JP4261830B2 - Method for determination of nucleic acid base sequence by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry - Google Patents

Description

【００１６】
化合物Ｉ
また、前記ニトロベンゼンを含む構造は、下記の一般式IIで示される化合物を用いて構築されていることもできる。
【００１７】
【化１１】

【００１８】
一般式II
（式中、ｎ＝３、または、４、X＝H、または、ＳＯ₃Ｎａである）
その際、前記基板は、表面に１級アミノ基が形成されたガラス基板であり、
前記プライマーの５’末端にスルファニル（ＳＨ）基が結合されており、
該アミノ基と、該スルファニル基との結合が、前記化合物Ｉ、あるいは、一般式IIで示される化合物を介して、アミノ基とこれら化合物のスクシイミドエステル部位との反応、及び、スルファニル基とこれら化合物のブロムベンジル部位との反応によって行われていることが望ましい。例えば、前記ガラス基板への１級アミノ基の形成は、１級アミノ基を有するシランカップリング剤を用いて行うことができる。あるいは、前記基板は、表面にスルファニル基が形成されたガラス基板であり、
前記プライマーの５’末端にアミノ基が結合されており、
該スルファニル基と、該アミノ基の結合が、化合物Ｉ、あるいは、一般式IIで示される化合物を介して、スルファニル基とこれら化合物のブロムベンジル部位との反応、及び、アミノ基とこれら化合物のスクシイミドエステル部位との反応によって行われている形態とすることも可能である。この時、例えば、前記ガラス基板へのスルファニル基の形成は、スルファニル基を有するシランカップリング剤を用いて行うことができる。
【００１９】
さらには、前記ニトロベンゼンを含む構造は、下記の一般式IIIで示される化合物を用いて構築されていることも可能である。
【００２０】
【化１２】

【００２１】
化合物III
（式中、ＤＭＴｒＯは、ジメトキシトリチルオキシ基、ＣＮＥｔは、２−シアノエチル基を表す。）
本発明の方法において、伸長反応に用いられる酵素が耐熱性であることが好ましい。加えて、特に、前記プライマーが固定されている基板は、複数のプライマー核酸がマトリクス状に配置された核酸チップの形態であり、
前記工程（３）において、該核酸チップ上で、該プライマー核酸の一部、または、全部について、それらの鋳型とともに酵素的核酸伸長反応を行ない、
前記工程（４）において、該伸長反応が行われたマトリクス部分についてＭＡＬＤＩ−ＴＯＦ ＭＳ法により分析する形態とすると、より好ましい。
【００２２】
【発明の実施の形態】
以下に、本発明をより詳しき説明する。
【００２３】
本発明の核酸塩基配列の決定方法では、以下の工程（１）〜（６）の各工程を行うことで核酸塩基配列の決定がなされる点に特長を有する。
工程（１）
基配列の解析を所望する核酸（ＤＮＡ）の、該塩基配列の解析を所望とする部位より３’側の塩基配列の一部、または、全部に相補的な核酸（ＤＮＡ）を、上記塩基配列の解析を所望する核酸を鋳型として酵素的核酸伸長反応するに必要なプライマーとして、光によって切断される部分構造を該プライマーの上記相補的な塩基配列よ５’側に含む構造で基板上に固定する工程；
工程（２）
上記塩基配列の解析を所望する核酸を、上記相補的な塩基配列部分で、上記基板上に固定されたプライマーとアニールしてハイブリッドを形成する工程；
工程（３）
酵素的核酸伸長反応に必要な４種の２’−デオキシヌクレオチド三リン酸（ｄＮＴＰ：ＮはＡ；アデニン、Ｇ；グアニン、Ｃ；シトシン、Ｔ；チミン）と、伸長反応のターミネーターとての４種の２’，３’−ジデオキシヌクレオチド三リン酸（ｄｄＮＴＰ）との適量共存下で、上記ハイブリッドを形成した基板上で、塩基配列を所望する核酸を鋳型として酵素的伸長反応を行う工程；
工程（４）
上記伸長反応が行われた基板から鋳型核酸を除去する工程；
工程（５）
光によって切断される部分構造を含む構造で基板に固定されているプライマー部分を含む、鎖長の異なる複数の伸長反応産物に、該切断される部分構造が切断される光を照射し、該光照射によって切断された上記伸長産物の分子量をＭＡＬＤＩ−ＴＯＦ ＭＳ法によって分析し、プライマーの分子量からの分子量増加量に基づき、上記伸長物の伸長部分の塩基配列を明らかにする工程；
工程（６）
上記伸長部分の塩基配列に基づいて、前記、塩基配列の解析を所望とする核酸の解析を所望する塩基配列の一部、または、全部を解析する工程。
【００２４】
本発明の方法によれば、伸長反応産物の基板からの切断を光照射によって行うので、上記文献の方法、すなわち、酸性物質を用いて切断する方法における課題である、デピュリネーションと呼ばれる現象によって、アデノシン、グアノシンのプリン部位で非選択的な切断の影響を本質的に回避することが可能となる。
【００２５】
その際、照射される光がＭＡＬＤＩ−ＴＯＦ ＭＳ法の分析時に使用されるレーザー光であれば、予め切断用の光照射を分析に先だって行う必要がないばかりか、切断と脱着、イオン化が同時に可能なのできわめて都合がよい。ＭＡＬＤＩ−ＴＯＦ ＭＳ法で用いられるレーザーは、一般的に波長３３７ｎｍの窒素レーザー光であるが、以下に示すようにこの波長で切断されうる有機構造はいくつかあり、従って窒素レーザーを本発明に利用することが可能である。もっとも、本発明は、この波長のレーザーに限定されるものではなく、例えば、Ｎｄ：ＹＡＧレーザーの波長５３２ｎｍ第二次高調波等も使用することができる。核酸は紫外線照射によりチミン二量体を形成するなどのダメージを受けることが知られているが、チミン二量体を形成を引き起こす紫外線の波長は、核酸塩基の吸収波長帯である、およそ、２５０〜２７０ｎｍであり、したがって、本発明で使用する上記レーザー波長は核酸にダメージを与えることなく好適に使用し得る。
【００２６】
本発明に使用される、光照射によって切断される構造は、特に限定されるものではないが、例として、ニトロベンゼン含む構造をあげることができる。より具体的な構造としては、下記の化合物Ｉ、または、一般式IIを用いて構築される構造をあげることができる。
【００２７】
【化１３】

【００２８】
化合物Ｉ
【００２９】
【化１４】

【００３０】
一般式II
（式中、ｎ＝３、または、４、X＝H、または、ＳＯ₃Ｎａである）
本発明で利用する、プライマーが結合した基板の具体的な作製方法としては、一例として、基板が表面に１級アミノ基が形成されたガラス基板であり、プライマーの５’末端にスルファニル（ＳＨ）基が結合されており、該アミノ基と、該スルファニル基の結合が、化合物Ｉ、あるいは、一般式ＩＩで示される化合物を介して、すなわち、アミノ基とこれら化合物のスクシイミドエステル部位との反応、及び、スルファニル基とこれら化合物のブロムベンジル部位との反応によって行われる方法をあげることができる。この際、ガラス基板への１級アミノ基の形成は、１級アミノ基を有するシランカップリング剤を用いる方法を例とすることができる。
【００３１】
【化１５】

【００３２】
上記の方法で、構築された基板に結合するプライマーの構造の一例を、上の化学式（左）に示す。また、光照射によって切断された構造の例を、同じく、上の化学式（右）に示す（Ｂｉｏｃｈｅｍｉｓｔｒｙ ＩｎｔｅｒｎａｔｉｏｎａｌＶｏｌ． ２６ Ｎｏ． ５， １９９２）。例示するように、切断された後の核酸の切断された部位の構造は、切断される前の構造と同じであることがわかる。すなわち、この場合、ＭＡＬＤＩ−ＴＯＦ ＭＳ法によるシークエンシング時には、伸長反応によって、プライマーの元の分子量から一塩基ずつの増加が観察されることが期待される。
【００３３】
基板作製方法の他の例としては、基板が表面にスルファニル基が形成されたガラス基板であり、プライマーの５’端にアミノ基が結合されており、該スルファニル基と、該アミノ基の結合が、化合物Ｉ、あるいは、一般式IIで示される化合物を介して、すなわち、スルファニル基とこれら化合物の部ブロムベンジル部位との反応、及び、アミノ基とこれら化合物のスクシイミドエステル部位との反応によって行われる方法をあげることができる。その際、ガラス基板へのスルファニル基の形成は、スルファニル基を有するシランカップリング剤を用いて行われる方法が例示できる。
【００３４】
また、本発明の方法に利用可能な、ニトロベンゼンを含む構造の他の例として、下記の化合物IIIで構築される構造をあげることができる。この場合、プライマー核酸を核酸自動合成機で合成する際に、５’末端に基板に結合させるべき官能基を有するユニットを導入する直前に化合物IIIにより、光開裂が可能な構造を導入することができる。この場合、基板に結合させるべき官能基としては、アミノ基、スルファニル基を例としてあげることができ、そのような官能基を核酸自動合成機中で導入する試薬は、例えば、グレンリサーチ社から販売されているので、それらを適宜利用すればよい。また、基板側の処理は、既述のシランカップリング剤を利用した方法を、この場合でも採用可能である。
【００３５】
【化１６】

【００３６】
化合物III
（式中、ＤＭＴｒＯは、ジメトキシトリチルオキシ基、ＣＮＥｔは、２−シアノエチル基を表す。）
これまで述べてきたように、本発明の方法では、固相上に固定されたプライマーとアニールした核酸を鋳型として、酵素的核酸伸長反応を行うが、その際、伸長反応に用いる酵素と、プライマーが結合した基板、あるいは、鋳型核酸それぞれの共存下でアニール、ついで、伸長反応を行うことができれば効率がよいが、アニールの際には、場合によりプライマー結合基板、鋳型核酸を９０℃程度に昇温させる必要がある。その場合、一般的な酵素では、このような高温に耐えることができない。このような場合、耐熱性の酵素を用いると都合がよい。そのような酵素の例として、Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ ＤＮＡ Ｐｏｌｙｍｅｒａｓｅ（アマシャムファルマシアバイオテク）をあげることができる。
【００３７】
さて、従来技術の有する問題点のひとつとして、伸長反応等の反応を、シークエンシングを所望する核酸について個別に行う必要がある点をすでにあげた。また、それは、プライマーが固相上に結合されていても同様である点もすでに述べた。
【００３８】
本発明ではこの問題点を解決する手段として、複数のプライマー核酸が、これまで述べた手段を用いて、マトリクス状に固定配置された、言謂、核酸チップ上で、該プライマー核酸の一部、または、全部について、それらの鋳型とともに酵素的核酸伸長反応を行ない、該伸長反応が行われたマトリクス部分について、既述したＭＡＬＤＩ−ＴＯＦ ＭＳ法により分析する方法を採用することを特長のひとつとする。この際、プライマーの塩基配列を、すくなくとも一回の伸長反応液中に含まれるシークエンシングを所望とする各々の核酸についてユニークな配列とすれば、各々の核酸は、それぞれの伸長反応に必要なプライマーのみを認識するので、これらの全ての核酸を鋳型とする伸長反応を一枚のチップ上で、一度の反応で行うことが可能である。
【００３９】
核酸チップの作製方法の例として、特開平１１−１８７９００号公報には、１級アミノ基が形成された基板に、スルファニル基を有する核酸との結合に、下記の化学式ＩＶで示す、Ｎ−（６−マレイミドカプロイルオキシ）スクシイミドを用いる例の記載があるが、本発明では、この化学式ＩＶの化合物に代えて、化合物Ｉ、あるいは、一般式IIで示される化合物を用いることで、各マトリクスにおいて、それぞれ核酸塩基配列の解析が可能となる。
【００４０】
【化１７】

【００４１】
化合物ＩＶ
さて、ＭＡＬＤＩ−ＴＯＦ ＭＳ法では、先に述べたように、測定したい物質（被検物質、本発明ではプライマー部分を含む伸長産物）を脱離、及び、イオン化させるために、マトリクスという物質を被検物質と共存させる。その方法として、よく行われる方法は、マトリクスの飽和溶液に被検物質を適当な濃度で溶解し、この共存溶液の、例えば、数μｌを、適宜アドレスが施されたステンレスプレートに滴下、乾燥させ、マトリクスの結晶内に被検物質が適当な濃度で共存している状態とする。この場合、マトリクスの結晶状態、三次元形態、被検物質の濃度によっては、スペクトルが得られなかったり、得られたとしても、Ｓ／Ｎ比、精度が良好でない場合がある。また、マトリクスの結晶の表面に微小ではあれ、凹凸、傾斜がある場合には、マトリクス中に被検物資が存在するために、場合によっては飛行時間に影響を与える結果、マススペクトルのマス精度に悪影響を与えることがある。本発明では、伸長産物は平滑な基板表面に固定化されているので、飛行時間に影響を与える懸念は比較的小さい。また、被検物質に対するマトリクス物質の供給も、場合により、基板に対してマトリクスを薄膜状にコーティングすることにより、均一に行うことができ、また、マトリクスの結晶状態も良好となるために、良好なマス精度を得ることが可能となり、結果として、精度のよい塩基配列の決定が行われる。
【００４２】
上記、マトリクス物質のコーティングは、ディッピング、スピンコーティング法等を適宜利用することができる。また、コーティングの厚さに関しては、厚すぎると、場合によっては、マトリクス物質の層内部に被検物質が埋め込まれた状態となるため、被検物質の基板表面からの切断、脱離、イオン化が良好に行われない場合がある。また、薄すぎると、被検物質がマトリクスから顔を出した状態となり、やはり、基板表面からの切断、脱離、イオン化が良好に行われない場合がある。従って、コーティングの厚さは、被検物質の切断、脱離、イオン化に必要、且つ、十分な厚さに選択することが最も好ましい。その際、上記、被検物質の切断、脱離、イオン化に必要、且つ、十分なマトリクス物質のコーティングの厚さは、被検物質が存在する基板からの高さと同程度から千倍程度が好適であるが、もちろん、この厚さに限定されるものではない。
【００４３】
【実施例】
以下に、実施例を挙げて、本発明をより具体的に説明する。
【００４４】
（実施例１）
dT40プライマーによる核酸結合基板の作製
特開平１１−１８７９００号公報に記載の方法に準じて、核酸プライマーが一様に結合した基板を作製した。前記公開公報に記載の方法と異なる点は、核酸プライマーの固定に利用する二官能性架橋剤を、Ｎ−（６−マレイミドカプロイルオキシ）スクシイミド（化合物IV）から、一般式IIで示される化合物のひとつである下記化合物V、すなわち、スクシイミジル６−（４−ブロモメチル−３−ニトロベンゾイル）アミノヘキサノエートに変更したことと、基板上に、マトリクス状ではなく、一様に核酸プライマーを結合させている点である。
【００４５】
【化１８】

【００４６】
化合物Ｖ
（１）基板洗浄
２５．４ｍｍ×２５．４ｍｍ×１ｍｍの合成石英基板をラックに入れ、純水で１０％に稀釈した超音波洗浄用洗剤（ブランソン：ＧＰIII）に一晩浸した。その後、洗剤中で２０分間超音波洗浄を行い、その後、水洗により洗剤を除去した。純水ですすいだ後、純水の入った容器中でさらに超音波処理を２０分間行った。次に、予め８０℃に加温した１Ｎ水酸化ナトリウム水溶液に基板を１０分間浸した。引き続き水洗、純水洗浄を行って、洗浄済基板をそのまま次の表面処理工程に供した。
【００４７】
（２）表面処理
アミノ基を結合したシランカップリング剤、Ｎ−β−（アミノエチル）−γ−アミノプロピルトリメトキシシラン ＫＢＭ６０３（信越化学工業）の１ｗｔ％水溶液を室温下で２時間攪拌し、上記シラン化合物の分子内のメトキシ基を加水分解した。次いで、この溶液に（１）で得た洗浄済基板を室温で1時間浸漬した後、純水で洗浄し、窒素ガスを基板の両面に吹き付けて乾燥させた。次に基板を１２０℃に加熱したオーブン中で１時間ベークして最終的に基板表面にアミノ基を導入した。
【００４８】
次いで、化合物Ｖ（同仁化学研究所）５ｍｇを、ジメチルスルホキシド（ＤＭＳＯ）／エタノールの１：１（容量比）混合溶媒に濃度が０．５ｍｇ／ｍｌとなる様に溶解した。シランカップリング処理を行った石英基板を、この化合物Ｖの溶液に室温で２時間浸漬して、シランカップリング処理によって基板表面に担持されているアミノ基と化合物Ｖのスクシイミド基を反応させた。この段階で基板表面に化合物Ｖ由来のブロモエチル基が存在することになる。化合物Ｖの溶液から引き上げた基板は、ＤＭＳＯ／エタノール混合溶媒、及びエタノールで順次洗浄した後、窒素ガスを吹き付けて乾燥させた。
【００４９】
（３）プローブＤＮＡの合成
ＤＮＡ合成業者（ベックス）に依頼して、配列番号１の一本鎖核酸（ｄＴの４０量体）を合成した。なお、配列番号１の一本鎖ＤＮＡの５’末端には、合成時にチオールモディファイア（グレンリサーチ）を用いることによって、スルファニル（ＳＨ）基を導入した。なお、脱保護、ＤＮＡの回収は定法により行い、また、精製にはＨＰＬＣを用いた。合成から精製までの一連の工程は、全て合成業者に依頼して行った。また、配列番号１の核酸の分子量計算値は、１２３０２．１７Ｄａである。
配列番号：１
５’ ＨＳ−（ＣＨ₂）₆−Ｏ−ＰＯ₂−Ｏ−ＴＴＴＴＴＴＴＴＴＴ ＴＴＴＴＴＴＴＴＴＴ ＴＴＴＴＴＴＴＴＴＴ ＴＴＴＴＴＴＴＴＴＴ ３’
（４）ＤＮＡの基板への結合
（３）に記載の配列番号１の一本鎖ＤＮＡを、８μＭの濃度で、グリセリン７．５ｗｔ％、尿素７．５ｗｔ％、チオジグリコール７．５ｗｔ％、アセチレンアルコール（商品名：アセチレノールＥＨ；川研ファインケミカル（株）社製）１ｗｔ％を含む溶液に溶解した。
【００５０】
次に、上記ＤＮＡ溶液２５μｌを、（２）で表面処理したガラス基板にのせ、１８ｍｍ×１８ｍｍのカバーガラスで覆い、ガラス板表面のブロモエチル基と核酸プローブ末端のスルファニル基とを室温で反応させた。３０分後、基板を純水で洗浄し、純水中で保存した。
（実施例２）
伸長反応と、ＭＡＬＤＩ−ＴＯＦ ＭＳによる分析
（１）伸長反応
予め、ＤＮＡシーケンシング用ＤＮＡポリメラーゼ（Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ：アマシャムファルマシアバイオテク）を、添付の希釈用バッファーで１ｕｎｉｔ／μｌに希釈しておく。
【００５１】
０．５ｍｌのエッペンドルフチューブに、Ｔｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅに添付の反応バッファー４．５μｌ、Ｐｏｌｙ（ｄＡ）溶液（アマシャムファルマシアバイオテク：平均鎖長３００を０．２５μｇ／μｌの濃度で純水に溶解したもの）５μｌを採り、４種デオキシリボヌクレオチド混液（Ｓｅｑｕｅｎｃｉｎｇ Ｇｒａｄｅ Ｓｏｌｕｔｉｏｎ ｄＮＴＰｓ：アマシャムファルマシアバイオテク；１００ｍＭ）１μｌ、４種ジデオキシリボヌクレオチド混液（Ｓｅｑｕｅｎｃｉｎｇ Ｇｒａｄｅ Ｓｏｌｕｔｉｏｎ ｄｄＮＴＰｓ：アマシャムファルマシアバイオテク；５ｍＭ）１μｌを加え、さらに純水を加えて、総容量を４３μｌに調製する。
【００５２】
この溶液に、予め希釈しておいたＴｈｅｒｍｏ Ｓｅｑｕｅｎａｓｅ溶液を２μｌ加え、よく混合した後、２０μｌをハイブリダイゼーション用インキュベーション・チャンバー（ＣｏｖｅｒＷｅｌｌ ２０μｌ Ｃｈａｎｂｅｒ：フナコシ）のウェル内に注ぎ、この上に、実施例１で作製したＤＮＡ結合基板の処理をほどこした側が液に触れるようにおき、液が漏れないように密着させる。温度調節が可能なホットプレートに、チャンバーをおいて、８５℃で１０分間加熱し、ついで、５０℃のインキュベーター中で３０分間放置して、伸長反応を行った。反応終了後、基板をチャンバーから剥して、８５℃の純水で２分間洗い、次に、流純水で１分間リンスして、基板から鋳型ＤＮＡ、未反応のモノマーなどを洗い流した。つぎに、基板に窒素ガスを吹き付けて水を除去した後、イオン交換樹脂ＡＧ ５０Ｗ−Ｘ８（ＢＩＯ−ＲＡＤ）の適当量を１００μｌに分散して、チップ表面の分析対象部分を含む領域にのせ、５分間、室温に放置して脱塩処理を行った。ついで、純水で洗浄し、窒素ガスで水を除いた後、真空デシケーター中で保存した。
（２）ＭＡＬＤＩ−ＴＯＦ ＭＳによる分析
伸長反応を行った基板を、以下の条件でＭＡＬＤＩ−ＴＯＦ ＭＳによる分析を行った。なお、分析時、基板はＭＡＬＤＩ−ＴＯＦ ＭＳ分析用のステンレスプレート（日本ブルカー・ダルトニクス）に、ステンレスピンとステンレス製の基板押さえを用いて固定した。
装置名：ａｕｔｏｆｌｅｘ Ｒｅｆｌｅｃｔｒｏｎ（日本ブルカー・ダルトニクス）
レーザー：窒素レーザー
加速電圧：２０ＫＶ
測定モード：リニアモード
イオン化：ポジティブ
内部標準：オリゴヌクレオチド；６１１７、９１９１Ｄａ
仕様マトリクス：３−ヒドロキシ−２−プロピオン酸（３−ＨＰＡ）
（３）分析結果
分子量１２５８８．９５Ｄａを最小として、３０４．１９Ｄａの分子量が順次増加していく質量スペクトルが観察された。配列番号１のＤＮＡが光照射によって切断された場合の分子量計算値は１２３０２．１７であり、ここに、ｄｄＴＴＰが結合した（結合部分は単純なリン酸ジエステルとなる）場合の分子量計算値は１２５９０．３６Ｄａとなる。また、ｄｄＴＴＰによってターミネートされる前に、ｄＮＴＰが順次付加されていくと、その分子量増加計算値は３０４．１９Ｄａである。よって、観察された順次分子量が増加していくピークはｄＮＴＰが一分子ずつ増加しているものと考えられ、ここから逆に、モデル的に塩基配列を求めようとした鋳型のＤＮＡがアデノシンの連続配列であることを知ることができる。すなわち、本発明の方法により核酸の塩基配列の解析が可能であることがわかる。
【００５３】
なお、観察された分子量の絶対値が、理論分子量からズレを生じている理由は、質量較正に用いた内部標準と実際に分析したＤＮＡの分子量差が大きかったため、測定値に系統的なズレを生じた結果と考えられる。
【００５４】
（実施例３）
ファージDNA塩基配列のMALDI-TOF MSによる解析
（１）DNAチップの作製
基板として、スライドグラス上に黒色テフロン（登録商標）樹脂のウェル（直径２ｍｍ）が３０個形成されたテフロンプリント・スライドグラス（ＥＲ−２１２：フナコシ薬品）を用い、実施例１の基板洗浄前に、ＵＶ−オゾン処理を施した後に、実施例１の基板洗浄、および、表面処理を行った。
配列番号：２
５’ ＨＳ−（ＣＨ₂）₆−Ｏ−ＰＯ₂−Ｏ−ＴＧＴＡＡＡＡＣＧＡＣＧＧＣＣＡＧＴ ３’
次に、配列番号２のＤＮＡを実施例１と同様に溶解し、上記表面処理を施したスライドグラスのウェルに４μｌずつ供給し、保湿チャンバー中に室温下３０分間放置し、ＤＮＡと基板を反応させた。次いで、純水で洗浄し、純水中に保存した。なお、配列番号２の核酸は、一本鎖（＋ｓｔｒａｎｄ）バクテリオファージＭ１３の６３１８から６３０１の塩基配列に相補的な塩基配列であり、分子量計算値は５７２８．８４である。
（２）伸長反応
実施例２の鋳型ＤＮＡをＭ１３ｍｐ１８（＋）Ｓｔｒａｎｄ ＤＮＡ（アマシャムファルマシアバイオテク）２．５μｇとする以外は全く同様な条件で伸長反応と後処理を行った。また、脱塩処理も各ウェルについて行った。
【００５５】
（３）ＭＡＬＤＩ−ＴＯＦ ＭＳによる分析
伸長反応を行ったＤＮＡチップをＭＡＬＤＩ−ＴＯＦ ＭＳにより分析した。なお、分析はウェルの内部にレーザーのスポットを照射して行った。また、使用した内部標準核酸は３６４５Ｄａ、６１１７Ｄａ、９１９１Ｄａである。
【００５６】
（４）分析結果
分子量６０４２．１０Ｄａを最小として、分子量が順次増加していく質量スペクトルが観察された。配列番号２のＤＮＡにｄｄＧＴＰが結合した場合、その分子量計算値は、６０４２．０５Ｄａなので、最初にプライマーに付加したのはＧ、すなわち、鋳型ではＣの配列であることがわかる。同様にして、順次分子量増分に基づき解析を進め、鋳型の塩基配列は、最初のＣからＣＧＧＴＴＣＧＡＡＣＧＴＡＣＧＧＡＣＧＴ ＣＣＡＧＣＴＧＡＧＡ ＴＣＴＣＣＴＡＧＧＧ ＧＣＣＣＡＴＧＧＣＴ ＣＧＡＧＣＴＴＡＡＧ・・・と約１８０塩基まで解析された。解析された塩基配列は、文献に記載されている塩基配列と完全に一致した。これにより、本発明の方法により、プライマー・チップを用いて鋳型の塩基配列の解析が可能であることがわかる。
（実施例４）
Ｐｏｌｙ（ｄＡ）、ファージＤＮＡ塩基配列の同時解析
実施例３と同様な方法で、実施例２と実施例３で用いた二種のプライマーをチップ上の異なるマトリクスに搭載したＤＮＡチップを作製し、実施例２で用いたＰｏｌｙ（ｄＡ）、実施例３で用いたＭ１３ｍｐ１８（＋）Ｓｔｒａｎｄ ＤＮＡの共存下で伸長反応を行ない、これまで述べた方法より、各マトリクスについてＭＡＬＤＩ−ＴＯＦ ＭＳによる解析を行った。その結果、各マトリクスから、それぞれ、実施例２と実施例３と同様な塩基配列情報がえられた。すなわち、プライマー・チップの各マトリクスに、対象とする鋳型核酸にユニークなプライマーを搭載し、これを一括した伸長反応に供することにより、それぞれの鋳型について、その塩基配列の解析を並行して進めることが可能であることがわかる。
【００５７】
【発明の効果】
本発明の方法を用いることで、基板上に結合された核酸をプライマーとして利用し、解析対象の核酸を鋳型とする伸長反応を行い、その後、伸長産物のＭＡＬＤＩ−ＴＯＦ ＭＳによる分析により、前記伸長反応の鋳型とする、解析対象核酸の塩基配列の解析を高い作業効率で行うことが可能となった。また、複数の核酸を、基板上に配置されたプライマー・チップを用いることで、簡便に同様の塩基配列の解析も可能となった。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for determining a nucleic acid base sequence, and in particular, in base sequence analysis based on the dideoxy method, matrix-assisted laser desorption / ionization flight time for specifying the type of single base added to a sequence primer in association with an extension reaction. The present invention relates to a method for determining a nucleobase sequence using a type mass spectrometry.
[0002]
[Prior art]
Under an international project, work on elucidation of the entire base sequence of human genomic DNA has progressed. In 2000, a draft sequence of more than 90% of bases was published, and human genes were released. The elucidation of the gene information that has been included has become widely known to the public as one of the major achievements of science and technology development in recent years.
[0003]
Nucleic acid base sequence reading, i.e., sequencing, began in the early 1970s and has become the mainstream after the early Maxam-Gilbert method (Processing of the National Academy of Sciences, USA 74, 560, 1977). The sequencing method is based on the dideoxy method (Proceeding of the National Academy of Sciences, USA 74, 5463, 1977) developed by Sanger et al.
[0004]
The dideoxy method subsequently changed, and is currently divided into three different types, ie, four types labeled with four fluorescent dyes, ie, 2 ', 3'-dideoxynucleotides of adenosine, guanosine, cytidine, and uridine. A technique combining a dye (dye) termination method using phosphoric acid (ddNTP) and a capillary electrophoresis method is mainly used as a sequencing method. The following is a brief description of this method.
(1) A nucleic acid (DNA) complementary to a part or all of the base sequence 3 'from the site desired to analyze the base sequence of the nucleic acid (DNA) for which base sequence analysis is desired, As a sequence primer to be used in the enzymatic nucleic acid extension reaction using the nucleic acid whose base sequence analysis is desired as a template, anneal with the nucleic acid whose base sequence analysis is desired, and hybridize with the complementary base sequence part. Form.
(2) Four kinds of 2′-deoxynucleotide triphosphates (dNTP: N is A; adenine, G: guanine, C: cytosine, T; thymine) necessary for enzymatic nucleic acid elongation reaction, and as a terminator of the elongation reaction In the presence of an appropriate amount of the above-described four kinds of fluorescently labeled 2 ′, 3′-dideoxynucleotide triphosphates (ddNTPs), the nucleotide sequence is derived from the sequence primer that has formed the hybrid, and the desired nucleic acid is used as a template. Perform an elongation reaction.
(3) The hybrid body subjected to the extension reaction is dissociated into single strands.
(4) For the solution containing the obtained extension products of a plurality of chain lengths, capillary electrophoresis is performed to separate the chain lengths, and the extended base is A, from the fluorescence of the part extended by one base from the sequence primer. Knowing whether it is G, C, or T, a part of the extended part or the remaining base sequence is obtained.
(5) Based on the base sequence of the extended portion, a part or all of the base sequence desired to analyze the nucleic acid whose base sequence is desired is analyzed.
[0005]
Recently, for example, an apparatus that can finish the same number of electrophoresiss in 2 to 3 hours using 96 or 384 capillary bundles has been developed. Automation using robots is also being planned. Large amounts of data analysis can also be processed at high speed by high-performance computers. As a result, the whole genome analysis of humans already described has been carried out by these fluorescent dideoxy methods and capillary electrophoresis methods.
[0006]
[Problems to be solved by the invention]
However, these widely used base sequence analysis methods based on the fluorescent dideoxy method and capillary electrophoresis also have the following problems. That is, as already mentioned,
2 to 3 hours are required for one electrophoresis,
The gel in the gel capillary needs to be changed after each run,
The capillaries are expensive and need to be regularly replaced,
It is not always possible to read a single base difference reliably, and for this reason, it may be necessary to perform multiple runs on one extension product,
Enzyme extension reactions must be performed individually, etc.
[0007]
Among these problems, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (especially as a new separation and analysis method that may solve the problems related to electrophoresis, M atrix- A sisted L aser D esorpion / I onization T ime-of- F light M ass S (hereinafter, abbreviated as MALDI-TOF MS) has been proposed.
[0008]
In the MALDI-TOF MS method, a substance called a matrix that absorbs specific light and a test substance are mixed and placed (adsorbed) on a stainless steel substrate for analysis, for example, where the matrix absorbs. By irradiating light, the test substance is desorbed / ionized by energy transfer from the matrix to the test substance. The basic principle of the MALDI-TOF MS method is to perform time-of-flight mass analysis of the desorbed ions. According to this MALDI-TOF MS method, one analysis can be completed in a few seconds to a few tens of seconds, and, for example, 384 kinds of samples are placed on one plate and automatically analyzed. Dozens of plates can be automatically analyzed as well. Therefore, for example, tens of thousands of samples can be automatically measured overnight, and the time efficiency is extremely high. Further, since the molecular weight can be known as an absolute value, ambiguity is eliminated, and the plate can be used any number of times.
[0009]
That is, it is considered that nucleic acid sequencing with extremely high work efficiency is possible if the nucleic acid extension product described above can be analyzed by the MALDI-TOF MS method. However, the extension product is a mixture of an enzyme, each nucleotide monomer, each dye-labeled terminator, a salt, and the like. In such a case, there is a problem in a method for efficiently desorbing / ionizing the desired extension product (Genomics Vol. 19, 417, 1994). Further, even if the extension product is purified, there is a problem that a lot of labor is required particularly when trying to sequence many nucleic acids.
[0010]
As a means to solve these problems, a primer is immobilized on a solid phase, an enzyme extension reaction is performed here, and then the extension product is cut out from the solid phase and subjected to mass spectrometry by MALDI-TOF MS method, and sequencing is performed. Has been devised (Nucleic Acid Research Vol. 29, No. 18, 3864, 2001). In this method, an acid-decomposable NP internucleotide bond is introduced at an arbitrary nucleotide site extending from a linker, and an acidic substance such as trifluoroacetic acid is mixed in an acidic matrix to thereby form the NP bond. The fragment is cleaved and analyzed for the cleaved fragment. By using the dideoxy method in combination, in principle, base sequence information after the cleaving point can be obtained. In addition, since the extension product including the primer portion is immobilized on the solid phase before cleavage, it is relatively easy to remove other impurities such as the template and nucleotide monomers. However, this method has the following problems. That is, the nucleic acid causes non-selective cleavage at the purine site of adenosine and guanosine due to a phenomenon called depuration in acidity, so that the acidity at the time of cleavage of the NP bond cannot be increased. As a result, the efficiency of cutting, desorption, and ionization is poor, so that sufficient sensitivity of mass spectrometry cannot be obtained. In addition, in the method described in the above document, since only one kind of primer is bonded to one substrate, in order to simultaneously sequence a plurality of nucleic acids, a corresponding number of substrates are used. On the other hand, there was also a problem that an extension reaction was necessary.
[0011]
The present invention solves the above-mentioned problems, and an object of the present invention is to fix a primer on a solid phase, perform an enzyme extension reaction, and then cut out the extension product from the solid phase to obtain a MALDI-TOF MS. When adopting the method of mass spectrometry and sequencing, the extension product is cleaved from the solid phase, NP internucleotide bond capable of acid degradation is introduced, and acidic substances such as trifluoroacetic acid are mixed In addition, a means capable of cleaving site-selectively in the portion of the primer linked to the solid phase without using an acidic substance, instead of a means for cleaving such an NP bond, An object of the present invention is to provide a method for analyzing a nucleic acid base sequence capable of maintaining the efficiency of desorption and ionization when mass spectrometry is performed by the MALDI-TOF MS method.
[0012]
[Means for Solving the Problems]
As a result of diligent studies to solve the above-mentioned problems, the present inventors have ligated the primer to the solid phase as a means capable of site-selective cleavage in the primer portion linked to the solid phase. As a means that can be selectively cleaved at the part, a partial structure that is cleaved by light is provided on the 5 ′ side in the primer so as not to affect hybridization with the template, and this is applied by predetermined light irradiation after the extension reaction. The principle of the conventional method of causing non-selective cleavage at the purine site of adenosine and guanosine by adopting a method of selective cleavage with a partial structure that is cleaved by light, which is called depuration in acidity. The present inventors have found that nucleic acid sequencing can be performed with extremely high work efficiency while avoiding such problems, and the present invention has been completed.
[0013]
That is, the method for determining a nucleobase sequence according to the present invention includes:
A method for determining a nucleobase sequence comprising:
(1) A nucleic acid (DNA) complementary to a part or all of the base sequence 3 'from the site desired to analyze the base sequence of the nucleic acid (DNA) for which base sequence analysis is desired, A structure comprising a partial structure that is cleaved by light as a primer used in the enzymatic nucleic acid extension reaction using a nucleic acid whose analysis of the base sequence is desired as a template, on the 5 ′ side of the complementary base sequence in the primer Fixing on the substrate with
(2) A step of annealing a nucleic acid desired to be analyzed for the base sequence with a primer fixed on the substrate at the complementary base sequence portion to form a hybrid;
(3) Four kinds of 2′-deoxynucleotide triphosphates (dNTP: N is A; adenine, G: guanine, C: cytosine, T; thymine) necessary for enzymatic nucleic acid extension reaction, and as a terminator of extension reaction In the presence of an appropriate amount of four kinds of 2 ′, 3′-dideoxynucleotide triphosphates (ddNTPs), an enzymatic extension reaction is performed on the hybrid-formed substrate using the nucleic acid having the desired base sequence as a template. Process;
(4) A step of removing the template nucleic acid from the substrate on which the extension reaction has been performed;
(5) Light with which a partial structure to be cleaved is cleaved with respect to a plurality of extension reaction products having different chain lengths, including a primer portion that is fixed to a substrate with a structure that includes a partial structure that is cleaved by light. Irradiated,
Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry analysis of the molecular weight of the extension product cleaved by the light irradiation ( M atrix- A sisted L aser D esorpion / I onization T ime-of- F light M ass S (hereinafter, abbreviated as MALDI-TOF MS) method,
Clarifying the base sequence of the extension of the extension based on the molecular weight increase from the molecular weight of the primer in the extension product;
(6) a step of analyzing a part or all of a base sequence desired to analyze a nucleic acid desired to analyze the base sequence based on the base sequence of the extended portion;
A method for determining a nucleobase sequence comprising at least the steps (1) to (6) above. At that time, in the step (5), it is preferable that the irradiated light is a laser beam used in the analysis of the MALDI-TOF MS method. More preferably, the laser beam used in the analysis of the MALDI-TOF MS method is a nitrogen laser beam having a wavelength of 337 nm.
[0014]
In the method of the present invention, in the step (5), a structure containing nitrobenzene can be selected as a partial structure to be cut by light irradiation. For example, the structure containing the nitrobenzene can be constructed using the following compound I.
[0015]
[Chemical Formula 10]

[0016]
Compound I
The structure containing nitrobenzene can also be constructed using a compound represented by the following general formula II.
[0017]
Embedded image

[0018]
Formula II
(Where n = 3 or 4, X = H or SO _Three Na)
At that time, the substrate is a glass substrate having a primary amino group formed on the surface,
A sulfanyl (SH) group is bound to the 5 ′ end of the primer,
The bond between the amino group and the sulfanyl group is obtained by reacting the amino group with a succinimide ester moiety of the compound via the compound I or the compound represented by the general formula II, and a sulfanyl group. It is desirable to be carried out by reaction with the bromobenzyl moiety of these compounds. For example, the primary amino group can be formed on the glass substrate using a silane coupling agent having a primary amino group. Alternatively, the substrate is a glass substrate having a sulfanyl group formed on the surface,
An amino group is bound to the 5 ′ end of the primer;
The bond between the sulfanyl group and the amino group is bonded to the sulfanyl group and the compound via the compound represented by Formula I or General Formula II. Brombenzyl moiety It is also possible to adopt a form that is carried out by the reaction with, and the reaction between the amino group and the succinimide ester moiety of these compounds. At this time, for example, the sulfanyl group can be formed on the glass substrate using a silane coupling agent having a sulfanyl group.
[0019]
Furthermore, the structure containing the nitrobenzene can be constructed using a compound represented by the following general formula III.
[0020]
Embedded image

[0021]
Compound III
(In the formula, DMTrO represents a dimethoxytrityloxy group, and CNEt represents a 2-cyanoethyl group.)
In the method of the present invention, the enzyme used for the extension reaction is preferably heat resistant. In addition, in particular, the substrate on which the primer is fixed is in the form of a nucleic acid chip in which a plurality of primer nucleic acids are arranged in a matrix,
In the step (3), an enzymatic nucleic acid extension reaction is performed on the nucleic acid chip together with their templates for some or all of the primer nucleic acids,
In the step (4), the matrix portion subjected to the extension reaction is more preferably analyzed by the MALDI-TOF MS method.
[0022]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described in detail below.
[0023]
The nucleobase sequence determination method of the present invention is characterized in that the nucleobase sequence is determined by performing the following steps (1) to (6).
Process (1)
A nucleic acid (DNA) complementary to a part or all of the 3'-side base sequence of the nucleic acid (DNA) for which the base sequence analysis is desired is compared with the base sequence described above. As a primer necessary for the enzymatic nucleic acid extension reaction using the nucleic acid desired to be analyzed as a template, a partial structure that is cleaved by light is fixed on the substrate with a structure that is 5 'to the complementary base sequence of the primer. The step of:
Process (2)
Annealing the nucleic acid desired to be analyzed for the base sequence at the complementary base sequence portion with a primer fixed on the substrate to form a hybrid;
Process (3)
Four 2′-deoxynucleotide triphosphates necessary for enzymatic nucleic acid elongation reaction (dNTP: N is A; adenine, G; guanine, C; cytosine, T; thymine) and 4 as terminators of the elongation reaction A step of performing an enzymatic extension reaction using a nucleic acid having a desired base sequence as a template on the hybrid-formed substrate in the presence of an appropriate amount of 2 ', 3'-dideoxynucleotide triphosphate (ddNTP) of the species;
Step (4)
Removing the template nucleic acid from the substrate on which the extension reaction has been performed;
Step (5)
Irradiating a plurality of extension reaction products having different chain lengths with light that cleaves the partial structure to be cleaved, including a primer portion that is fixed to the substrate in a structure that includes the partial structure that is cleaved by light, Analyzing the molecular weight of the extension product cleaved by irradiation by the MALDI-TOF MS method and clarifying the base sequence of the extension part of the extension product based on the molecular weight increase from the molecular weight of the primer;
Step (6)
A step of analyzing a part or all of the base sequence desired to analyze the nucleic acid whose base sequence is desired based on the base sequence of the extended portion.
[0024]
According to the method of the present invention, the elongation reaction product is cleaved from the substrate by light irradiation. Therefore, due to a phenomenon called depurination, which is a problem in the method of the above literature, that is, a method of cleaving using an acidic substance. It is possible to essentially avoid the effects of non-selective cleavage at the purine sites of adenosine and guanosine.
[0025]
At that time, if the irradiated light is a laser light used in the analysis of the MALDI-TOF MS method, it is not necessary to perform the light irradiation for cutting prior to the analysis in advance, and cutting, desorption and ionization can be performed simultaneously. So it is very convenient. The laser used in the MALDI-TOF MS method is generally a nitrogen laser beam having a wavelength of 337 nm, but there are several organic structures that can be cut at this wavelength as shown below, and therefore the nitrogen laser is used in the present invention. Is possible. However, the present invention is not limited to the laser having this wavelength, and for example, a second harmonic of a Nd: YAG laser having a wavelength of 532 nm can also be used. Nucleic acids are known to suffer damage such as the formation of a thymine dimer by ultraviolet irradiation, but the wavelength of ultraviolet light that causes the formation of a thymine dimer is the absorption wavelength band of nucleobases, approximately 250 Therefore, the laser wavelength used in the present invention can be preferably used without damaging the nucleic acid.
[0026]
The structure used in the present invention that is cleaved by light irradiation is not particularly limited, but a structure containing nitrobenzene can be given as an example. As a more specific structure, the structure constructed | assembled using the following compound I or general formula II can be mention | raise | lifted.
[0027]
Embedded image

[0028]
Compound I
[0029]
Embedded image

[0030]
Formula II
(Where n = 3 or 4, X = H or SO _Three Na)
As a specific method for producing a primer-bonded substrate used in the present invention, as an example, the substrate is a glass substrate having a primary amino group formed on the surface, and sulfanyl (SH) is used at the 5 ′ end of the primer. The amino group and the sulfanyl group are bonded to each other via the compound I or the compound represented by the general formula II, that is, between the amino group and the succinimide ester site of these compounds. Examples thereof include a reaction and a method performed by a reaction between a sulfanyl group and a bromobenzyl moiety of these compounds. At this time, the formation of the primary amino group on the glass substrate can be exemplified by a method using a silane coupling agent having a primary amino group.
[0031]
Embedded image

[0032]
An example of the structure of a primer that binds to the substrate constructed by the above method is shown in the above chemical formula (left). An example of the structure cut by light irradiation is also shown in the above chemical formula (right) (Biochemistry International Vol. 26 No. 5, 1992). As illustrated, the structure of the cleaved site of the nucleic acid after cleaving is the same as the structure before cleaving. That is, in this case, at the time of sequencing by the MALDI-TOF MS method, it is expected that an increase by one base from the original molecular weight of the primer is observed by the extension reaction.
[0033]
As another example of the substrate manufacturing method, the substrate is a glass substrate having a sulfanyl group formed on the surface, an amino group is bonded to the 5 ′ end of the primer, and the bond between the sulfanyl group and the amino group is Through a compound of formula I or a compound of general formula II, i.e., a reaction of a sulfanyl group with a partial bromobenzyl moiety of these compounds and a reaction of an amino group with a succinimide ester moiety of these compounds. The way it is done can be mentioned. In that case, formation of the sulfanyl group to a glass substrate can illustrate the method performed using the silane coupling agent which has a sulfanyl group.
[0034]
Another example of a structure containing nitrobenzene that can be used in the method of the present invention is a structure constructed by the following compound III. In this case, when synthesizing a primer nucleic acid with an automatic nucleic acid synthesizer, a structure capable of photocleavage may be introduced by compound III immediately before introducing a unit having a functional group to be bonded to the substrate at the 5 ′ end. it can. In this case, examples of functional groups to be bonded to the substrate include amino groups and sulfanyl groups. Reagents for introducing such functional groups in an automatic nucleic acid synthesizer are sold by, for example, Glen Research. These may be used as appropriate. Further, for the substrate-side treatment, the above-described method using a silane coupling agent can be adopted even in this case.
[0035]
Embedded image

[0036]
Compound III
(In the formula, DMTrO represents a dimethoxytrityloxy group, and CNEt represents a 2-cyanoethyl group.)
As described above, in the method of the present invention, an enzymatic nucleic acid extension reaction is performed using a primer immobilized on a solid phase and an annealed nucleic acid as a template. At that time, the enzyme used for the extension reaction and the primer It is efficient if annealing can be carried out in the presence of the substrate bound to each of the template nucleic acid and the template nucleic acid, followed by extension reaction, but the primer binding substrate and the template nucleic acid may be raised to about 90 ° C. depending on the case. It needs to be warmed. In that case, ordinary enzymes cannot withstand such high temperatures. In such a case, it is convenient to use a thermostable enzyme. An example of such an enzyme is Thermo Sequenase DNA Polymerase (Amersham Pharmacia Biotech).
[0037]
As one of the problems of the prior art, it has already been pointed out that a reaction such as an extension reaction needs to be performed individually for a nucleic acid for which sequencing is desired. It has also been mentioned that it is the same even if the primer is bound on a solid phase.
[0038]
In the present invention, as a means for solving this problem, a plurality of primer nucleic acids are fixedly arranged in a matrix using the means described above, so-called a nucleic acid chip, a part of the primer nucleic acids, Alternatively, it is one of the features that the enzymatic nucleic acid extension reaction is performed on all of them together with their templates, and the matrix portion subjected to the extension reaction is analyzed by the MALDI-TOF MS method described above. . At this time, if the nucleotide sequence of the primer is a unique sequence for each nucleic acid desired to be sequenced in at least one extension reaction solution, each nucleic acid is a primer necessary for each extension reaction. Therefore, it is possible to carry out an extension reaction using all these nucleic acids as a template on a single chip in a single reaction.
[0039]
As an example of a method for producing a nucleic acid chip, Japanese Patent Application Laid-Open No. 11-187900 discloses N- (shown by the following chemical formula IV for bonding with a nucleic acid having a sulfanyl group on a substrate on which a primary amino group is formed. 6-maleimidocaproyloxy) succinimide is described as an example. In the present invention, instead of the compound of the chemical formula IV, the compound represented by the general formula II or the compound represented by the general formula II is used. , It is possible to analyze the nucleic acid base sequence.
[0040]
Embedded image

[0041]
Compound IV
In the MALDI-TOF MS method, as described above, in order to desorb and ionize a substance to be measured (test substance, in the present invention, an extension product including a primer portion), a substance called a matrix is coated. Coexist with the test substance. As a method often used, a test substance is dissolved in a saturated solution of a matrix at an appropriate concentration, and several μl of this coexisting solution, for example, is dropped on an appropriately addressed stainless steel plate and dried. The test substance coexists in an appropriate concentration in the matrix crystal. In this case, depending on the crystal state of the matrix, the three-dimensional form, and the concentration of the test substance, a spectrum may not be obtained, or even if it is obtained, the S / N ratio and accuracy may not be good. In addition, if the surface of the matrix crystal is minute, but has irregularities and slopes, the test substance is present in the matrix, which may affect the time of flight in some cases, resulting in the mass accuracy of the mass spectrum. May cause adverse effects. In the present invention, since the extension product is immobilized on the smooth substrate surface, there is relatively little concern that affects the flight time. In addition, in some cases, the matrix substance can be supplied to the test substance evenly by coating the matrix in a thin film on the substrate, and the matrix crystal state is also good. As a result, accurate base sequence determination is performed.
[0042]
For the coating of the matrix material, dipping, spin coating or the like can be used as appropriate. Further, regarding the thickness of the coating, if it is too thick, the test substance is embedded in the matrix substance layer in some cases, so that the test substance can be cut, desorbed, or ionized from the substrate surface. It may not be performed well. On the other hand, if it is too thin, the test substance is exposed from the matrix, and cutting, desorption, and ionization from the substrate surface may not be performed satisfactorily. Therefore, it is most preferable that the thickness of the coating is selected so as to be necessary and sufficient for cutting, desorption, and ionization of the test substance. At that time, the thickness of the coating of the matrix material necessary and sufficient for cutting, desorption and ionization of the test substance is preferably about the same as the height from the substrate on which the test substance exists to about 1000 times. However, of course, it is not limited to this thickness.
[0043]
【Example】
Hereinafter, the present invention will be described more specifically with reference to examples.
[0044]
(Example 1)
Preparation of nucleic acid binding substrate with dT40 primer
In accordance with the method described in JP-A-11-187900, a substrate on which nucleic acid primers were uniformly bound was prepared. The difference from the method described in the publication is that a bifunctional cross-linking agent used for immobilizing a nucleic acid primer is changed from N- (6-maleimidocaproyloxy) succinimide (compound IV) to a compound represented by the general formula II The following compound V, ie, succinimidyl 6- (4-bromomethyl-3-nitrobenzoyl) aminohexanoate, was added to the substrate, and nucleic acid primers were bound uniformly on the substrate, not in a matrix form. It is a point.
[0045]
Embedded image

[0046]
Compound V
(1) Substrate cleaning
A synthetic quartz substrate of 25.4 mm × 25.4 mm × 1 mm was put in a rack and immersed in an ultrasonic cleaning detergent (Branson: GPIII) diluted to 10% with pure water overnight. Thereafter, ultrasonic cleaning was performed in a detergent for 20 minutes, and then the detergent was removed by washing with water. After rinsing with pure water, sonication was further performed for 20 minutes in a container containing pure water. Next, the substrate was immersed in an aqueous 1N sodium hydroxide solution preheated to 80 ° C. for 10 minutes. Subsequently, washing with water and pure water were performed, and the cleaned substrate was directly subjected to the next surface treatment step.
[0047]
(2) Surface treatment
A 1 wt% aqueous solution of an amino group-bonded silane coupling agent, N-β- (aminoethyl) -γ-aminopropyltrimethoxysilane KBM603 (Shin-Etsu Chemical Co., Ltd.) was stirred at room temperature for 2 hours, and the molecules of the silane compound The methoxy group was hydrolyzed. Next, the washed substrate obtained in (1) was immersed in this solution at room temperature for 1 hour, washed with pure water, and dried by blowing nitrogen gas on both sides of the substrate. Next, the substrate was baked in an oven heated to 120 ° C. for 1 hour to finally introduce amino groups onto the substrate surface.
[0048]
Next, 5 mg of Compound V (Dojindo Laboratories) was dissolved in a 1: 1 (volume ratio) mixed solvent of dimethyl sulfoxide (DMSO) / ethanol to a concentration of 0.5 mg / ml. The quartz substrate subjected to the silane coupling treatment was immersed in the compound V solution at room temperature for 2 hours to react the amino group supported on the substrate surface with the succinimide group of the compound V by the silane coupling treatment. At this stage, a bromoethyl group derived from compound V exists on the substrate surface. The substrate pulled up from the compound V solution was sequentially washed with a DMSO / ethanol mixed solvent and ethanol, and then dried by blowing nitrogen gas.
[0049]
(3) Synthesis of probe DNA
A DNA synthesizer (Bex) was requested to synthesize a single-stranded nucleic acid (a 40-mer of dT) of SEQ ID NO: 1. A sulfanyl (SH) group was introduced into the 5 ′ end of the single-stranded DNA of SEQ ID NO: 1 by using a thiol modifier (Glen Research) during synthesis. Deprotection and DNA recovery were performed by conventional methods, and purification was performed using HPLC. A series of steps from synthesis to purification was performed by a synthesizer. The calculated molecular weight of the nucleic acid of SEQ ID NO: 1 is 12302.17 Da.
SEQ ID NO: 1
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT 3TT
(4) Binding of DNA to substrate
The single-stranded DNA of SEQ ID NO: 1 described in (3) is glycerin 7.5 wt%, urea 7.5 wt%, thiodiglycol 7.5 wt%, acetylene alcohol (trade name: acetylenol EH; Dissolved in a solution containing 1 wt% of Kawaken Fine Chemical Co., Ltd.
[0050]
Next, 25 μl of the DNA solution was placed on the glass substrate surface-treated in (2), covered with a cover glass of 18 mm × 18 mm, and the bromoethyl group on the glass plate surface and the sulfanyl group at the end of the nucleic acid probe were reacted at room temperature. . After 30 minutes, the substrate was washed with pure water and stored in pure water.
(Example 2)
Elongation reaction and analysis by MALDI-TOF MS
(1) Elongation reaction
In advance, DNA sequencing DNA polymerase (Thermo Sequenase: Amersham Pharmacia Biotech) is diluted to 1 unit / μl with the attached dilution buffer.
[0051]
In a 0.5 ml Eppendorf tube, 4.5 μl of reaction buffer attached to Thermo Sequenase, Poly (dA) solution (Amersham Pharmacia Biotech: average chain length 300 dissolved in pure water at a concentration of 0.25 μg / μl) 5 μl 4 μl of deoxyribonucleotide mixed solution (Sequencing Grade Solution dNTPs: Amersham Pharmacia Biotech; 100 mM) Prepare a total volume of 43 μl.
[0052]
To this solution, 2 μl of a previously diluted Thermo Sequenase solution was added and mixed well, and then 20 μl was poured into wells of a hybridization incubation chamber (CoverWell 20 μl Chamber: Funakoshi). The DNA-bonded substrate prepared in step 1 is placed in contact with the liquid so that the liquid does not leak. A chamber was placed on a hot plate capable of adjusting the temperature, heated at 85 ° C. for 10 minutes, and then allowed to stand in an incubator at 50 ° C. for 30 minutes to carry out an extension reaction. After completion of the reaction, the substrate was peeled off from the chamber, washed with pure water at 85 ° C. for 2 minutes, and then rinsed with flowing pure water for 1 minute to wash away template DNA, unreacted monomers and the like from the substrate. Next, nitrogen gas is blown onto the substrate to remove water, and then an appropriate amount of ion exchange resin AG 50W-X8 (BIO-RAD) is dispersed in 100 μl and placed on an area including the analysis target portion on the chip surface, The desalting treatment was performed by leaving it at room temperature for 5 minutes. Subsequently, it was washed with pure water, water was removed with nitrogen gas, and it was stored in a vacuum desiccator.
(2) Analysis by MALDI-TOF MS
The substrate subjected to the elongation reaction was analyzed by MALDI-TOF MS under the following conditions. During analysis, the substrate was fixed to a stainless plate for MALDI-TOF MS analysis (Nippon Bruker Daltonics) using a stainless steel pin and a stainless steel substrate holder.
Device name: autoflex Reflecton (Nippon Bruker Daltonics)
Laser: Nitrogen laser
Accelerating voltage: 20KV
Measurement mode: Linear mode
Ionization: Positive
Internal standard: oligonucleotide; 6117, 9191 Da
Specification matrix: 3-hydroxy-2-propionic acid (3-HPA)
(3) Analysis results
A mass spectrum was observed in which the molecular weight of 304.19 Da gradually increased with the molecular weight of 12588.95 Da as the minimum. When the DNA of SEQ ID NO: 1 is cleaved by light irradiation, the calculated molecular weight is 12302.17, and the calculated molecular weight when ddTTP is bound (the binding portion is a simple phosphodiester) is 12590. .36 Da. In addition, when dNTPs are sequentially added before being terminated by ddTTP, the calculated increase in molecular weight is 304.19 Da. Therefore, it is considered that the observed peak of increasing molecular weight is that dNTP is increasing one molecule at a time, and conversely, the template DNA for which the base sequence is to be obtained in a model manner is a continuous sequence of adenosine. You can know that it is an array. That is, it can be seen that the nucleotide sequence of a nucleic acid can be analyzed by the method of the present invention.
[0053]
The reason why the observed absolute value of the molecular weight is deviated from the theoretical molecular weight is because the difference in molecular weight between the internal standard used for mass calibration and the actually analyzed DNA is large. Probable result.
[0054]
(Example 3)
Analysis of phage DNA base sequence by MALDI-TOF MS
(1) Preparation of DNA chip
Black as a substrate on the slide glass Teflon (registered trademark) Using Teflon-printed slide glass (ER-212: Funakoshi Chemical) in which 30 resin wells (2 mm in diameter) were formed, Substrate cleaning Before, after applying UV-ozone treatment, Example 1 of Substrate cleaning And surface treatment.
SEQ ID NO: 2
5 'HS- (CH ₂ ) ₆ -O-PO ₂ -O-TGTAAAACGACGGGCCAGT 3 '
Next, the DNA of SEQ ID NO: 2 was dissolved in the same manner as in Example 1, 4 μl each was supplied to the well of the slide glass subjected to the above surface treatment, and left in a moisturizing chamber at room temperature for 30 minutes to react the DNA and substrate I let you. Subsequently, it was washed with pure water and stored in pure water. The nucleic acid of SEQ ID NO: 2 is a base sequence complementary to the base sequence from 6318 to 6301 of single-strand (+ strand) bacteriophage M13, and the calculated molecular weight is 5728.84.
(2) Elongation reaction
The extension reaction and post-treatment were carried out under exactly the same conditions except that the template DNA of Example 2 was 2.5 μg of M13mp18 (+) Strand DNA (Amersham Pharmacia Biotech). A desalting treatment was also performed for each well.
[0055]
(3) Analysis by MALDI-TOF MS
The DNA chip subjected to the extension reaction was analyzed by MALDI-TOF MS. The analysis was performed by irradiating a laser spot inside the well. The internal standard nucleic acids used are 3645 Da, 6117 Da, and 9191 Da.
[0056]
(4) Analysis results
A mass spectrum was observed in which the molecular weight increased gradually with the molecular weight of 604.10 Da as the minimum. When ddGTP binds to the DNA of SEQ ID NO: 2, the calculated molecular weight is 6042.05 Da, so that it is understood that the first sequence added to the primer is G, that is, the C sequence in the template. Similarly, the analysis was sequentially performed based on the molecular weight increment, and the base sequence of the template was analyzed from the first C to about 180 bases as CGGTTCGAACGTACGGACGT CCAGCTGAGA TCTCTCTAGGG GCCCCATGGCT CGAGCTTAAG. The analyzed base sequence completely matched the base sequence described in the literature. Thus, it can be seen that the base sequence of the template can be analyzed by using the primer chip by the method of the present invention.
(Example 4)
Poly (dA), simultaneous analysis of phage DNA base sequence
In the same manner as in Example 3, a DNA chip was prepared by mounting the two types of primers used in Example 2 and Example 3 on different matrices on the chip, and the Poly (dA) used in Example 2 was carried out. An extension reaction was performed in the presence of M13mp18 (+) Strand DNA used in Example 3, and each matrix was analyzed by MALDI-TOF MS by the method described so far. As a result, the same base sequence information as in Example 2 and Example 3 was obtained from each matrix. In other words, each primer matrix is equipped with a unique primer for the target template nucleic acid, and this is subjected to a batch extension reaction, allowing the base sequence analysis of each template to proceed in parallel. It is understood that is possible.
[0057]
【The invention's effect】
By using the nucleic acid bound on the substrate as a primer by using the method of the present invention, an extension reaction is performed using the nucleic acid to be analyzed as a template, and then the extension product is analyzed by MALDI-TOF MS. Analysis of the base sequence of the nucleic acid to be analyzed as a reaction template can be performed with high work efficiency. In addition, by using a primer chip on which a plurality of nucleic acids are arranged on a substrate, the same base sequence can be easily analyzed.

Claims (12)

A method for determining the base sequences of a plurality of types of nucleic acids,
(1)
For the plurality of types of nucleic acids (DNA) for which analysis of the base sequence is desired, a plurality of nucleic acids that are complementary to all or part of the base sequence 3 ′ from the site for which each base sequence is desired to be analyzed As a primer to be used for nucleic acid extension reaction of each kind of nucleic acid (DNA) using the plurality of kinds of nucleic acids whose base sequence analysis is desired as a template, a partial structure cleaved by light is used as the complementary in the primer. A step of arranging them in a matrix by fixing each of them on a smooth substrate with a structure included 5 'from the base sequence;
(2)
A step of annealing a plurality of types of nucleic acids for which analysis of the base sequence is desired with each primer fixed on the substrate at the complementary base sequence portion to form a hybrid; (3)
Four kinds of 2′-deoxynucleotide triphosphates necessary for the nucleic acid extension reaction (dNTP: N is A; adenine, G; guanine, C; cytosine, T; thymine) and four kinds of terminators for the nucleic acid extension reaction In the presence of 2 ', 3'-dideoxynucleotide triphosphate (ddNTP), an enzyme is used on the substrate on which the hybrid is formed, using the plurality of nucleic acids whose base sequence analysis is desired as a template. Performing a nucleic acid extension reaction;
(4)
Removing the template nucleic acid from the substrate on which the nucleic acid extension reaction has been performed;
(5)
For each of the fixed positions of each primer for a plurality of nucleic acids for which analysis of the base sequence is desired,
Comprising said primer portion fixed to the substrate structure including a portion structure to be disconnected by light for different elongation reaction product chain length, irradiated with light partial structure the cutting is cut ,
Perform mass analysis of each of the plurality of extension reaction products cleaved by the light irradiation,
Is obtained by nucleic acid extension on the 3 'side of the primer, in the plurality of elongation reaction product, based on the molecular weight increase of the molecular weight of the primer, the nucleotide sequence of the extension portion of the plurality of elongation reaction product Revealing steps;
(6)
Analyzing a part or all of the base sequence desired to be analyzed for a plurality of types of nucleic acids desired to be analyzed based on the base sequence of the extended portion;
At least the steps (1) to (6) above,
The means for performing mass spectrometry is a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (Matrix Assisted Laser Deposition Ionization Time-of-Flight Mass Spectrometry, hereinafter abbreviated as MALDI-TOF MS) method,
The partial structure cleaved by light is constructed using any of the following compounds I to III,

Compound I

Formula II
(Wherein n = 3 or 4, X = H or SO ₃ Na)

Compound III
(In the formula, DMTrO represents a dimethoxytrityloxy group, and CNEt represents a 2-cyanoethyl group.)
Furthermore,
(A) Prior to the step (1),
A step of coating a substance for binding and fixing the plurality of primers on the smooth substrate surface; and (b) after the step (4), prior to the step (5),
When mass spectrometry is performed by MALDI-TOF MS method, a substance (matrix substance) supporting the desorption and ionization of the extension reaction product including the primer, which is fixed on the substrate, is at least the smooth substrate surface A method for determining a nucleobase sequence, comprising a step of coating the region to be subjected to mass spectrometry. A substance for binding and fixing the plurality of primers on the smooth substrate surface is:
Corresponds to any one of the compound I, the compound represented by the general formula II, and the compound III, which are used for the construction of a structure contained on the 5 ′ side of the complementary base sequence in each primer to be immobilized. The compound I, the succinimide ester moiety in the compound represented by the general formula II, the bromobenzyl moiety in the compound I, the compound represented by the general formula II, or the dimethoxytrityloxy group in the compound III The method according to claim 1, wherein the method is selected from the group consisting of silane coupling agents having a functional group capable of reacting with any of the sites. The method according to claim 1 or 2 , wherein in the step (5), the irradiated light is nitrogen laser light having a wavelength of 337 nm. The laser beam used in the analysis of the MALDI-TOF MS method is
The method according to claim 1 , wherein the method is nitrogen laser light having a wavelength of 337 nm. The method according to claim 1, wherein the partial structure cleaved by light is constructed using the following compound I.

Compound I The method according to any one of claims 1 to 4, wherein the partial structure cleaved by light is constructed using a compound represented by the following general formula II.

Formula II
(Wherein n = 3 or 4, X = H or SO ₃ Na) The substrate is a glass substrate having a primary amino group formed on the surface,
A sulfanyl (SH) group is bound to the 5 ′ end of the primer,
The bond between the amino group and the sulfanyl group is obtained by reacting the amino group with a succinimide ester moiety of the compound via the compound I or the compound represented by the general formula II, and a sulfanyl group. The method according to claim 5 or 6 , which is carried out by a reaction with a bromobenzyl moiety of these compounds. The method according to claim 7 , wherein the primary amino group is formed on the glass substrate by using a silane coupling agent having a primary amino group. The substrate is a glass substrate having a sulfanyl group formed on the surface,
An amino group is bound to the 5 ′ end of the primer;
The bond between the sulfanyl group and the amino group is obtained by reacting the sulfanyl group with the bromobenzyl moiety of these compounds via the compound I or the compound represented by the general formula II, and the amino group and the compounds of these compounds. The method according to claim 5 or 6 , wherein the method is carried out by reaction with a succinimide ester moiety. The method according to claim 9 , wherein the sulfanyl group is formed on the glass substrate using a silane coupling agent having a sulfanyl group. The method according to claim 1, wherein the partial structure cleaved by light is constructed using a compound represented by the following general formula III.

Compound III
(In the formula, DMTrO represents a dimethoxytrityloxy group, and CNEt represents a 2-cyanoethyl group.) The method according to any one of claims 1 to 11, wherein the enzyme used in the nucleic acid extension reaction is heat-resistant.