeable support are polyethylene terephthalate and polystyrene. In order to firmly adhere the hydrophilic layer, an undercoat layer is usually provided or a hydrophilic treatment is performed.
[0068]
When a porous layer is used as the reagent layer, the porous medium may be fibrous or non-fibrous. As the fibrous material, for example, filter paper, non-woven fabric, woven fabric (for example, plain woven fabric), knitted fabric (for example, tricot knitted fabric), glass fiber filter paper, and the like can be used. Examples of non-fibrous materials include membrane filters made of cellulose acetate described in JP-A-49-53888, JP-A-49-53888, JP-A-55-90859 (corresponding to US Pat. No. 4,258). , 001) Japanese Patent Application Laid-Open No. 58-70163 (corresponding US Pat. No. 4,486,537) and the like may be any of a continuous void-containing granular structure layer composed of inorganic or organic fine particles. JP 61-4959 A (corresponding European published EP 0166365A), JP 62-116258 A, JP 62-138756 A (corresponding European published EP 0226465A), JP 62-138757 ( Corresponding European publication EP 0226465A), JP-A-62-138758 (corresponding European publication EP 0226465A) and the like are also suitable.
[0069]
The porous layer may be a spreading layer having a so-called metering action that spreads the liquid in an area approximately proportional to the amount of liquid supplied. Of these, a woven fabric, a knitted fabric and the like are preferable as the spreading layer. A woven fabric or the like may be subjected to glow discharge treatment as described in JP-A-57-66359. In the development layer, in order to adjust the development area, the development speed, etc., JP-A-60-222770 (corresponding to: EP 0162301A), JP-A-62-219397 (corresponding to West German Patent Publication DE 3717913A), JP-A. A hydrophilic polymer or a surfactant as described in JP-A-63-112999 (corresponding: DE 3717913A) and JP-A-62-182652 (corresponding: DE 3717913A) may be contained.
[0070]
For example, after impregnating or applying the reagent of the present invention to a porous film made of paper, cloth, polymer or the like in advance, another water-permeable layer provided on the support, for example, a detection layer, is disclosed in JP-A-55. It is also a useful method to adhere by the method as described in Japanese Patent No. 1645356.
[0071]
The thickness of the reagent layer thus produced is not particularly limited, but when it is provided as a coating layer, a range of about 1 μm to 50 μm, preferably 2 μm to 30 μm is appropriate. In the case of a method other than coating, such as lamination by lamination, the thickness can vary greatly in the range of several tens to several hundreds of μm.
[0072]
When the reagent layer is composed of a water-permeable layer made of a hydrophilic polymer binder, examples of the hydrophilic polymer that can be used include the following. Gelatin and derivatives thereof (for example, phthalated gelatin), cellulose derivatives (for example, hydroxyethyl cellulose), agarose, sodium alginate, acrylamide copolymers and methacrylamide copolymers (for example, acrylamide or copolymers of methacrylamide and various vinyl monomers) Polymer), polyhydroxyethyl methacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, sodium polyacrylate, copolymers of acrylic acid and various vinyl monomers, and the like.
[0073]
The reagent layer composed of a hydrophilic polymer binder is disclosed in JP-B-53-21777 (corresponding US Pat. No. 3,992,158), JP-A-55-164356 (corresponding US Pat. No. 4,292,272), An aqueous solution or aqueous dispersion containing the reagent composition of the present invention and a hydrophilic polymer according to the method described in the specification of Kokai 54-101398 (corresponding US Pat. No. 4,132,528) or the like is used as a support or a detection layer. It can be provided by coating on another layer and drying. The dry thickness of the reagent layer containing a hydrophilic polymer as a binder is in the range of about 2 μm to about 50 μm, preferably about 4 μm to about 30 μm, and the coating amount is about 2 g / m ² to about 50 g / m ² , preferably about It is in the range of 4 g / m ² to about 30 g / m ² .
[0074]
In addition to the reagent composition of formula 2 or formula 3, the reagent layer has an enzyme activator, a coenzyme for the purpose of improving various properties such as coating properties, diffusibility of the diffusible compound, reactivity, and storage stability. In addition, surfactants, pH buffer compositions, fine powders, antioxidants, and other various additives made of organic or inorganic substances can be added. Examples of the buffering agent that can be contained in the reagent layer include “Chemical Handbook Basic” edited by the Chemical Society of Japan (Maruzen Co., Ltd., 1966), pages 1312-1320, R.C. M.M. C. Dawson et al, "Data for Biochemical Research" 2nd edition (Oxford at the Clarendon Press, published 1969), pages 476-508, "Biochemistry", pages 467-477 (1966), "Analytical 104" There is a pH buffer system described in pages 300-310 (1980). Examples of pH buffer systems include buffers containing borate; buffers containing citric acid or citrate; buffers containing glycine; buffers containing bicine; buffers containing HEPES; Good buffering agents such as buffering agents. A buffer containing phosphate cannot be used for a dry analytical element for detecting pyrophosphate.
[0075]
The dry analytical element for quantification of pyrophosphoric acid that can be used in the present invention can be prepared by known methods described in the aforementioned patent specifications. The pyrophosphoric acid quantification dry analytical element is cut into small pieces such as a square of about 5 mm to about 30 mm on one side or a circle of about the same size, Japanese Patent Publication No. 57-283331 (corresponding US Pat. No. 4,169,751), Akira Akira. No. 56-142454 (corresponding U.S. Pat. No. 4,387,990), JP-A-57-63452, JP-A-58-32350, JP-T-58-501144 (corresponding international publication: WO083 / 00391). It is preferable to use it as a chemical analysis slide by placing it in a slide frame as described above in terms of manufacturing, packaging, transportation, storage, measurement operation, and the like. Depending on the purpose of use, it is used in a long tape form in a cassette or magazine, or a small piece is placed in a container with an opening, or a small piece is affixed or placed in an open card, or a cut piece is used. It can be used as it is.
[0076]
The dry analytical element for quantifying pyrophosphoric acid that can be used in the present invention can quantitatively detect pyrophosphoric acid, which is an analyte in a liquid sample, by the same operations as those described in the above-mentioned patent specifications. For example, an aqueous liquid sample solution in the range of about 2 μL to about 30 μL, preferably 4 μL to 15 μL is spotted on the reagent layer. The spotted analytical element is incubated at a constant temperature in the range of about 20 ° C to about 45 ° C, preferably at a constant temperature in the range of about 30 ° C to about 40 ° C for 1 to 10 minutes. The color development or discoloration in the analytical element is reflected and photometrically measured from the side of the light-transmitting support, and the amount of pyrophosphoric acid in the sample can be determined by the principle of the colorimetric measurement method using a calibration curve prepared in advance. Quantitative analysis can be performed with high accuracy by keeping the amount of liquid sample to be spotted, incubation time and temperature constant.
[0077]
The measuring operation is disclosed in JP-A-60-125543, JP-A-60-220862, JP-A-61-294367, JP-A-58-161867 (corresponding to US Pat. No. 4,424,191). With the described chemical analyzer, highly accurate quantitative analysis can be performed with extremely easy operation. Depending on the purpose and required accuracy, semi-quantitative measurement may be performed by visually judging the degree of color development.
[0078]
In the pyrophosphoric acid quantitative dry analytical element that can be used in the present invention, it is stored and stored in a dry state until analysis is performed, so there is no need to prepare a reagent at the time of use, and generally in a dry state. Because of the high stability of the reagent, it is superior to the so-called wet method in which the reagent solution must be prepared at the time of use, and is superior in simplicity and speed. Moreover, it is also excellent as an inspection method capable of quickly performing a high-accuracy inspection with a small amount of liquid sample.
[0079]
The dry analytical element for inorganic phosphorus determination that can be used in the second embodiment of the present invention can be prepared by removing pyrophosphatase from the reagent layer in the pyrophosphoric acid quantitative dry analytical element. It is also possible to use a dry analytical element described in JP-A-7-197. The dry analytical element for quantitative determination of inorganic phosphorus is the same as the dry analytical element for quantitative determination of pyrophosphoric acid in the layer configuration, production method and method of use, except that the reagent layer does not contain pyrophosphatase.
Hereinafter, the present invention will be described in detail with reference to examples. However, the technical scope of the present invention is not limited by this embodiment.
[0080]
【Example】
<Reference Example 1 (Comparative Example)>
Single nucleotide polymorphism (SNPs) detection of aldehyde dehydrogenase gene (ALDH2 gene) related site using dry analytical element for quantification of pyrophosphate (Example in which the portion corresponding to single nucleotide polymorphism is set near the 3 'end of the primer) )
(1) Preparation of nucleic acid sample solution containing target nucleic acid fragment ALDH2 active type, ALDH2 low active type, inactive type due to different specific one base type of ALDH2 gene related site by sequencing of base sequence in advance 1 mL of a genomic nucleic acid fragment extracted and purified from each blood sample collected from one individual using a commercially available nucleic acid extraction and purification kit (QIAAMP DNA BloodMini Kit) By recovering in water, a nucleic acid sample solution containing the target nucleic acid fragment was prepared.
[0081]
(2) Preparation of dry analytical element for quantification of pyrophosphoric acid Composition shown in Table 1 on a colorless transparent polyethylene terephthalate (PET) smooth film sheet (support) having a thickness of 180 μm provided with a gelatin subbing layer The aqueous solution of (a) was applied so as to have the following coverage, and dried to provide a reagent layer.
[0082]
[Table 1]

[0083]
On this reagent layer, an adhesive layer aqueous solution having the composition (b) described in Table 2 below was applied so as to have the following coverage, and dried to provide an adhesive layer.
[0084]
[Table 2]

[0085]
Next, water is supplied over the entire surface of the adhesive layer at a rate of 30 g / m ² to swell the gelatin layer, and a pure polyester bronze woven fabric is applied almost uniformly and lightly on the laminating layer. To provide a porous spreading layer.
[0086]
Next, an aqueous solution of the composition (c) shown in Table 3 below is applied almost uniformly on the spread layer so as to have the following coverage, dried, cut into 13 mm × 14 mm, and placed in a plastic mounting material. As a result, a dry analytical element for quantifying pyrophosphoric acid was prepared.
[0087]
[Table 3]

[0088]
(3) PCR amplification Using the nucleic acid sample solution containing the target nucleic acid fragment obtained by extracting and purifying from the human whole blood sample of ALDH2 active type or ALDH2 inactive type in (1) above as it is, the following PCR amplification was performed under conditions.
[0089]
<Primer>
The primer is a common primer (upper) in the ALDH2 gene-related site on chromosome 12, and a portion corresponding to the single nucleotide polymorphism that determines the activity of ALDH2 is set near the 3 ′ end (lower-1 and A set of two primers (lower-1) and primer (lower-2) corresponding to ALDH2 active type and inactive type, which were underlined in the primer base sequence described in lower-2), were used. . Note that. The base 5 ′ upstream of the sequence corresponding to the single nucleotide polymorphism of the lower primer was changed (T → A) to artificially create a mismatch.
[0090]
Active detection primer (upper):
5′-AACGAAGCCCCAGCAAATGA-3 ′ (SEQ ID NO: 1)
Primer (lower-1):
5'-GGGCTGCAGGCATACACA G A-3 ' ( SEQ ID NO: 2)
Inactive detection primer primer (upper):
5′-AACGAAGCCCCAGCAAATGA-3 ′ (SEQ ID NO: 1)
Primer (lower-2):
5′-GGGCTGCAGGGCATACACA A A-3 ′ (SEQ ID NO: 3)
[0091]
PCR amplification was carried out by repeating 35 cycles of [denaturation: 94 ° C. for 20 seconds, annealing: 60 ° C. for 30 seconds, polymerase extension reaction: 72 ° C. for 1 minute 30 seconds] with the composition of the reaction solution shown below.
[0092]
<Composition of reaction solution>
10 x PCR buffer-5 μL
2.5 mM dNTP 5 μL
5 μM primer (upper) 2 μL
5 μM primer (lower-1 or -2) 2 μL
Taq 0.5μL
Nucleic acid fragment sample solution obtained in (1) 0.5 μL
Purified water 35μL
[0093]
(4) Detection using an analytical element for quantifying pyrophosphate The solution after the PCR amplification reaction in (3) is spotted on the dry analytical element for quantifying pyrophosphoric acid produced in (2) as it is. After incubating the dry analytical element for quantitative determination of phosphoric acid at 37 ° C. for 5 minutes, the reflection optical density (OD _R ) was measured from the support side at a wavelength of 650 nm. The results obtained are shown in Table 4 below.
[0094]
[Table 4]

[0095]
From the results of Reference Example 1, the relationship between the known active form of ALDH2 in the sample and the reflection optical density (ODr) measured using a dry analytical element for pyrophosphoric acid determination is consistent. That is, it can be seen that single nucleotide polymorphisms (SNPs) of aldehyde dehydrogenase gene (ALDH2 gene) related sites have been detected. However, it can be seen that it is difficult to determine the active type and the low activity type because the amount of pyrophosphate produced in each low activity type allele is different.
[0096]
<Example 1>
Single nucleotide polymorphism (SNPs) detection of aldehyde dehydrogenase gene (ALDH2 gene) related site using dry analytical element for quantification of pyrophosphate (example of primer bases set according to each allele)
[0097]
(1) Preparation of Nucleic Acid Sample Solution Containing Target Nucleic Acid Fragment Similar to Reference Example 1, one person each known to be ALDH2 active type, ALDH2 low active type, and inactive type in advance by sequencing the base sequence From the blood sample, a nucleic acid sample solution containing the target nucleic acid fragment was prepared.
[0098]
(2) Preparation of dry analytical element for quantification of pyrophosphoric acid The same procedure as in Reference Example 1 was performed.
[0099]
(3) PCR amplification Using the nucleic acid sample solution containing the target nucleic acid fragment obtained by extracting and purifying from the human whole blood sample of ALDH2 active type or ALDH2 inactive type in (1) above as it is, the following PCR amplification was performed under conditions.
[0100]
<Primer>
The primer is a common primer (upper) in the ALDH2 gene-related site on chromosome 12, and a portion corresponding to the single nucleotide polymorphism that determines the activity of ALDH2 is set near the 3 ′ end (lower-1 and A set of two primers (lower-1) and primer (lower-2) corresponding to ALDH2 active type and inactive type, which were underlined in the primer base sequence described in lower-2), were used. . In addition, in order to artificially create a mismatch, the base corresponding to one nucleotide polymorphism of the lower primer was changed from (T → A) to one base 5 ′ upstream and (C → A). Each of the things was used.
[0101]
○ Primer primer for active detection (upper):
5′-AACGAAGCCCCAGCAAATGA-3 ′ (SEQ ID NO: 1)
Primer (lower-1A):
5′-GGGCTGCAGGGCATACAC AG A-3 ′ (SEQ ID NO: 4)
Primer (lower-1C):
5′-GGGCTGCAGGGCATACAC CG A-3 (SEQ ID NO: 5)
○ Inactive detection primer primer (upper):
5′-AACGAAGCCCCAGCAAATGA-3 ′ (SEQ ID NO: 1)
Primer (lower-2A):
5′-GGGCTGCAGGGCATACAC AA A-3 ′ (SEQ ID NO: 6)
Primer (lower-2C):
5′-GGGCTGCAGGGCATACAC CA A-3 ′ (SEQ ID NO: 7)
[0102]
PCR amplification was carried out by repeating 35 cycles of [denaturation: 94 ° C. for 20 seconds, annealing: 60 ° C. for 30 seconds, polymerase extension reaction: 72 ° C. for 1 minute 30 seconds] with the composition of the reaction solution shown below.
[0103]
<Composition of reaction solution>
10 x PCR buffer-5 μL
2.5 mM dNTP 5 μL
5 μM primer (upper) 2 μL
5 μM primer (lower-1A, C or −2A, C) 2 μL
Taq 0.5μL
Nucleic acid fragment sample solution obtained in (1) 0.5 μL
Purified water 35μL
[0104]
(4) Detection using an analytical element for quantifying pyrophosphate The solution after the PCR amplification reaction in (3) is spotted on the dry analytical element for quantifying pyrophosphoric acid produced in (2) as it is. After incubating the dry analytical element for quantitative determination of phosphoric acid at 37 ° C. for 5 minutes, the reflection optical density (OD _R ) was measured from the support side at a wavelength of 650 nm. The results obtained are shown in Table 5 below.
[0105]
[Table 5]

[0106]
From the result of Example 1, the reflection optical density (ODr) measured using a dry analytical element for quantifying pyrophosphate is 1A as an active primer and 2C as an inactive primer in a low activity sample. Are equal. As a result, the distinction between the active / inactive type and the low active type can be made clear.
[0107]
【The invention's effect】
According to the present invention, single nucleotide polymorphisms can be detected accurately, simply and rapidly. In particular, according to the present invention, it is possible to clearly distinguish between heterozygous and homozygous alleles.
[0108]
[Sequence Listing]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[Brief description of the drawings]
FIG. 1 is a conceptual diagram illustrating an embodiment of the present invention.

Claims (12)

A method for detecting a single nucleotide polymorphism using two types of allele-specific primers designed so that the amount of amplification of each allele in a heterozygote is substantially the same. The method according to claim 1, wherein the allele-specific primer designed so that the polymorphic site is present within 4 bases from the 3 'end of the allele-specific primer. The method according to claim 1 or 2, wherein an allele-specific primer having a mismatch base introduced into the adjacent base of the polymorphic site is used. The method according to any one of claims 1 to 3, wherein an allele-specific primer in which a mismatch base adjacent to a polymorphic site is selected according to each allele is used. The method according to any one of claims 1 to 4, wherein a single nucleotide polymorphism is detected using a polymerase reaction. The method according to any one of claims 1 to 5, wherein a single nucleotide polymorphism is detected using a polymerase reaction product. The method according to claim 6, wherein the single nucleotide polymorphism is detected using electrophoresis, chromatography or HPLC as a detection means. The method according to any one of claims 1 to 5, wherein a single nucleotide polymorphism is detected using a side reaction product during a polymerase reaction. The method of claim 8, wherein the side reaction product is pyrophosphoric acid. The method according to claim 9, wherein pyrophosphoric acid is detected using a dry analytical element. The method according to any one of claims 1 to 10, wherein the detection of a single nucleotide polymorphism comprises discriminating a homozygous / heterojunction of a single nucleotide polymorphism. The primer set for performing the method in any one of Claim 1 to 11 containing the 2 types of allele-specific primer designed so that the amplification amount of each allele may become substantially the same.

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