JP3974441B2 - Nucleic acid synthesis method - Google Patents

Description

【００４５】
ここで、Ｆ２は鋳型となる核酸の領域Ｆ２ｃに相補的な塩基配列である。またＲ２はＦ２をプライマーとして合成される相補鎖に含まれる任意の領域Ｒ２ｃに相補的な塩基配列である。Ｆ１ｃとＲ１ｃはそれぞれ、Ｆ２ｃおよびＲ２ｃのそれぞれ下流に位置する任意の塩基配列である。ここでＦ２−Ｒ２間の距離は任意であって良い。相補鎖合成を行うDNAポリメラーゼの合成能力にも依存するが、好適な条件では1kbp程度の長さであっても十分に合成が可能である。より具体的には、Bst DNAポリメラーゼを用いた場合、F2/R2c間で800bp、望ましくは500bp以下の長さであれば確実に合成される。温度サイクルを伴うPCRでは、温度変化ストレスによる酵素活性の低下が長い塩基配列の合成効率を下げるとされている。本発明における望ましい態様では、核酸増幅工程における温度サイクルが不要となるので、長い塩基配列であっても合成、ならびに増幅を確実に達成することができる。
【００４６】
まず鋳型となる核酸に対してＦＡのＦ２をアニールさせ、これを合成起点として相補鎖合成を行う。以下、図１の(4)にいたるまでは先に説明した本発明の基本的な態様（図５）と同様の反応工程となっている。図１の(2)でＦ３としてアニールしている配列は、先に説明したアウタープライマーである。このプライマーを合成起点として鎖置換型の相補鎖合成を行うDNAポリメラーゼで行うことにより、ＦＡから合成した相補鎖は置換され、塩基対結合が可能な状態となる。
【００４７】
(4)でＲ２ｃが塩基対結合が可能な状態となったところで、リバースプライマーとしてのＲＡがＲ２ｃ／Ｒ２の組み合わせでアニールする。これを合成起点とする相補鎖合成は、ＦＡの5'側末端であるＦ１ｃに至る部分まで行われる。この相補鎖合成反応に続いて、やはり置換用のアウタープライマーＲ３がアニールし、鎖置換を伴って相補鎖合成を行うことにより、ＲＡを合成起点として合成された相補鎖が置換される。このとき置換される相補鎖は、ＲＡを5'側に持ちＦＡに相補的な配列が3'末端に位置する。
【００４８】
さて、こうして置換された１本鎖核酸の3'側には、同一鎖上のＦ１ｃに相補的な配列Ｆ１が存在する。Ｆ１は、同一分子内に並ぶＦ１ｃに速やかにアニールし、相補鎖合成が始まる。3'末端（Ｆ１）が同一鎖上のＦ１ｃにアニールするときに、Ｆ２ｃを含むループが形成されている。このループ部分は塩基対結合が可能な状態で維持されていることは、図２-(7)からも明らかである。Ｆ２ｃに相補的な塩基配列を持つ本発明のオリゴヌクレオチドＦＡは、このループ部分にアニールして相補鎖合成の起点となる(7)。ループ部分からの相補鎖合成は、先に開始したＦ１からの相補鎖合成の反応生成物を置換しながら進む。その結果、自身を鋳型として合成された相補鎖は、再び3'末端において塩基対結合が可能な状態となる。この3'末端は、同一鎖上のＲ１ｃにアニールしうる領域Ｒ１を3'末端に備えており、やはり同一分子内の速やかな反応により両者は優先的にアニールする。こうして、先に説明したＦＡを鋳型として合成された3'末端からの反応と同様の反応が、この領域でも進行する。結果として、本発明による１本鎖上に相補的な塩基配列が交互に連結された核酸は次々と相補鎖合成と置換とを継続し、その3'末端Ｒ１を起点とする伸長を続けることになる。3'末端Ｒ１の同一鎖へのアニールによって形成されるループには常にＲ２ｃが含まれることから、以降の反応で3'末端のループ部分にアニールするのは常にＲ２を備えたオリゴヌクレオチド（すなわちＲＡ）となる。
【００４９】
一方、自分自身を鋳型として伸長を継続する１本鎖の核酸に対して、そのループ部分にアニールするオリゴヌクレオチドを合成起点として相補鎖合成される核酸に注目すると、ここでも本発明による１本鎖上に相補的な塩基配列が交互に連結された核酸の合成が進行している。すなわち、ループ部分からの相補鎖合成は、たとえば図２−(7)においては、ＲＡに達した時点で完了する。そして、この核酸の合成によって置換された核酸が相補鎖合成を開始（図３−(8)）すると、やがてその反応はかつて合成起点であったループ部分に達して再び置換が始まる。こうしてループ部分から合成を開始した核酸も置換され、その結果同一鎖上にアニールすることができる3'末端Ｒ１を得る（図３−(10)）。この3'末端Ｒ１は同一鎖のＲ１ｃにアニールして相補鎖合成を開始する。さて、この反応のＦとＲを読みかえれば、図２−(7)で起きている反応と同じである。したがって図３−(10)に示す構造は、自身の伸長と新たな核酸の生成を継続する新しい核酸として機能することができる。
【００５０】
なお図３−(10)に示す核酸から開始する核酸の合成反応は、ここまで述べてきたものとは逆に常に3'末端Ｆ１を合成起点とする伸長となる。すなわち本発明においては、１つの核酸の伸長に伴って、これとは別に伸長を開始する新たな核酸を供給しつづける反応が進行する。更に鎖が伸長するのに従い、末端のみならず、同一鎖上に複数のループ形成配列がもたらされる。これらのループ形成配列は、鎖置換合成反応により塩基対形成可能な状態となると、オリゴヌクレオチドがアニールし、新たな核酸の生成反応の基点となる。末端のみならず鎖の途中からの合成反応も組み合わされることにより、さらに効率のよい増幅反応が達成されるのである。以上のようにリバースプライマーとして本発明に基づくオリゴヌクレオチドＲＡを組み合わせることによって、伸長とそれに伴う新たな核酸の生成が起きる。更に本発明においては、この新たに生成した核酸自身が伸長し、それに付随する更に新たな核酸の生成をもたらす。一連の反応は、理論的には永久に継続し、きわめて効率的な核酸の増幅を達成することができる。しかも本発明の反応は、等温条件のもとで行うことができる。
【００５１】
このとき蓄積する反応生成物は、Ｆ１−Ｒ１間の塩基配列とその相補配列が交互に連結された構造を持つ。ただし繰り返し単位となっている配列の両端には、Ｆ２−Ｆ１（Ｆ２ｃ−Ｆ１ｃ）、またはＲ２−Ｒ１（Ｒ２ｃ−Ｒ１ｃ）の塩基配列で構成される領域が連続している。たとえば図３−(9)では、5'側から（Ｒ２−Ｆ２ｃ）−（Ｆ１−Ｒ２ｃ）−（Ｒ１−Ｆ１ｃ）−（Ｆ２−Ｒ２ｃ）という順序で連結された状態となる。これは、本発明に基づく増幅反応が、オリゴヌクレオチドを合成起点としてＦ２（またはＲ２）から開始し、続いて自身の3'末端を合成起点とするＦ１（またはＲ１）からの相補鎖合成反応によって伸長するという原理のもとに進行しているためである。
【００５２】
さて、ここでは最も望ましい態様としてループ部分にアニールするオリゴヌクレオチドに本発明によるオリゴヌクレオチドＦＡ、およびＲＡを用いた。しかし本発明による核酸の増幅反応は、これらの限られた構造を持ったオリゴヌクレオチドのみならず、ループからの相補鎖合成を開始できるオリゴヌクレオチドを利用しさえすれば実施することができる。つまり、伸長を続ける3'末端はループからの相補鎖合成によって置換されさえすれば、再びループ部分を与える。ループ部分から開始する相補鎖合成は、常に１本鎖上に相補的な塩基配列が交互に連結された核酸を鋳型としていることから、本発明で目的としている核酸の合成が可能なことは自明である。ただし、ここで合成される核酸は、置換後にループを形成して相補鎖合成は行うものの、以降のループを形成するための3'末端を持たないため、新たな鋳型としては機能できなくなる。したがって、ＦＡ、あるいはＲＡによって合成を開始した核酸と違って指数的な増幅は期待できない。このような理由から、ＦＡやＲＡのような構造を持ったオリゴヌクレオチドは、本発明に基づく高度に効率的な核酸の合成に有用なのである。
【００５３】
一連の反応は、鋳型となる１本鎖の核酸に対して、以下の成分を加え、ＦＡおよびＲＡを構成する塩基配列が相補的な塩基配列に対して安定な塩基対結合を形成することができ、かつ酵素活性を維持しうる温度でインキュベートするだけで進行する。
・４種類のオリゴヌクレオチド：
ＦＡ、
ＲＡ、
アウタープライマーＦ３、
およびアウタープライマーＲ３、
・鎖置換型の相補鎖合成を行うDNAポリメラーゼ、
・DNAポリメラーゼの基質となるヌクレオチド
【００５４】
したがって、PCRのような温度サイクルは必要無い。なおここでいう安定な塩基対結合とは、反応系に存在するオリゴヌクレオチドの少なくとも一部が相補鎖合成の起点を与えうる状態を意味する。安定な塩基対結合をもたらす望ましい条件は、たとえば融解温度(Tm)以下に設定することである。一般に融解温度(Tm)は、互いに相補的な塩基配列を持つ核酸の50%が塩基対結合した状態となる温度とされている。融解温度(Tm)以下に設定することは本発明の必須の条件ではないが、高度な合成効率を達成するためには考慮すべき反応条件の一つである。鋳型とすべき核酸が２本鎖である場合には、少なくともオリゴヌクレオチドがアニールする領域を塩基対結合が可能な状態とする必要がある。そのためには一般に加熱変性が行われるが、これは反応開始前の前処理として１度だけ行えば良い。
【００５５】
この反応は、酵素反応に好適なpHを与える緩衝剤、酵素の触媒活性の維持やアニールのために必要な塩類、酵素の保護剤、更には必要に応じて融解温度(Tm)の調整剤等の共存下で行う。緩衝剤としては、Tris-HCl等の中性から弱アルカリ性に緩衝作用を持つものが用いられる。pHは使用するDNAポリメラーゼに応じて調整する。塩類としてはKCl、NaCl、あるいは(NH₄)₂SO₄等が、酵素の活性維持と核酸の融解温度(Tm)調整のために適宜添加される。酵素の保護剤としては、ウシ血清アルブミンや糖類が利用される。更に融解温度(Tm)の調整剤には、ジメチルスルホキシド(DMSO)やホルムアミドが一般に利用される。融解温度(Tm)の調整剤を利用することによって、前記オリゴヌクレオチドのアニールを限られた温度条件の下で調整することができる。更にベタイン(N,N,N,-trimethylglycine)やテトラアルキルアンモニウム塩は、そのisostabilize作用によって鎖置換効率の向上にも有効である。ベタインは、反応液中0.2〜3.0Ｍ、好ましくは0.5〜1.5 M程度の添加により、本発明の核酸増幅反応の促進作用を期待できる。これらの融解温度の調整剤は、融解温度を下げる方向に作用するので、塩濃度や反応温度等のその他の反応条件を考慮して、適切なストリンジェンシーと反応性を与える条件を経験的に設定する。
【００５６】
本発明においては、一連の反応が常に複数の領域の位置関係を維持した状態でなければ進行しないことが重要な特徴である。この特徴によって、非特異的な相補鎖合成に伴う非特異的な合成反応が効果的に防止できるのである。すなわち、たとえ何らかの非特異的な反応が起きたとしても、その生成物が以降の増幅工程に対して出発材料となる可能性を低く押さえることにつながるのである。またより多くの領域によって反応の進行が制御されているということは、類似した塩基配列の厳密な識別を可能とする検出系を自由に構成できる可能性をもたらす。
【００５７】
この特徴を遺伝子変異の検出に利用することができる。本発明におけるアウタープライマーを用いる態様においては、このアウタープライマー２種、本発明のオリゴヌクレオチドからなるプライマー２種の合計４種のプライマーが用いられている。すなわち４種のオリゴヌクレオチドに含まれる６領域が設計通りに働かなければ本発明の合成反応は進行しない。 特に、相補鎖合成の起点となる各オリゴヌクレオチドの3'末端、および相補配列が合成起点となるＸ１ｃ領域の5'末端の配列は重要である。そこで、この重要な配列を検出すべき変異に対応するように設計すれば、本発明による合成反応生成物を観察することによって、塩基の欠失や挿入といった変異の有無、あるいはSNPsのような遺伝子多型を総合的に分析することができる。より具体的には、変異や多型が予想される塩基が、相補鎖合成の起点となるオリゴヌクレオチドの3'末端付近（相補鎖が起点となる場合には5'末端付近）に相当するように設計するのである。相補鎖の合成起点となる3'末端や、その付近にミスマッチが存在すると核酸の相補鎖合成反応は著しく阻害される。本発明においては、反応初期の生成物における末端構造が繰り返し反応を行わなければ高度な増幅反応に結びつかない。したがって、たとえ誤った合成が行われたとしても、増幅反応を構成する相補鎖合成がいずれかの段階で常に妨げられるのでミスマッチを含んだままでは高度な増幅は起きない。結果的にミスマッチが増幅反応を効果的に抑制し、最終的には正確な結果をもたらすことになる。つまり本発明に基づく核酸の増幅反応は、より完成度の高い塩基配列のチェック機構を備えていると言うことができる。これらの特徴は、たとえば単純に２つの領域で増幅反応を行っているPCR法などでは期待しにくい利点である。
【００５８】
更に本発明に用いるオリゴヌクレオチドを特徴付ける領域Ｘ１ｃは、相補配列が合成されてはじめて合成起点となり、この相補配列が、新たに合成された同一鎖内の配列Ｘ１にアニールすることにより、自己を鋳型とする合成反応が進行する。このため、たとえ先行技術でしばしば重要な問題となるいわゆるプライマーダイマーを生成しても、本オリゴヌクレオチドはループを形成しない。したがって、プライマーダイマーに起因する非特異的な増幅は原理的に生じ得ず、反応の特異性向上に貢献している。
【００５９】
更に本発明によれば、Ｆ３（図１-(2)）やＲ３（図２-(5)）で示したアウタープライマーを組み合わせることによって、上記の一連の反応を等温条件下で行うことができる。すなわち本発明は、前記〔９〕に示した工程を含む１本鎖上に相補的な塩基配列が交互に連結された核酸を増幅する方法を提供するものである。この方法では、Ｆ２ｃ／Ｆ２間、Ｒ２ｃ／Ｒ２間、Ｆ１ｃ／Ｆ１間、そしてＲ１ｃ／Ｒ１間で安定なアニールが起きる温度条件が選択され、そして望ましくはＦ３ｃ／Ｆ３間、ならびにＲ３ｃ／Ｒ３間は、それぞれＦ２ｃ／Ｆ２間、ならびにＲ２ｃ／Ｒ２間のアニールに助けられるコンティギュアス スタッキング現象によってアニールするように設定される。
【００６０】
本発明においては核酸の合成(synthesis)と増幅(amplification)という用語を用いる。本発明における核酸の合成とは、合成起点となったオリゴヌクレオチドからの核酸の伸長を意味する。合成に加えて、更に他の核酸の生成と、この生成された核酸の伸長反応とが連続して起きるとき、一連の反応を総合して増幅という。
【００６１】
さて、3'末端に同一鎖上の一部Ｆ１ｃにアニールすることができる領域Ｆ１を備え、この領域Ｆ１が同一鎖上のＦ１ｃにアニールすることによって、塩基対結合が可能な領域Ｆ２ｃを含むループを形成することができる１本鎖核酸は、本発明の重要な構成要素である。このような１本鎖核酸は、次のような原理に基づいて供給することもできる。すなわち、予め次のような構造を持ったプライマーに基づいて相補鎖合成を進めるのである。
5'-［プライマー内に位置する領域X１ｃにアニールする領域X１］−［塩基対結合が可能な状態にあるループ形成配列］−［領域X１ｃ］−［鋳型に相補的な配列を持つ領域］-3'
【００６２】
鋳型に相補的な配列を持つ領域には、Ｆ１に相補的な塩基配列（プライマーＦＡ）およびＲ１ｃに相補的な塩基配列（プライマーＲＡ）の２種類を用意する。なお、このとき合成すべき核酸を構成する塩基配列は、領域Ｆ１から領域Ｒ１ｃにいたる塩基配列と、この塩基配列に相補的な塩基配列を持つ領域Ｒ１から領域Ｆ１ｃにいたる塩基配列とを含むものである。一方、プライマー内部でアニールすることができるＸ１ｃとＸ１は、任意の配列とすることができる。ただしプライマーＦＡとＲＡの間では、領域X１ｃ／X１の配列を異なるものとするのが望ましい。
【００６３】
まず鋳型核酸の領域Ｆ１から前記プライマーＦＡによる相補鎖合成を行う。次いで合成された相補鎖の領域Ｒ１ｃを塩基対結合が可能な状態とし、ここに一方のプライマーをアニールさせて相補鎖合成の起点とする。このとき合成される相補鎖の3'末端は、最初に合成された鎖の5'末端部分を構成するプライマーＦＡに相補的な塩基配列を持つので、3'末端には領域X１を持ち、これが同一鎖上の領域X１ｃにアニールするとともにループを形成する。こうして、前記本発明による特徴的な3'末端構造が提供され、以降の反応は最も望ましい態様として示した先の反応系そのものとなる。なおこのときループ部分にアニールするオリゴヌクレオチドは、3'末端にループ内に存在する領域X２ｃに相補的な領域X２を持ち、5'側には領域X１を持つものとする。先の反応系ではプライマーＦＡとＲＡを使って鋳型核酸に相補的な鎖を合成することによって核酸の3'末端にループ構造をもたらした。この方法は、短いプライマーで効果的に本発明に特徴的な末端構造を提供する。一方、本態様においては、プライマーとしてはじめからループを構成する塩基配列全体を提供しており、より長いプライマーの合成が必要となる。
【００６４】
リバースプライマーに制限酵素認識領域を含む塩基配列を利用すれば、本発明による異なった態様を構成することができる。図６に基づき、リバースプライマーが制限酵素認識配列を含む場合について具体的に説明する。図６−(D)が完成したところで、リバースプライマー内の制限酵素認識部位に対応する制限酵素によりニックが生じる。このニックを合成起点として鎖置換型の相補鎖合成反応が開始する。リバースプライマーは(D)を構成する２本鎖核酸の両端に位置しているので、相補鎖合成反応も両端から開始することになる。基本的には先行技術として記載したSDA法の原理に基づくが、鋳型となる塩基配列が本発明によって相補的な塩基配列を交互に連結した構造となっているので、本発明に特有の核酸合成系が構成されるのである。なお、ニックを入れるリバースプライマーの相補鎖となる部分には制限酵素による２本鎖の切断が生じないようにヌクレアーゼ耐性となるようにdNTP誘導体が取りこまれるように設計しなければならない。
【００６５】
リバースプライマーにRNAポリメラーゼのプロモーターを挿入しておくこともできる。この場合もSDA法を応用した先の態様と同様に、図６−(D)の両端からこのプロモーターを認識するRNAポリメラーゼにより転写が行われる。
【００６６】
本発明によって合成された核酸は、１本鎖とは言え相補的な塩基配列から構成されるため、その大部分が塩基対結合を形成している。この特徴を利用して、合成生成物の検出が可能である。エチジウムブロマイド、SYBR Green I、あるいはPico Greenのような２本鎖特異インターカレーターである蛍光色素の存在下で本発明による核酸の合成方法を実施すれば、生成物の増加に伴って蛍光強度の増大が観察される。これをモニターすれば、閉鎖系でリアルタイムな合成反応の追跡が可能である。この種の検出系はPCR法への応用も考えられているが、プライマーダイマー等によるシグナルの発生と区別がつかないことから問題が多いとされている。しかし本発明に応用した場合には、非特異的な塩基対結合が増加する可能性が非常に低いことから、高い感度と少ないノイズが同時に期待できる。２本鎖特異インターカレーターと同様に、均一系の検出系を実現する方法として、蛍光エネルギー転移の利用が可能である。
【００６７】
本発明による核酸の合成方法を支えているのは、鎖置換型の相補鎖合成反応を触媒するDNAポリメラーゼである。上記反応中には、必ずしも鎖置換型のポリメラーゼを要しない反応ステップも含まれてはいる。しかし、構成試薬の単純化、そして経済性の点で、１種類のDNAポリメラーゼを利用するのが有利である。この種のDNAポリメラーゼには、以下のようなものが知られている。また、これらの酵素の各種変異体についても、それが配列依存型の相補鎖合成活性と鎖置換活性を有する限り、本発明に利用することができる。ここで言う変異体とは、酵素の必要とする触媒活性をもたらす構造のみを取り出したもの、あるいはアミノ酸の変異等によって触媒活性、安定性、あるいは耐熱性を改変したもの等を示すことができる。
Bst DNAポリメラーゼ
Bca(exo-)DNAポリメラーゼ
DNA ポリメラーゼＩのクレノウ・フラグメント
Vent DNAポリメラーゼ
Vent(Exo-)DNAポリメラーゼ（Vent DNAポリメラーゼからエクソヌクレアーゼ活性を除いたもの）
DeepVent DNAポリメラーゼ
DeepVent(Exo-)DNAポリメラーゼ（DeepVent DNAポリメラーゼからエクソヌクレアーゼ活性を除いたもの）
Φ29ファージDNAポリメラーゼ
MS-2ファージDNAポリメラーゼ
Z-Taq DNAポリメラーゼ（宝酒造）
KOD DNAポリメラーゼ（東洋紡績）
【００６８】
これらの酵素の中でもBst DNAポリメラーゼやBca(exo-)DNAポリメラーゼは、ある程度の耐熱性を持ち、触媒活性も高いことから特に望ましい酵素である。本発明の反応は、望ましい態様においては等温で実施することができるが、融解温度(Tm)の調整などのために必ずしも酵素の安定性にふさわしい温度条件を利用できるとは限らない。したがって、酵素が耐熱性であることは望ましい条件の一つである。また、等温反応が可能とは言え、最初の鋳型となる核酸の提供のためにも加熱変性は行われる可能性があり、その点においても耐熱性酵素の利用はアッセイプロトコールの選択の幅を広げる。
【００６９】
Vent(Exo-)DNAポリメラーゼは、鎖置換活性と共に高度な耐熱性を備えた酵素である。ところでDNAポリメラーゼによる鎖置換を伴う相補鎖合成反応は、１本鎖結合タンパク質(single strand binding protein)の添加によって促進されることが知られている(Paul M.Lizardi et al, Nature Genetics 19, 225-232, July,1998)。この作用を本発明に応用し、１本鎖結合タンパク質を添加することによって相補鎖合成の促進効果を期待することができる。たとえばVent(Exo-)DNAポリメラーゼに対しては、１本鎖結合タンパク質としてT4 gene 32が有効である。
【００７０】
なお3'-5'エクソヌクレアーゼ活性を持たないDNAポリメラーゼには、相補鎖合成が鋳型の5'末端に達した部分で停止せず、１塩基突出させた状態まで合成を進める現象が知られている。本発明では、相補鎖合成が末端に至ったときの3'末端の配列が次の相補鎖合成の開始につながるため、このような現象は望ましくない。しかし、DNAポリメラーゼによる3'末端への塩基の付加は、高い確率でAとなる。したがって、dATPが誤って１塩基付加しても問題とならないように、3'末端からの合成がAで開始するように配列を選択すれば良い。また、相補鎖合成時に3'末端がたとえ突出してしまっても、これを消化してblunt endとする3'→5'エクソヌクレアーゼ活性を利用することもできる。たとえば、天然型のVent DNAポリメラーゼはこの活性を持つことから、Vent(Exo-)DNAポリメラーゼと混合して利用することにより、この問題を回避することができる。
【００７１】
本発明による核酸の合成方法、あるいは増幅方法に必要な各種の試薬類は、あらかじめパッケージングしてキットとして供給することができる。具体的には、本発明のために、相補鎖合成のプライマーとして、あるいは置換用のアウタープライマーとして必要な各種のオリゴヌクレオチド、相補鎖合成の基質となるdNTP、鎖置換型の相補鎖合成を行うDNAポリメラーゼ、酵素反応に好適な条件を与える緩衝液、更に必要に応じて合成反応生成物の検出のために必要な試薬類で構成されるキットが提供される。特に、本発明の望ましい態様においては、反応途中で試薬の添加が不要なことから、１回の反応に必要な試薬を反応容器に分注した状態で供給することにより、サンプルの添加のみで反応を開始できる状態とすることができる。発光シグナルや蛍光シグナルを利用して、反応生成物の検出を反応容器のままで行えるようなシステムとすれば、反応後の容器の開封を全面的に廃止することができる。これは、コンタミネーションの防止上、たいへん望ましいことである。
【００７２】
さて、本発明によって合成される、１本鎖上に相補的な塩基配列が交互に連結された核酸には、たとえば次のような有用性がある。第一には、相補的な塩基配列を１分子内に備えた特殊な構造に伴う利点の活用である。この特徴により、検出が容易となることが期待される。すなわち、相補的な塩基配列との塩基対結合に伴って、シグナルを増減する核酸の検出系が公知である。たとえば先に述べたような２本鎖特異インターカレーターを検出剤として利用する方法などを組み合わせれば、本発明の合成生成物の特徴を生かした検出系を実現することができる。このような検出系において本発明の合成反応生成物を一度熱変性して元の温度に戻すと、分子内のアニールが優先的に起きるため速やかに相補的配列の間で塩基対結合を構成する。一方、もしも非特異的反応生成物が存在していても、それは分子内に相補的配列を持っていないので熱変性により２分子以上に分離してしまい、すぐにはもとの二本鎖には戻れない。こうして、検出前に加熱変性工程を追加することによって、非特異反応に伴うノイズを軽減することができる。熱に対して耐性を持たないDNAポリメラーゼを使用しているときには、加熱変性工程は反応停止の意味も持ち、反応時間の制御の点で有利である。
【００７３】
第二の特徴は、塩基対結合が可能な状態にあるループを常に形成することである。塩基対結合が可能な状態にあるループの構造を、図４に示した。図４からわかるように、ループはプライマーのアニールが可能な塩基配列Ｆ２ｃ（Ｘ２ｃ）と、Ｆ２ｃ−Ｆ１ｃ（Ｘ１ｃ）の間に介在する塩基配列とで構成される。Ｆ２ｃ−Ｆ１ｃ間（普遍的に示せばＸ２ｃ−Ｘ１ｃ間）の配列は、鋳型に由来する塩基配列である。したがってこの領域に対して相補的な塩基配列を持つプローブをハイブリダイズさせれば、鋳型特異的な検出を行うことができる。しかも、この領域は常に塩基対結合が可能な状態にあることから、ハイブリダイズに先だって加熱変性する必要がない。なお本発明の増幅反応生成物におけるループを構成する塩基配列は、任意の長さとすることができる。したがって、プローブのハイブリダイズを目的とする場合には、プライマーがアニールすべき領域とプローブがハイブリダイズすべき領域を別々にして両者の競合を避けることにより理想的な反応条件を構成することができる。
【００７４】
本発明の望ましい態様によれば、1本の核酸鎖上に塩基対結合が可能な多数のループがもたらされる。このことは、核酸１分子に多数のプローブがハイブリダイズ可能なことを意味しており、感度の高い検出を可能とする。また感度のみならず、たとえば凝集反応のような特殊な反応原理に基づく核酸の検出方法を可能とするものでもある。たとえばポリスチレンラテックスのような微粒子に固定したプローブを本発明による反応生成物に加えると、プローブとのハイブリダイゼーションに伴ってラテックス粒子の凝集が観察される。凝集の強度を光学的に測定すれば、高感度に、しかも定量的な観察が可能である。あるいは、凝集反応を肉眼的に観察することもできるので、光学的な測定装置を使わない反応系を構成することもできる。
【００７５】
更に、１核酸分子当たり多くの標識を結合できる本発明の反応生成物は、クロマトグラフィックな検出をも可能とする。イムノアッセイの分野では、肉眼的に検出可能な標識を利用したクロマト媒体を用いた分析方法（イムノクロマトグラフィー法）が実用化されている。この方法は、クロマト媒体に固定した抗体と標識抗体でアナライトをサンドイッチし、未反応の標識成分を洗い去る原理に基づいている。本発明の反応生成物は、この原理を核酸の分析にも応用可能とする。すなわち、ループ部分に対する標識プローブを用意し、これをクロマト媒体に固定化した捕捉用プローブでトラップすることによってクロマト媒体中での分析が行われる。捕捉用プローブには、ループ部分に対する相補配列を利用することができる。本発明の反応生成物は、多数のループ部分を伴っていることから、多数の標識プローブを結合し、肉眼的に認識可能なシグナルをもたらす。
【００７６】
ループとして常に塩基対結合が可能な領域を与える本発明による反応生成物は、この他にもさまざまな検出系を可能とする。たとえば、このループ部分に対するプローブを固定した表面プラズモン共鳴を利用した検出系が可能である。また、ループ部分に対するプローブを２本鎖特異的なインターカレーターで標識しておけば、より高感度な蛍光分析を行うことができる。あるいは、本発明によって合成される核酸が3'側と5'側の両方に塩基対結合が可能なループを形成することを積極的に利用することもできる。たとえば、一方のループを正常型と異常型で共通の塩基配列となる部分とし、他方のループに両者の違いが生じる領域となるように設計しておくのである。共通部分に対するプローブで遺伝子の存在を確認し、他方の領域で異常の有無を確認するといった特徴的な分析系を構成することができる。本発明による核酸の合成反応は、等温で進めることも可能なことから、一般的な蛍光光度計によってリアルタイムな分析が可能となることも特筆すべき利点である。これまでにも同一鎖上にアニールする核酸の構造は公知である。しかし本発明によって得ることができる１本鎖上に相補的な塩基配列が交互に連結された核酸は、他のオリゴヌクレオチドが塩基対結合することができる多数のループ部分を含む点において新規である。
【００７７】
一方、本発明による反応生成物によって与えられる多数のループ部分そのものをプローブとして利用することも可能である。たとえば、DNAチップにおいては、限られたエリアに高密度にプローブを集積する必要がある。しかし現在の技術では、一定の面積に固定することができるオリゴヌクレオチドの数は制限される。そこで本発明の反応生成物を利用すれば、アニールが可能な多数のプローブを高密度に固定化することができる。すなわち、本発明による反応生成物をプローブとしてDNAチップ上に固定すればよい。反応生成物は、増幅後に公知の手法によって固定することもできるし、あるいは固定化したオリゴヌクレオチドを本発明の増幅反応におけるオリゴヌクレオチドとして利用することにより結果的に固定化された反応生成物とすることもできる。このようにして固定化されたプローブを用いれば、限られたエリア中に多くの試料DNAをハイブリダイズすることができ、結果的に高いシグナルを期待することができる。
【００７８】
【実施例】
〔実施例１〕 M13mp18内の領域の増幅
M13mp18を鋳型として、本発明による１本鎖上に相補的な塩基配列が交互に連結された核酸の合成方法を試みた。実験に使用したプライマーは、M13FA、M13RA、M13F3、そしてM13R3の４種類である。M13F3とM13R3は、それぞれM13FAとM13RAを合成起点として得られた第１の核酸を置換するためのアウタープライマーである。アウタープライマーはM13FA（あるいはM13RA）よりも後から相補鎖合成の起点となるべきプライマーなので、M13FA（あるいはM13RA）と隣接する領域にコンティギュアス スタッキング現象を利用してアニールするように設計した。また、M13FA（あるいはM13RA）のアニールが優先的に起こるようにこれらのプライマー濃度を高く設定した。
【００７９】
各プライマーを構成する塩基配列は配列表に示したとおりである。プライマーの構造的な特徴を以下にまとめた。また標的塩基配列(target)に対する各領域の位置関係を図７に示した。

【００８０】
このようなプライマーによって、M13mp18の領域F1cからR1cにいたる領域とその相補的な塩基配列とが、F2cを含むループ形成配列を挟んで１本鎖上に交互に連結した核酸が合成される。これらのプライマーによる本発明による核酸の合成方法のための反応液組成を以下に示す。
【００８１】
反応液組成（２５μL中）
20mM Tris-HCl pH8.8
10mM KCl
10mM (NH₄)₂SO₄
6mM MgSO₄
0.1% Triton X-100
5% ジメチルスルホキシド(DMSO)
0.4mM dNTP
プライマー：
800nM M13FA／配列番号：１
800nM M13RA／配列番号：２
200nM M13F3／配列番号：３
200nM M13R3／配列番号：４
ターゲット：M13mp18 dsDNA ／配列番号：５
反応：上記反応液を９５℃で５分間加熱し、ターゲットを変性させて１本鎖とした。反応液を氷水上に移し、Bst DNA ポリメラーゼ(NEW ENGLAND BioLabs)を４U 添加し、６５℃で１時間反応させた。反応後、８０℃１０分間で反応を停止し再び氷水上に移した。
反応の確認：上記反応液の５μL に１μLのloading bufferを添加し、２％アガロースゲル（0.5% TBE）を使って、１時間、80mVで電気泳動した。分子サイズマーカーとして、XIV(100bp ladder, Boehringer Mannheim製)を使用した。泳動後のゲルをSYBR Green I (Molecular Probes,Inc.)で染色して核酸を確認した。結果は図８に示すとおりである。各レーンは次のサンプルに対応している。
1. XIV size marker
2. 1fmol M13mp18 dsDNA
3. targetなし
【００８２】
レーン３では未反応のプライマーが染色されている以外にバンドは確認されなかった。レーン２はターゲットが存在する場合、低サイズのバンドのラダーと高サイズでのスメアな染色、およびゲル内でほとんど泳動されていないバンドとして生成物が確認された。低サイズのバンドのうち、290bp、450bp付近のバンドは、それぞれ本発明の合成反応により予想される産物である、配列番号：１１および配列番号：１２が２本鎖となったもの（図２−(7)および図２−(10)が２本鎖となったものに相当）および配列番号：１３（図３−(9)にある長い１本鎖に相当）とサイズが一致することから、反応が予想されるとおりに進行していることが確認された。高サイズのスメアなパターン、および泳動されていないバンドは、本反応が基本的に連続的な反応であることから、反応産物が一定のサイズにはならないこと、そして部分的な１本鎖、あるいは２本鎖の複合体を形成した複雑な構造をともなっているため、結果としてこのような泳動結果を与えるものと考えられた。
【００８３】
〔実施例２〕 反応産物の制限酵素消化確認
実施例１で得られた本発明による１本鎖上に相補的な塩基配列が交互に連結された核酸の構造を明らかにすることを目的として、制限酵素による消化を行った。制限酵素を使った消化によって理論どおりの断片を生じる一方、実施例１で観察された高サイズのスメアなパターンや泳動されないバンドが消滅すれば、これらがいずれも本発明によって合成された１本鎖上に相補的な塩基配列を交互に連結した核酸であることが推定できる。
【００８４】
実施例１の反応液を８本分（200μL）プールし、フェノール処理後、エタノール沈でんを行って精製した。この沈でんを回収して200μLのTE緩衝液で再溶解し、その10μLを制限酵素BamHI、PvuII、およびHindIIIでそれぞれ３７℃２時間消化した。消化物を2%アガロースゲル（0.5% TBE）を使って、１時間、80mVで電気泳動した。分子サイズマーカーとして、SUPER LADDER-LOW (100bp ladder)（Gensura Laboratories,Inc.製）を使用した。泳動後のゲルをSYBR Green I (Molecular Probes,Inc.)で染色して核酸を確認した。結果は図９に示すとおりである。各レーンは次のサンプルに対応している。
1. XIV size marker
2. 精製物のBamHI消化物
3. 精製物のPvuII消化物
4. 精製物のHindIII消化物
【００８５】
ここで比較的鎖長の短い増幅生成物を構成している塩基配列は、配列番号：１３、配列番号：１４、配列番号：１５、および配列番号：１６等と推定される。これらの塩基配列から、推測される各制限酵素消化断片のサイズは表１のとおりである。表中のLは、ループ（１本鎖）を含む断片なので泳動位置が未確定であることを示す。
【００８６】
【表１】
本発明による増幅生成物の制限酵素消化断片

【００８７】
未消化でのバンドのほとんどが消化後には推定されるサイズのバンドへ変化したことから、目的の反応産物が増幅されていることが確認された。また非特異的産物はほとんどないことも示された。
【００８８】
〔実施例３〕 ベタイン添加による増幅反応の促進
増幅反応液中へのベタイン(betaine: N,N,N,-trimethylglycine、SIGMA)添加による、核酸の増幅反応に対する効果を調べる実験を行った。実施例１と同様に、M13mp18を鋳型とし、本発明による１本鎖上に相対的な塩基配列が交互に連結された核酸の合成方法を、種々の濃度のベタイン存在下で行った。実験に使用したプライマーは、実施例１で使用したものと同じである。鋳型DNA量は、10^-21 mol (M13mp18)で、陰性対照として水を用いた。添加するベタインは、0、0.5、1、2 Mの濃度になるように反応液に加えた。反応液組成を以下に示す。
反応液組成（２５μL中）
20mM Tris-HCl pH8.8
4mM MgSO₄
0.4mM dNTPs
10mM KCl
10mM (NH₄)₂SO₄
0.1% TritonX-100
プライマー：
800nM M13FA／配列番号：１
800nM M13RA／配列番号：２
200nM M13F3／配列番号：３
200nM M13R3／配列番号：４
ターゲット：M13mp18 dsDNA ／配列番号：５
使用したポリメラーゼ、反応条件、反応後の電気泳動条件は、実施例１に記載したものと同じである。
【００８９】
結果を図１０に示す。ベタイン濃度0.5、1.0M存在下での反応では、増幅産物量が増大した。また2.0Mまで増やすと逆に増幅産物は確認されなかった。これにより、適度のベタイン存在で、増幅反応が促進されることが示された。ベタイン濃度2Mの場合に、増幅産物が低下したことは、Tmが低下しすぎたのが原因と考えられた。
【００９０】
〔実施例４〕 HBV遺伝子配列の増幅
HBV遺伝子の部分配列を組み込んだM13mp18 dsDNAを鋳型として、本発明による核酸の合成方法を試みた。実験に使用したプライマーは、HB65FA（配列番号：６）、HB65RA（配列番号：７）、HBF3（配列番号：８）、そしてHBR3（配列番号：９）の４種類である。HBF3とHBR3は、それぞれHB65FAとHB65RAを合成起点として得られた第１の核酸を置換するためのアウタープライマーである。アウタープライマーはHB65FA（あるいはHB65RA）よりも後から相補鎖合成の起点となるべきプライマーなので、HB65FA（あるいはHB65RA）と隣接する領域にコンティギュアススタッキング現象を利用してアニールするように設計した。また、HB65FA（あるいはHB65RA）のアニールが優先的に起こるようにこれらのプライマー濃度を高く設定した。M13mp18に組みこまれたHBVに由来する本実施例のターゲット配列(430bp)を配列１０に示した。
【００９１】
各プライマーを構成する塩基配列は配列表に示したとおりである。プライマーの構造的な特徴を以下にまとめた。また標的塩基配列(target)に対する各領域の位置関係を図１１に示した。

【００９２】
このようなプライマーによって、HBV遺伝子の部分配列を組み込んだM13mp18(HBV-M13mp18)の領域F1cからR1cにいたる領域とその相補的な塩基配列とが、F2cを含むループ形成配列を挟んで１本鎖上で交互に連結した核酸が合成される。上記プライマーを用いる他は実施例１と同じ条件で反応させ、その反応液をアガロース電気泳動により分析した。結果は図１２に示すとおりである。各レーンは次のサンプルに対応している。
1. XIV size marker
2. １fmol HBV-M13mp18 dsDNA
3. targetなし
【００９３】
実施例１と同様に、targetが存在するときにのみ、低サイズのバンドのラダーと高サイズでのスメアな染色、およびゲル内でほとんど泳動されていないバンドとして生成物が確認された（レーン２）。低サイズのバンドのうち、310bp、および480bp付近のバンドはそれぞれ、本反応により予想される産物である、配列番号：１７および配列番号：１８の２本鎖とサイズが一致することから、反応が予想されるとおりに進行していることが確認された。高サイズのスメアなパターン、および泳動されていないバンドは、実施例１の結果で述べたように、本発明に特徴的な合成生成物の構造が原因となっているものと推定された。この実験により、増幅する配列(target)が異なっても本発明を実施可能であることが確認された。
【００９４】
〔実施例５〕 合成反応生成物のサイズの確認
本発明に基づいて合成された核酸の構造を確認するために、その長さをアルカリ変性条件下での電気泳動によって分析した。実施例１と実施例４のターゲット存在下での反応液の５μLに、それぞれ１μLのalkaline loading bufferを添加し、0.7％アガロースゲル(50mM NaOH, 1mM EDTA)を使って、14時間、50mAで電気泳動した。分子サイズマーカーとして、ラムダファージのHindIII消化断片を使用した。泳動後のゲルを1M Tris pH 8で中和後、SYBR Green I (Molecular Probes,Inc.)で染色して核酸を確認した。結果は図１３に示す。各レーンは以下のサンプルに対応している。
1. ラムダファージのHindIII消化断片
2. 実施例１の反応生成物
3. 実施例４の反応生成物
【００９５】
反応生成物をアルカリ変性条件で泳動すると１本鎖状態でのサイズ確認が可能である。実施例１（レーン２）、実施例４（レーン３）ともに主な生成物は2 kbase内であることが確認された。また、本発明による生成物はこの分析によって確認できる範囲で少なくとも6 kbase以上にまで伸びていることが判明した。加えて、実施例１や実施例４の未変性条件下で泳動されなかったバンドは、変性状態では個々の１本鎖に分離されサイズが小さくなることが改めて確認された。
【００９６】
〔実施例６〕 M-13mp13内の領域の増幅における、ターゲット濃度依存的増幅の確認
本発明による核酸の合成方法に及ぼす、ターゲットの濃度変化の影響を観察した。ターゲットであるM13mp18 dsDNAを０〜1 fmolとし、反応時間を１時間および３時間とする他は、実施例１と同じ条件で本発明による核酸の合成方法を実施した。実施例１と同様に、２％アガロースゲル（0.5% TBE）で電気泳動し、SYBR Green I (Molecular Probes,Inc.)染色により核酸を確認した。分子サイズマーカーとして、XIV(100bp ladder, Boehringer Mannheim)を使用した。結果は図１４（上：反応時間１時間、下：反応時間３時間）に示した。各レーンは、次のサンプルに対応する。
1. M13mp18 dsDNA 1x10^-15mol/tube
2. M13mp18 dsDNA 1x10^-16mol/tube
3. M13mp18 dsDNA 1x10^-17mol/tube
4. M13mp18 dsDNA 1x10^-18mol/tube
5. M13mp18 dsDNA 1x10^-19mol/tube
6. M13mp18 dsDNA 1x10^-20mol/tube
7. M13mp18 dsDNA 1x10^-21mol/tube
8. M13mp18 dsDNA 1x10^-22mol/tube
9. target なし
10. XIV size marker
【００９７】
泳動像の下部に見られる各レーンに共通のバンドは未反応のプライマーが染色されたものである。反応時間にかかわらず、ターゲットが存在しないときは全く増幅産物は観察されない。ターゲット存在下でのみ、ターゲットの濃度依存的に増幅産物の染色パターンが得られた。また、反応時間を長くすることにより、より低濃度まで増幅産物が確認できた。
【００９８】
〔実施例７〕 点変異（ポイントミューテーション）の検出
（１）M13mp18FM（変異型）の作製
ターゲットDNAとして、M13mp18（野生型）、およびM13mp18FM（変異型）を用いた。変異型であるM13mp18FMの作製は、LA PCR^TM in vitro Mutagenesis Kit（宝酒造）を使用し、1塩基置換を導入した。その後、シークエンシングにより配列を確認した。F1領域での配列を以下に示す。
野生型：CCGGGGATCCTCTAGAGTCG(配列番号：１９)
変異型：CCGGGGATCCTCTAGAGTCA（配列番号：２０）
【００９９】
（２）プライマーのデザイン
使用するプライマーは、FAプライマーのF1c領域の5'末端に野生型、変異型で配列の異なる塩基となるようにした。変異の位置、および標的塩基配列(target)に対する各領域の位置関係を図１５に示す。
【０１００】
（３）増幅反応
M13mp18（野生型）、およびM13mp18FM（変異型）を鋳型にして、以下に示すそれぞれに特異的なプライマーの組み合わせで鋳型特異的な増幅反応が起きるかどうか実験を行った。
野生型増幅用プライマーセット： FAd4, RAd4, F3, R3
変異型増幅用プライマーセット： FAMd4, RAd4, F3, R3
各プライマーの塩基配列は以下の通りである。
FAd4 :CGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT（配列番号：２１）
FAMd4： TGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT（配列番号：２２）
RAd4 ： CGTCGTGACTGGGAAAACCCTTTTTGTGCGGGCCTCTTCGCTATTAC（配列番号：２３）
F3 ： ACTTTATGCTTCCGGCTCGTA（配列番号：２４）
R3 ： GTTGGGAAGGGCGATCG（配列番号：２５）
【０１０１】
（４）M13mp18の点突然変異の検出
反応液の組成は以下のとおりである。

【０１０２】
上記反応液にターゲットM13mp18、またはM13mp18FM 1 fmol (2μl)を添加し、９５℃で５分間加熱し、ターゲットを変性させて１本鎖とした。反応液を氷水上に移し、Bst DNA ポリメラーゼ(NEW ENGLAND BioLabs)を1μL(8U) 添加し、６８℃または68.5℃で１時間反応させた。反応後、８０℃１０分間で反応を停止し再び氷水上に移した。
図１６で示すように、FAプライマーとして野生型用のFAd4を用いたときは、野生型の鋳型存在のみ効果的に増幅が観察された。一方、FAプライマーとして変異型用のFAMd4を用いたときは、野生型の鋳型存在のみ効果的に増幅が観察された。
以上の結果から、本発明の増幅反応を利用することにより、点変異を効率的に検出できることが示された。
【０１０３】
〔実施例８〕 mRNAをターゲットとした増幅反応
ターゲットとなる核酸をmRNAとして、本発明による核酸の合成方法を試みた。ターゲットとなるmRNAは、前立腺特異抗原(Prostate specific antigen; PSA)を発現した細胞である前立腺癌細胞株LNCaP cell (ATCC No. CRL-1740)と、非発現細胞である慢性骨髄性白血病細胞株K562 cell (ATCC No. CCL-243)を、1:10⁶〜100:10⁶で混合し、Qiagen社（ドイツ）のRNeasy Mini kitを用いて全RNAを抽出した。実験に使用したプライマーは、PSAFA、PSARA、PSAF3、そしてPSAR3の4種類である。PSAF3とPSAR3は、それぞれPSAFAとPSARAを合成起点として得られた第一の核酸を置換するためのアウタープライマーである。また、PSAFA(あるいはPSARA)のアニールが優先的に起こるようにこれらのプライマー濃度を高く設定した。各プライマーを構成する塩基配列は以下のとおりである。
プライマー：
PSAFA: TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG （配列番号：２６）
PSARA: TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG（配列番号：２７）
PSAF3: TGCTTGTGGCCTCTCGTG（配列番号：２８）
PSAR3: GGGTGTGGGAAGCTGTG（配列番号：２９）
【０１０４】
プライマーの構造的な特徴を以下にまとめた。また、標的のmRNAをコードするDNA塩基配列に対する各プライマーの位置関係、および制限酵素Sau3A Iの認識部位を図１７に示した。

【０１０５】
本発明による核酸の合成方法のための反応液組成を以下に示す。
反応液組成（２５μL中）
20mM Tris-HCl pH8.8
4mM MgSO₄
0.4mM dNTPs
10mM KCl
10mM (NH₄)₂SO₄
0.1% TritonX-100
0.8M betaine
5mM DTT
1600nM PSAFA & PSARA プライマー
200nM PSAF3 & PSAR3 プライマー
8U Bst DNAポリメラーゼ
100U ReverTra Ace (TOYOBO, 日本)
5μg 全RNA
【０１０６】
全ての成分は氷上で混合した。本実験においてはmRNA（１本鎖）をtargetとしているので、加熱変性によって１本鎖とする工程は不要である。反応は、65℃で45分間行い、85℃、5分で、反応を停止させた。反応終了後、5μLの反応液を2%アガロースを使って電気泳動し、SYBR Green Iで検出した。
結果を図１８に示す。各レーンは、以下のサンプルに対応している。
【０１０７】
レーン Bst RT LNCaP細胞数／10⁶個のK562
１ − ＋ 0
２ − ＋ 10
３ ＋ − 0
４ ＋ − 10
５ ＋ ＋ 0
６ ＋ ＋ 1
７ ＋ ＋ 10
８ レーン６の反応液１μL分をSau3A Iで消化したもの
９ レーン７の反応液１μL分をSau3A Iで消化したもの
１０ サイズマーカー 100bpラダー(New England Biolabs)
【０１０８】
Bst DNAポリメラーゼ、ReverTra Aceのいずれか一方がないと、増幅産物が得られなかった（レーン１〜４）。両方の酵素存在下では、LNCaP由来のRNAが存在すると、増幅産物が検出された（レーン５〜７）。100万個のK562細胞に1個のLNCaPからの抽出RNAでも検出可能であった（レーン６）。増幅産物は、ターゲット内部の配列にある制限酵素部位Sau3A Iで消化したところ、予想される大きさの断片に消化された（レーン８，９）。
以上の結果から、本発明による核酸の合成方法において、ターゲットとしてRNAを用いた場合でも、目的の反応産物が得られることが確認された。
【０１０９】
【発明の効果】
本発明による新規なオリゴヌクレオチドとそれを用いた核酸の合成方法により、複雑な温度制御の不要な１本鎖上に交互に相補的な塩基配列が連結した核酸の合成方法が提供される。本発明に基づくオリゴヌクレオチドをプライマーとして合成される相補鎖は、常にその3'末端が自身を鋳型とする新たな相補鎖合成の合成起点となる。このとき、新たなプライマーのアニールをもたらすループの形成を伴い、この部分からの相補鎖合成によって先に合成された自身を鋳型とする相補鎖合成反応の生成物は再び置換され塩基対結合が可能な状態となる。このようにして得ることができる自身を鋳型として合成された核酸は、たとえばSDAのような公知の核酸合成方法との組み合わせによって、それらの核酸合成効率の向上に貢献する。
【０１１０】
更に本発明の望ましい態様によれば、単に公知の核酸合成方法の効率向上を達成するのみならず、複雑な温度制御を必要としない、しかも高度な増幅効率を期待でき、更には高い特異性を達成できる新規な核酸の合成方法が提供される。すなわち、本発明に基づくオリゴヌクレオチドを鋳型鎖とその相補鎖に対して適用することによって、１本鎖上に相補的な塩基配列が交互に連結された核酸が連続して合成されるようになる。この反応は、原理的には合成に必要な出発材料が枯渇するまで続き、その間にループ部分から合成を開始した新たな核酸を生成し続ける。こうして、ループにアニールしたオリゴヌクレオチドからの伸長が、長い１本鎖核酸（すなわち、複数組の相補的な塩基配列が連結したもの）の伸長のための3'-OHを供給する鎖置換を行う。一方、長い１本鎖の3'-OHは自身を鋳型とする相補鎖合成反応を行うことによって自身の伸長を達成すると同時に、ループから合成開始した新たな相補鎖の置換を行う。このような増幅反応工程が、高い特異性を維持しながら等温条件下で進行する。
【０１１１】
本発明におけるオリゴヌクレオチドは、２つの連続した領域が設計どおりに配置されているときにはじめて本発明による核酸合成反応のためのプライマーとして機能する。このことが、特異性の維持に大きく貢献する。たとえばPCRでは、２つのプライマーの意図した位置関係とは無関係に、非特異的なミスアニールにより、非特異的増幅反応が開始してしまうことと比べれば、本発明では高い特異性が期待できることは容易に説明できる。この特徴を利用してSNPsを高い感度で精確に検出することができる。
【０１１２】
本発明の特徴は、このような反応がごく単純な試薬構成で容易に達成できることにある。たとえば本発明によるオリゴヌクレオチドは、特殊な構造を持つとは言えそれは塩基配列の選択の問題であって、物質としては単なるオリゴヌクレオチドである。また、望ましい態様においては、鎖置換型の相補鎖合成反応を触媒するDNAポリメラーゼのみで反応を進めることができる。更に、RNAを鋳型として本発明を実施する場合には、Bca DNAポリメラーゼのような逆転写酵素活性と鎖置換型のDNAポリメラーゼ活性を併せ持つDNAポリメラーゼを利用することによって、全ての酵素反応を単一の酵素によって行うことができる。このようなシンプルな酵素反応で高度な核酸の増幅反応を実現する反応原理はこれまでに知られていない。あるいは、SDA等の公知の核酸合成反応に適用するとしても、本発明との組み合わせによって新たな酵素が必要となるようなことはなく、単に本発明に基づくオリゴヌクレオチドを組み合わせるだけで各種反応系への適応が可能である。したがて、本発明による核酸合成方法は、コストの点においても有利といえる。
以上述べたように、本発明の核酸の合成方法とそのためのオリゴヌクレオチドは、操作性（温度制御不要）、合成効率の向上、経済性、そして高い特異性という、複数の困難な課題を同時に解決する新たな原理を提供する。
【０１１３】
【配列表】

【図面の簡単な説明】
【図１】 本発明の望ましい態様の反応原理の一部(1)-(4)を示す模式図である。
【図２】 本発明の望ましい態様の反応原理の一部(5)-(7)を示す模式図である。
【図３】 本発明の望ましい態様の反応原理の一部(8)-(10)を示す模式図である。
【図４】 本発明による１本鎖核酸が形成するループの構造を示す模式図である。
【図５】 本発明による基礎的な態様の一部(A)-(B)を示す模式図である。
【図６】 本発明による基礎的な態様の一部(C)-(D)を示す模式図である。
【図７】 M13mp18の標的塩基配列における、オリゴヌクレオチドを構成する各塩基配列の位置関係を示す図である。
【図８】 M13mp18を鋳型として本発明による１本鎖核酸の合成方法によって得られた生成物のアガロース電気泳動の結果を示す写真である。
レーン１：XIV size marker
レーン２：１fmol M13mp18 dsDNA
レーン３：targetなし
【図９】 実施例１によって得られた本発明による核酸合成反応の生成物を制限酵素で消化しアガロース電気泳動した結果を示す写真である。
レーン１：XIV size marker
レーン２：精製物のBamHI消化物
レーン３：精製物のPvuII消化物
レーン４：精製物のHindIII消化物
【図１０】 M13mp18を鋳型として、ベタイン添加による本発明の１本鎖核酸の合成方法によって得られた生成物のアガロース電気泳動の結果を示す写真である。０、0.5、1、２は反応液中に添加したベタイン濃度(M)を表す。また、Nは陰性対照を、-21は鋳型DNAの濃度10^-21molを表す。
【図１１】 HBV由来の標的塩基配列における、オリゴヌクレオチドを構成する各塩基配列の位置関係を示す図である。
【図１２】 M13mp18に組みこまれたHBV-M13mp18を鋳型として本発明による１本鎖核酸の合成方法によって得られた生成物のアガロース電気泳動の結果を示す写真である。
レーン１：XIV size marker
レーン２：１fmol HBV-M13mp18 dsDNA
レーン３：targetなし
【図１３】 本発明による１本鎖核酸の合成方法によって得られた生成物のアルカリ変性ゲル電気泳動の結果を示す写真である。
レーン１：ラムダファージのHindIII消化断片
レーン２：実施例１の反応生成物
レーン３：実施例３の反応生成物
【図１４】 ターゲットであるM13mp18の濃度を変えてときに、本発明による１本鎖核酸の合成方法によって得られた生成物のアガロース電気泳動の結果を示す写真である。上は反応時間１時間、下は反応時間３時間の結果である。
レーン１：M13mp18 dsDNA 1x10^-15mol/tube
レーン２：M13mp18 dsDNA 1x10^-16mol/tube
レーン３：M13mp18 dsDNA 1x10^-17mol/tube
レーン４：M13mp18 dsDNA 1x10^-18mol/tube
レーン５：M13mp18 dsDNA 1x10^-19mol/tube
レーン６：M13mp18 dsDNA 1x10^-20mol/tube
レーン７：M13mp18 dsDNA 1x10^-21mol/tube
レーン８：M13mp18 dsDNA 1x10^-22mol/tube
レーン９：target なし
レーン１０：XIV size marker
【図１５】 変異の位置、および標的塩基配列(target)に対する各領域の位置関係を表す図である。下線で示したグアニンが、変異型ではアデニンに置換されている。
【図１６】 本発明の増幅反応による生成物のアガロース電気泳動の結果を示す写真である。
M: 100bp ladder（New England Biolabs）
N: 鋳型なし（精製水）
WT: 野生型鋳型M13mp18 1 fmol
MT: 変異型鋳型M13mp18FM 1 fmol
【図１７】 標的mRNAをコードする塩基配列における、オリゴヌクレオチドを構成する各塩基配列の位置関係を示す図である。
【図１８】 mRNAをターゲットとして本発明による１本鎖核酸の合成方法によって得られた生成物のアガロース電気泳動の結果を示す写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for synthesizing a nucleic acid composed of a specific base sequence, which is useful as a method for amplifying a nucleic acid.
[0002]
[Prior art]
Analysis methods based on complementarity of nucleobase sequences can directly analyze genetic characteristics. Therefore, it is a very effective means for genetic diseases, canceration, identification of microorganisms, and the like. In addition, in order to make the gene itself a detection target, for example, a time-consuming and laborious operation such as culture may be omitted.
[0003]
However, detection when the amount of the target gene present in the sample is small is generally not easy, and it is necessary to amplify the target gene itself or the detection signal. A PCR (Polymerase Chain Reaction) method is known as one method for amplifying a target gene (Science, 230, 1350-1354, 1985). The PCR method is currently the most common method for nucleic acid amplification in vitro. It has become established as an excellent detection method due to its high sensitivity based on its exponential amplification effect. In addition, since the amplified product can be recovered as DNA, it is widely applied as an important tool supporting genetic engineering techniques such as gene cloning and structure determination. However, the PCR method requires a special temperature control device for the implementation; the amplification reaction proceeds exponentially, and there is a problem with the quantitativeness; It has been pointed out that the nucleic acid mixed in is easily affected by contamination that functions as a template.
[0004]
With the accumulation of genome information, analysis of single nucleotide polymorphisms (SNPs) has attracted attention. By designing SNPs to be included in the primer base sequence, it is possible to detect SNPs using PCR. That is, the presence / absence of a base sequence complementary to the primer can be determined from the presence / absence of the reaction product. However, in PCR, if the complementary strand synthesis is mistakenly performed, the product functions as a template for the subsequent reaction and gives an incorrect result. Actually, it is said that it is difficult to strictly control PCR only by a single base difference at the end of the primer. Therefore, there is a need for improved specificity to utilize PCR for the detection of SNPs.
[0005]
On the other hand, nucleic acid synthesis methods based on ligase have also been put into practical use. The LCR method (Ligase Chain Reaction, Laffler TG; Carrino JJ; Marshall RL; Ann. Biol. Clin. (Paris), 1993, 51: 9, 821-6) uses two adjacent probes on the sequence to be detected. The basic principle is a reaction in which the two are hybridized and linked together by ligase. Since the two probes cannot be linked when the target base sequence does not exist, the presence of the ligation product serves as an indicator of the target base sequence. The LCR method also has the same problems as the PCR method because temperature control is required to separate the synthesized complementary strand from the template. Regarding LCR, a method of improving the specificity by adding a step of filling a gap between adjacent probes and filling it with DNA polymerase has also been reported. However, only the specificity can be expected by this improved method, and there remains a problem with respect to requiring temperature control. Moreover, it can be said that the cost is sacrificed because the necessary enzymes increase.
[0006]
A method for amplifying DNA having a complementary sequence using a detection target sequence as a template is SDA (Strand Displacement Amplification) [Proc. Natl. Acad. Sci. USA, 89, 392-396; 1992] [Nucleic Acid. Res. , 20, 1691-1696; 1992] is also known. In the SDA method, when complementary strand synthesis is performed using a complementary primer on the 3 'side of a base sequence as a starting point, if a double-stranded region is present on the 5' side, the complementary strand is synthesized while replacing that strand. It is a method using a special DNA polymerase that performs the above. In the following description, when simply expressed as the 5 ′ side or the 3 ′ side, it means the direction in the strand that is the template. This is called the SDA method because the 5 'double-stranded portion is displaced by a newly synthesized complementary strand. In the SDA method, a restriction enzyme recognition sequence is inserted in advance into the annealed sequence as a primer, whereby the temperature change step that is essential in the PCR method can be omitted. In other words, the nick produced by the restriction enzyme gives a 3′-OH group that is the starting point for complementary strand synthesis, and by performing strand displacement synthesis from that, the previously synthesized complementary strand is released as a single strand. Reused as a template for complementary strand synthesis. Thus, the SDA method eliminates the need for complicated temperature control that was essential in the PCR method.
[0007]
However, in the SDA method, in addition to the strand displacement type DNA polymerase, it is necessary to combine restriction enzymes that cause nicks. An increase in the number of necessary enzymes is a factor in increasing costs. In addition, since the nick is introduced (ie, only one strand is cleaved) rather than the double strand is cleaved by the restriction enzyme to be used, α is used as a substrate during synthesis so that one strand is resistant to enzymatic digestion. DNTP derivatives such as thio dNTPs must be utilized. For this reason, the amplification product by SDA has a structure different from that of a natural nucleic acid, and its use such as cleavage by a restriction enzyme and application of the amplification product to gene cloning is limited. In this respect, it can be said that there is a factor of cost increase. In addition, when applying the SDA method to an unknown sequence, the possibility that the same base sequence as the restriction enzyme recognition sequence for nick introduction exists in the synthesized region cannot be denied. In such a case, there is a concern that the synthesis of a completely complementary strand is hindered.
[0008]
As a nucleic acid amplification method that does not require complicated temperature control, NASBA (also referred to as Nucleic Acid Sequence-based Amplification or TMA / Transcription Mediated Amplification method) is known. NASBA synthesizes DNA with DNA polymerase using a target RNA as a template and a T7 promoter-added probe. This is further converted into double strands with a second probe, and the resulting double-stranded DNA is used as a template for transcription with T7 RNA polymerase. Is a reaction system that amplifies a large amount of RNA (Nature, 350, 91-92, 1991). NASBA requires several heat denaturation steps to complete double-stranded DNA, but the subsequent transcription reaction with T7 RNA polymerase proceeds isothermally. However, since a combination of a plurality of enzymes such as reverse transcriptase, RNaseH, DNA polymerase, and T7 RNA polymerase is essential, it is disadvantageous in terms of cost as with SDA. In addition, since setting conditions for performing a plurality of enzyme reactions is complicated, it is difficult to disseminate it as a general analysis method. As described above, the known nucleic acid amplification reaction still has a problem of complicated temperature control or a need for a plurality of enzymes.
[0009]
Furthermore, there are few reports on attempts to further improve the efficiency of nucleic acid synthesis without sacrificing specificity or cost for these known nucleic acid synthesis reactions. For example, in a method called RCA (Rolling-circle amplification), it has been shown that single-stranded DNA having a continuous base sequence complementary to a padlock probe can be continuously synthesized in the presence of the target base sequence. (Paul M. Lizardi et al, Nature Genetics 19, 225-232, July, 1998). In RCA, a padlock probe having a special structure in which the 5 ′ end and 3 ′ end of one oligonucleotide constitute adjacent probes in LCR is used. By combining a polymerase that catalyzes a strand displacement type complementary strand synthesis reaction, a continuous complementary strand synthesis reaction is triggered using a padlock probe that has been ligated and cyclized in the presence of the target base sequence as a template. A single-stranded nucleic acid having a structure in which regions composed of the same base sequence are repeated continuously is generated. Primers are further annealed to the single-stranded nucleic acid to synthesize the complementary strand, thereby realizing high-level amplification. However, the need for multiple enzymes remains a challenge. In addition, the trigger for complementary strand synthesis depends on the ligation reaction of two adjacent regions, and its specificity is in principle the same level as LCR.
[0010]
For the problem of supplying 3′-OH, a method is known in which a 3 ′ end has a sequence complementary to the base sequence on the same strand to form a hairpin loop at the end (Gene 71, 29- 40,1988). From such a hairpin loop, complementary strand synthesis is performed using itself as a template, and a single-stranded nucleic acid composed of a complementary base sequence is generated. For example, PCT / FR95 / 00891 realizes a structure that anneals on the same strand at the terminal portion where complementary base sequences are linked. However, in this method, a step of eliminating the base pairing with the complementary strand at the end and reconstructing the base pairing on the same strand is essential. This step is said to proceed depending on a delicate equilibrium state at the ends of complementary base sequences with base pairing. That is, using the equilibrium state maintained between the base pair bond with the complementary strand and the base pair bond on the same strand, only the one annealed with the base sequence on the same strand is the starting point for the complementary strand synthesis. Become. Therefore, in order to achieve a high reaction efficiency, it is considered that strict reaction conditions are required. Furthermore, in this prior art, the primer itself forms a loop structure. Therefore, once the primer dimer is generated, the amplification reaction is automatically started regardless of the presence or absence of the target base sequence, and a non-specific synthesis product is formed. This is a serious problem. Furthermore, the generation of primer dimers and the consumption of primers due to the non-specific synthesis reaction associated therewith leads to a decrease in the amplification efficiency of the target reaction.
[0011]
In addition, there is a report (EP713922) that realizes a 3 ′ end structure that anneals to the same strand using a region that does not serve as a template for DNA polymerase. This report also has the same problems as the previous PCT / FR95 / 00891 in that it uses the dynamic equilibrium in the terminal part or in the possibility of non-specific synthesis reaction accompanying primer dimer formation. Furthermore, a special region that does not serve as a template for DNA polymerase must be prepared as a primer.
[0012]
In various signal amplification reactions applying the above-mentioned NASBA principle, an oligonucleotide with a hairpin-like structure at the end is often used to supply a double-stranded promoter region (Japanese Patent Laid-Open No. 5-21873). . However, these do not allow continuous supply of 3'-OH for complementary strand synthesis. Furthermore, in Japanese Patent Publication No. 10-510161 (WO96 / 17079), a hairpin loop structure in which the 3 ′ end is annealed on the same strand is used for the purpose of obtaining a DNA template to be transcribed by RNA polymerase. In this method, template amplification is performed using transcription to RNA and reverse transcription from RNA to DNA. However, this method cannot also constitute a reaction system unless a plurality of enzymes are combined.
[0013]
[Problems to be solved by the invention]
An object of the present invention is to provide a nucleic acid synthesis method based on a novel principle. More specifically, it is to provide a method capable of realizing nucleic acid synthesis depending on sequences efficiently at low cost. That is, it is an object of the present invention to provide a method capable of achieving nucleic acid synthesis and amplification using a single enzyme and under isothermal reaction conditions. Another object of the present invention is to provide a method for synthesizing a nucleic acid capable of realizing high specificity that is difficult to achieve by the known nucleic acid synthesis reaction principle, and a method for amplifying a nucleic acid using this synthesis method.
[0014]
[Means for Solving the Problems]
The present inventors first noticed that the use of a polymerase that catalyzes the synthesis of a strand displacement type complementary strand is useful for nucleic acid synthesis that does not depend on complicated temperature control. Such a DNA polymerase is an enzyme also used in SDA and RCA. However, even if such an enzyme is used, a known primer-based method always requires another enzyme reaction to supply 3′-OH as a starting point of synthesis, such as SDA.
[0015]
Therefore, the present inventors examined the supply of 3′-OH from a completely different angle from the known approach. As a result, the inventors have found that by using an oligonucleotide having a special structure, 3′-OH can be supplied without depending on an additional enzyme reaction, and the present invention has been completed. That is, the present invention relates to a method for synthesizing the following nucleic acids, a method for amplifying nucleic acids using the method for synthesizing nucleic acids, and novel oligonucleotides that enable these methods.
[1] A method for synthesizing a nucleic acid in which complementary base sequences are alternately linked on a single strand, including the following steps.
a) A region F1 that can be annealed to a part F1c on the same strand is provided at the 3 ′ end, and this region F1 anneals to F1c, thereby forming a loop including a region F2c capable of base pairing. Providing a nucleic acid capable of
b) A step of synthesizing complementary strands starting from the 3 ′ end of F1 annealed to F1c
c) An oligonucleotide containing F2 having a sequence complementary to the region F2c at its 3 ′ end is annealed, and this is used as a starting point for synthesis to carry out complementary strand synthesis using a polymerase that catalyzes a strand displacement complementary strand synthesis reaction. The step of substituting the complementary strand synthesized in
d) Annealing a polynucleotide containing a sequence complementary to an arbitrary region in the complementary strand that has been replaced in step c) and capable of base pairing, and complementing the strand using the 3 'end as a synthesis origin Performing complementary strand synthesis with a polymerase that catalyzes the strand synthesis reaction, and replacing the complementary strand synthesized in step c)
[2] In step d), the synthesis origin is a region R1 existing at the 3 ′ end on the same strand that can be annealed to the region R1c, and the region R2c capable of base pairing by annealing R1 to R1c The method according to [1], wherein a loop containing is formed.
[3] An oligonucleotide comprising at least the following two regions X2 and X1c, wherein X1c is linked to the 5 ′ side of X2.
X2: a region having a base sequence complementary to an arbitrary region X2c of a nucleic acid having a specific base sequence
X1c: region having substantially the same base sequence as region X1c located 5 ′ of region X2c in a nucleic acid having a specific base sequence
[4] The method according to [1], wherein the nucleic acid in step a) is a second nucleic acid provided by the following steps.
i) annealing the region F2 of the oligonucleotide according to [3], wherein the region X2 is the region F2 and the region X1c is the region F1c;
ii) A step of synthesizing a first nucleic acid having a base sequence complementary to a template using F2 of the oligonucleotide as a synthesis origin.
iii) making any region of the first nucleic acid synthesized in step ii) a state capable of base pairing;
iv) annealing an oligonucleotide having a base sequence complementary to the region capable of base pairing of the first nucleic acid in step iii), and synthesizing the second nucleic acid using it as a starting point for synthesis; Of F1 in a state capable of base pairing
[5] The region that enables base pairing in step iii) is R2c, and the oligonucleotide in step iv) is that the region X2c is region R2c and the region X1c is region R1c The method according to [4], which is an oligonucleotide.
[6] The step of enabling base pairing in steps iii) and iv) was used as the synthesis starting point in the outer primer that anneals further to the 3 ′ side of F2c in the template and in step iv) of the first nucleic acid. The method according to [4] or [5], wherein the method is carried out by strand displacement complementary strand synthesis using a polymerase that catalyzes strand displacement complementary strand synthesis reaction starting from an outer primer that anneals further 3 ′ to the region.
[7] The method according to [6], wherein the melting temperature of each oligonucleotide used in the reaction and its complementary region in the template has the following relationship under the same stringency. (3 ′ side region in outer primer / template) ≦ (F2c / F2 and R2c / R2) ≦ (F1c / F1 and R1c / R1)
[8] The method according to any one of [4] to [7], wherein the template nucleic acid is RNA, and the complementary strand synthesis in step ii) is performed with an enzyme having reverse transcriptase activity.
[9] A method for amplifying a nucleic acid in which complementary base sequences are alternately linked on a single strand by repeating the following steps.
A) At the 3 ′ end and 5 ′ end, a region composed of a base sequence complementary to the end region is provided on the same strand, and when this complementary base sequence anneals, a base pair bond is present between the two. Providing a mold in which a possible loop is formed
B) Complementary strand synthesis using the 3 ′ end of the template annealed to the same strand as a synthesis origin,
C) An oligonucleotide containing a complementary base sequence at the 3 ′ end in the loop located on the 3 ′ end side of the loop is annealed to the loop portion, and this is used as a starting point for synthesis to catalyze the strand displacement complementary strand synthesis reaction. Performing complementary strand synthesis with a polymerase to replace the complementary strand synthesized in step B) so that its 3 ′ end is capable of base pairing; and
D) A step in which a strand in which the 3 ′ end is capable of base pairing in step C) is used as a new template in step A)
[10] The amplification method according to [9], wherein the oligonucleotide in step C) is provided with a complementary base sequence at the 3 ′ end, which is the origin of synthesis in step B), at the 5 ′ end.
[11] The amplification method according to [10], further comprising the step of using the complementary strand synthesized from the oligonucleotide in step C) as a template in step A).
[12] The amplification method according to [9], wherein the template in step A) is synthesized by the method according to [5].
[13] The method according to [1] or [9], wherein the strand displacement complementary strand synthesis reaction is performed in the presence of a melting temperature adjusting agent.
[14] The method according to [13], wherein the melting temperature adjusting agent is betaine.
[15] The method according to [14], wherein 0.2 to 3.0 M betaine is present in the reaction solution.
[16] A method for detecting a target base sequence in a sample by performing the amplification method according to any one of [9] to [15] and observing whether an amplification reaction product has been generated.
[17] The method according to [16], wherein a probe containing a base sequence complementary to the loop is added to the amplification reaction product, and hybridization between the two is observed.
[18] The method according to [17], wherein the probe is particle-labeled and an agglutination reaction caused by hybridization is observed.
[19] The amplification method according to any one of [9] to [15] is performed in the presence of a nucleic acid detection agent, and observation is made as to whether an amplification reaction product has been generated based on a signal change of the detection agent [16]. The method described in 1.
[20] A method for detecting a mutation in a target base sequence by the detection method according to [16], wherein the mutation in the base sequence to be amplified prevents synthesis of any of the complementary strands constituting the amplification method. There is a way.
[21] A kit for synthesizing a nucleic acid, comprising the following elements, wherein complementary base sequences are alternately linked on a single strand.
i) The oligonucleotide according to [3], wherein the template nucleic acid region F2c is X2c, and F1c located on the 5 ′ side of F2c is X1c.
ii) an oligonucleotide comprising a base sequence complementary to any region in a complementary strand synthesized using the oligonucleotide of i) as a primer
iii) An oligonucleotide having a base sequence complementary to the region F3c located 3 ′ of the region F2c of the nucleic acid used as a template
iv) a DNA polymerase that catalyzes a strand displacement type complementary strand synthesis reaction; and
v) Nucleotides that are substrates for element iv)
[22] The oligonucleotide according to [3], wherein in the oligonucleotide ii), an arbitrary region R2c in the complementary strand synthesized from the oligonucleotide i) is defined as X2c, and R1c located 5 ′ of R2c is defined as X1c. [21] The kit according to [21].
[23] The kit according to [22], further comprising the following elements.
vi) An oligonucleotide having a base sequence complementary to a region R3c located 3 ′ of an arbitrary region R2c in a complementary strand synthesized using the oligonucleotide i) as a synthesis origin.
[24] A kit for detecting a target base sequence, which further comprises a detection agent for detecting a product of a nucleic acid synthesis reaction in any one of the kits according to [21] to [23].
[0016]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, a nucleic acid in which complementary base sequences are alternately linked on a single strand, which is the object of synthesis, means a nucleic acid in which complementary base sequences are linked side by side on a single strand. Furthermore, in the present invention, a base sequence for forming a loop between complementary base sequences must be included. In the present invention, this arrangement is called a loop-forming arrangement. The nucleic acid synthesized according to the present invention is substantially composed of complementary base sequences linked by the loop forming sequence. In general, a substance that does not separate into two or more molecules when the base pair bond is dissociated, regardless of whether it is partially accompanied by a base pair bond, is called a single strand. Complementary base sequences can form base pair bonds on the same strand. The intramolecular base-paired product obtained by base-pairing a nucleic acid having complementary base sequences alternately linked on one strand according to the present invention on the same strand is apparently a double-stranded product. A region to be constructed and a loop portion without base pairing are given.
[0017]
That is, the nucleic acid in which complementary base sequences are alternately linked on one strand in the present invention includes a complementary base sequence that can be annealed on the same strand, and the annealed product is a bent hinge. It can also be defined as a single-stranded nucleic acid that constitutes a loop that does not involve base pairing in the part. A nucleotide having a complementary base sequence can anneal to a loop that does not involve base pairing. The loop forming sequence can be any base sequence. Base pairing is possible so that complementary strand synthesis for substitution can be initiated, preferably with sequences that are distinguishable from base sequences present in other regions to achieve specific annealing. For example, in a desirable mode, the base sequence is substantially the same as the region F2c (or R2c) that is derived from the template nucleic acid and annealed on the same strand (ie, F1c or R1c) further 3 ′.
[0018]
In the present invention, substantially the same base sequence is defined as follows. That is, when a complementary strand synthesized using a certain sequence as a template anneals to the target base sequence and gives a starting point for complementary strand synthesis, the certain sequence is substantially identical to the target base sequence. For example, a base sequence substantially identical to F2 includes a base sequence that functions as a template that gives a base sequence that can anneal to F2 and serve as a starting point for complementary strand synthesis in addition to a base sequence that is exactly the same as F2. . The term “anneal” in the present invention means that a nucleic acid forms a double-stranded structure by base pairing based on Watson-Crick's law. Therefore, even if the nucleic acid strand constituting the base pair bond is a single strand, annealing is performed if a complementary base sequence in the molecule forms a base pair bond. In the present invention, annealing and hybridization are synonymous in that a nucleic acid forms a double-stranded structure by base pairing.
[0019]
The number of complementary base sequences constituting the nucleic acid according to the present invention is at least one set. According to a desirable aspect of the present invention, it may be an integer multiple thereof. In this case, theoretically, there is no upper limit to the number of pairs of complementary base sequences constituting the nucleic acid in the present invention. When the nucleic acid that is the synthetic product of the present invention is composed of a plurality of sets of complementary base sequences, the nucleic acid is composed of repetitions of the same base sequence.
[0020]
A nucleic acid in which complementary base sequences are alternately linked on a single strand synthesized according to the present invention does not necessarily have the same structure as a natural nucleic acid. It is known that a nucleic acid derivative can be synthesized by using a nucleotide derivative as a substrate when a nucleic acid is synthesized by the action of a nucleic acid polymerizing enzyme. Examples of such nucleotide derivatives include nucleotides labeled with radioisotopes and nucleotide derivatives labeled with binding ligands such as biotin and digoxin. By using these nucleotide derivatives, labeling of the product nucleic acid derivative is achieved. Alternatively, the product nucleic acid can be made into a fluorescent derivative by using a fluorescent nucleotide as a substrate. Furthermore, the product can be DNA or RNA. Which is produced is determined by the combination of the structure of the primer, the type of substrate for polymerization, and a polymerization reagent that performs polymerization of nucleic acid.
[0021]
The synthesis of a nucleic acid having the above structure comprises a DNA polymerase having strand displacement activity and a region F1 that can anneal to a part F1c on the same strand at the 3 ′ end, and this region F1 is on the same strand. Can be initiated by a nucleic acid capable of forming a loop containing a region F2c capable of base pairing. There are many reports of complementary strand synthesis reactions that form hairpin loops and use themselves as a template, but in the present invention, a region capable of base pairing is provided in the hairpin loop portion, and this region is used as a complementary strand. It is novel in that it is used for synthesis. By using this region as a starting point for synthesis, the complementary strand synthesized using itself as a template is replaced. Then, the region R1c (arbitrary region) existing on the 3 ′ side of the substituted chain is in a state capable of base pairing. A region having a base sequence complementary to R1c is annealed to synthesize a complementary strand, and as a result, a nucleic acid (2 that the base sequence from F1 to R1c and its complementary strand are alternately bonded via a loop-forming sequence. Molecule). In the present invention, for example, the arbitrarily selected region such as R1c can anneal a polynucleotide having a base sequence complementary to the region, and a complementary strand synthesized using the polynucleotide as a synthesis starting point. Can be selected from any region as long as it has a function necessary for the present invention.
[0022]
Furthermore, in the present invention, the term nucleic acid is used. In the present invention, the nucleic acid generally includes both DNA and RNA. However, the nucleic acid of the present invention can be used as long as it functions as a template for the synthesis of complementary strands, even if the constituent nucleotides are substituted with artificial derivatives, or natural DNA or RNA is modified. included. The nucleic acid of the present invention is generally contained in a biological sample. Biological samples can refer to animal, plant, or microbial tissue, cells, cultures, excreta or extracts thereof. The biological sample of the present invention includes genomic DNA or RNA of intracellular parasites such as viruses and mycoplasmas. The nucleic acid of the present invention may be derived from a nucleic acid contained in the biological sample. For example, cDNA synthesized based on mRNA and nucleic acid amplified based on nucleic acid derived from a biological sample are typical nucleic acids in the present invention.
[0023]
A region F1 that can be annealed to a part F1c on the same chain is provided at the 3 ′ end, which is a feature of the present invention, and this region F1 anneals to F1c on the same chain, thereby allowing base pairing to occur. Nucleic acids that can form a loop containing the possible region F2c can be obtained by various methods. In the most desirable embodiment, the structure can be provided based on a complementary strand synthesis reaction using an oligonucleotide having the following structure.
[0024]
That is, the oligonucleotide useful in the present invention is composed of an oligonucleotide having at least the following two regions X2 and X1c and having X1c linked to the 5 ′ side of X2.
X2: a region having a base sequence complementary to a region X2c of a nucleic acid having a specific base sequence
X1c: a region having substantially the same base sequence as region X1c located 5 ′ of region X2c in a nucleic acid having a specific base sequence
[0025]
Here, the nucleic acid having a specific base sequence that determines the structure of the oligonucleotide of the present invention means a nucleic acid that serves as a template when the oligonucleotide of the present invention is used as a primer. When nucleic acid is detected based on the synthesis method of the present invention, the nucleic acid having a specific base sequence is a detection target or a nucleic acid derived from the detection target. A nucleic acid having a specific base sequence means a nucleic acid in which at least a part of the base sequence is known or in a state that can be estimated. The part whose base sequence should be clarified is the region X2c and the region X1c located on the 5 ′ side thereof. The two regions can be assumed to be continuous and separated. The relative positional relationship between the two determines the state of the loop portion formed when the product nucleic acid self-anneals. Also, in order for the product nucleic acid to preferentially perform self-annealing rather than intermolecular annealing, it is desirable that the distance between the two is not unnecessarily separated. Therefore, it is desirable that the positional relationship between the two is usually continuous through a distance of 0 to 500 bases. However, in the loop formation by self-annealing described later, if both are too close, it may be disadvantageous to form a desired loop. In the loop, a structure capable of smoothly starting a complementary strand synthesis reaction involving annealing of a new oligonucleotide and strand substitution starting from the new oligonucleotide is required. Therefore, more preferably, the distance between the region X2c and the region X1c located on the 5 ′ side thereof is designed to be 0 to 100 bases, more preferably 10 to 70 bases. This numerical value indicates a length not including X1c and X2. The number of bases constituting the loop portion is a length obtained by adding a region corresponding to X2.
[0026]
It should be noted that the terms “identical” or “complementary” used for characterizing the base sequences constituting the oligonucleotide according to the present invention do not mean that they are completely identical or completely complementary. That is, the same as a certain sequence may include a sequence complementary to a base sequence that can be annealed to a certain sequence. On the other hand, complementary means a sequence that can anneal under stringent conditions and can provide a 3 ′ end from which complementary strand synthesis can begin.
[0027]
For the nucleic acid having the specific base sequence, the regions X2 and X1c constituting the oligonucleotide according to the present invention are usually arranged consecutively without overlapping. Alternatively, if both base sequences have a common part, they can be partially overlapped. Since X2 must function as a primer, it must always be at the 3 ′ end. On the other hand, as described later, X1c is arranged at the 5 ′ end because it needs to give a function as a primer to the 3 ′ end of the complementary strand synthesized using this as a template. In the next step, the complementary strand obtained using this oligonucleotide as a starting point of synthesis becomes a template for complementary strand synthesis in the reverse direction. Finally, the oligonucleotide portion according to the present invention is also copied to the complementary strand as a template. The 3 ′ end generated by copying has a base sequence X1, anneals to X1c on the same strand, and forms a loop.
[0028]
In the present invention, an oligonucleotide means a substance that satisfies the two conditions of being capable of forming a complementary base pair bond and providing an —OH group as a starting point for complementary strand synthesis at its 3 ′ end. Therefore, the backbone is not necessarily limited to that by phosphodiester bonds. For example, it may be composed of a phosphothioate having S as the backbone instead of P or a peptide nucleic acid based on a peptide bond. Moreover, the base should just be a thing which enables complementary base pair coupling | bonding. In the natural state, there are generally five types, ACTG and U, but can also be analogs such as bromodeoxyuridine. The oligonucleotide used in the present invention desirably functions not only as a starting point for synthesis but also as a template for complementary strand synthesis. In the present invention, a polynucleotide is used as a term including an oligonucleotide. The term polynucleotide is used when the chain length is not limited, and oligonucleotide is used as a term meaning a nucleotide polymer having a relatively short chain length.
[0029]
The oligonucleotide according to the present invention has a chain length that allows base pairing with a complementary strand in various nucleic acid synthesis reactions described below while maintaining the necessary specificity under a given environment. . Specifically, it is 5-200 bases, more preferably 10-50 base pairs. Since the chain length of a primer recognized by a known polymerase that catalyzes a sequence-dependent nucleic acid synthesis reaction is at least about 5 bases, the chain length of the portion to be annealed must be longer. In addition, in order to expect specificity as a base sequence, it is desirable to use a length of 10 bases or more stochastically. On the other hand, since it is difficult to prepare an excessively long base sequence by chemical synthesis, the chain length as described above is exemplified as a desirable range. In addition, the chain length illustrated here is the chain length of the part annealed with a complementary strand to the last. As will be described later, the oligonucleotides according to the invention can ultimately be annealed individually in at least two regions. Therefore, the chain length exemplified here should be understood as the chain length of each region constituting the oligonucleotide.
[0030]
Furthermore, the oligonucleotide according to the present invention can be labeled with a known labeling substance. Examples of labeling substances include binding ligands such as digoxin and biotin, enzymes, fluorescent substances, luminescent substances, and radioisotopes. Alternatively, a technique (WO95 / 05391, Proc. Natl. Acad. Sci. USA, 91, 6644-6648, 1994) for substituting a base constituting the oligonucleotide with a fluorescent analog is also known.
[0031]
In addition, the oligonucleotide according to the present invention can be bound to a solid phase. Alternatively, any part of the oligonucleotide can be labeled with a binding ligand such as biotin, and this can be indirectly immobilized by a binding partner such as immobilized avidin. In the case where a solid-phased oligonucleotide is used as a synthesis starting point, the synthesis reaction product of the nucleic acid is captured by the solid phase, so that separation becomes easy. Detection can also be performed by hybridizing a nucleic acid-specific indicator or a labeled probe to the separated product. Alternatively, the desired nucleic acid fragment can be recovered by digestion with an arbitrary restriction enzyme.
[0032]
The term template used in the present invention means a nucleic acid on the side serving as a template for complementary strand synthesis. A complementary strand having a base sequence complementary to the template has a meaning as a strand corresponding to the template, but the relationship between them is merely relative. That is, the strand synthesized as a complementary strand can function as a template again. That is, the complementary strand can be a template.
[0033]
Oligonucleotides useful in the present invention can include additional regions as well as the two regions. While X2 and X1c are located at the 3 ′ end and the 5 ′ end, respectively, it is possible to intervene any sequence between them. It can be, for example, a restriction enzyme recognition sequence, a promoter recognized by RNA polymerase, or a DNA encoding a ribozyme. By using a restriction enzyme recognition sequence, a nucleic acid in which complementary base sequences are alternately linked on a single strand, which is a synthetic product of the present invention, can be cut into a double-stranded nucleic acid having the same length. become. If a promoter sequence recognized by RNA polymerase is arranged, transcription to RNA is further performed using the synthetic product of the present invention as a template. At this time, if a DNA encoding a ribozyme is further arranged, a system for cleaving the transcription product itself can be realized. In addition, all of these accompanying base sequences function when it becomes a double strand. Therefore, these sequences do not function when the single-stranded nucleic acid according to the present invention forms a loop. It functions only when nucleic acid progresses and anneals with a strand having a complementary base sequence without a loop.
[0034]
When a promoter is combined with an oligonucleotide based on the present invention in a direction that allows transcription of the synthesized region, a reaction product based on the present invention that repeats the same base sequence has a highly efficient transcription system. Realize. By combining this with an appropriate expression system, translation into a protein is also possible. That is, it can be used for transcription and translation into protein in bacteria or animal cells or in vitro.
[0035]
The oligonucleotide according to the present invention having the above structure can be chemically synthesized. Alternatively, a natural nucleic acid can be cleaved with a restriction enzyme or the like, and modified or ligated so as to be composed of the above base sequence.
[0036]
The basic principle of the reaction in which the oligonucleotide useful in the nucleic acid synthesis method according to the present invention is used in combination with a DNA polymerase having strand displacement activity will be described below with reference to FIGS. 5-6. To do. The oligonucleotide (FA in FIG. 5) first anneals to nucleic acid using X2 (corresponding to F2) as a template and becomes a starting point for complementary strand synthesis. In FIG. 5, the complementary strand synthesized starting from FA is replaced by complementary strand synthesis (described later) from the outer primer (F3) to form a single strand (FIG. 5-A). When complementary strand synthesis is further performed on the obtained complementary strand, the 3 ′ end portion of the nucleic acid synthesized as the complementary strand in FIG. 5-A has a base sequence complementary to the oligonucleotide according to the present invention. . That is, since the oligonucleotide of the present invention has the same sequence as the region X1c (corresponding to F1c) at its 5 ′ end portion, the 3 ′ end portion of the nucleic acid synthesized at this time has its complementary sequence X1 (F1). Will have. FIG. 5 shows how the complementary strand synthesized starting from R1 is replaced by complementary strand synthesis starting from the outer primer R3. When the 3 ′ terminal part becomes capable of base pairing by substitution, X1 (F1) at the 3 ′ terminal anneals to X1c (F1c) on the same strand, and the extension reaction proceeds using self as a template (FIG. 5-B). Then, X2c (F2c) located on the 3 ′ side is left as a loop without base pairing. In this loop, the oligonucleotide X2 (F2) of the oligonucleotide according to the present invention anneals, and complementary strand synthesis is carried out using this as the synthesis starting point (FIG. 5-B). At this time, the product of the complementary strand synthesis reaction using the previously synthesized template as a template is replaced by the strand displacement reaction and becomes capable of base pairing.
[0037]
As shown in FIG. 6, there is a basic configuration using one kind of oligonucleotide according to the present invention and any reverse primer capable of performing nucleic acid synthesis using a complementary strand synthesized using this oligonucleotide as a primer. Multiple nucleic acid synthesis products can be obtained. As can be seen from FIG. 6, (D) is a nucleic acid in which complementary base sequences are alternately linked on a single strand which is the object of synthesis in the present invention. The other product (E) becomes a template for producing (D) again if it becomes a single strand by treatment such as heat denaturation. In addition, the product (D), which is a nucleic acid in a double-stranded state, is annealed within the same strand with a high probability if it is made into a single strand by heat denaturation or the like, instead of becoming the original double strand. Happens. This is because complementary sequences with the same melting temperature (Tm) are much more preferential in intramolecular reactions than in intermolecular reactions. The single strands derived from the product (D) annealed on the same strand each anneal within the same strand and return to the state of (B), so that each further has one molecule (D) and (E )give. By repeating these steps, it is possible to sequentially synthesize nucleic acids in which complementary base sequences are alternately linked on a single strand. Since the number of templates and products generated in one cycle increases exponentially, the reaction is very efficient.
[0038]
By the way, in order to realize the state of FIG. 5- (A), it is necessary to make the pair of complementary strands capable of base pairing at least at the part where the reverse primer anneals at first. This step can be accomplished by any method. That is, an outer primer (F3) that anneals to the region F3c on the 3 'side of the template further than the region F2c that the oligonucleotide of the present invention anneals to the first template is prepared separately. When complementary strand synthesis is performed using a polymerase that catalyzes the synthesis of a strand displacement type complementary strand using this outer primer as a starting point, the complementary strand synthesized using F2c of the present invention as a synthesis starting point is replaced, and R1 anneals soon. The power region R1c is brought into a state where base pairing is possible (FIG. 5). By utilizing the strand displacement reaction, the reaction so far can be allowed to proceed under isothermal conditions.
[0039]
When an outer primer is used, synthesis from the outer primer (F3) needs to be started after synthesis from F2c. The simplest method is to make the concentration of the inner primer higher than the concentration of the outer primer. Specifically, the reaction can be performed as expected by using primers with a concentration difference of usually 2 to 50 times, preferably 4 to 10 times. The timing of synthesis can also be controlled by setting the melting temperature (Tm) of the outer primer to be lower than the Tm of the X1 (equivalent to F1 or R1) region of the inner primer. That is, (outer primer F3: F3c) ≦ (F2c / F2) ≦ (F1c / F1), or (3 ′ side region in outer primer / template) ≦ (X2c: X2) ≦ (X1c: X1). Here, (F2c / F2) ≦ (F1c / F1) is set so that annealing between F1c / F1 is performed before F2 anneals to the loop portion. Since annealing between F1c / F1 is an intramolecular reaction, there is a high possibility that the annealing proceeds preferentially. However, it is meaningful to consider Tm to give more desirable reaction conditions. It goes without saying that similar conditions should be considered in the design of reverse primers. By adopting such a relationship, ideal reaction conditions can be achieved stochastically. The melting temperature (Tm) can be theoretically calculated based on the combination of the length of the complementary strand to be annealed and the base constituting the base pair bond if other conditions are constant. Accordingly, those skilled in the art can easily derive desirable conditions based on the disclosure of the present specification.
[0040]
Furthermore, in order to adjust the annealing timing of the outer primer, a phenomenon called contiguous stacking can be applied. Contiguous stacking is a phenomenon that allows annealing when oligonucleotides that cannot be annealed alone are adjacent to the double-stranded portion (Chiara Borghesi-Nicoletti et.al. Bio Techniques 12, 474-477). (1992) In other words, the outer primer is designed to be adjacent to F2c (X2c) so that it cannot be annealed alone, so that the outer primer can be annealed only when F2c (X2c) is annealed. Therefore, F2c (X2c) annealing is inevitably given priority.Based on this principle, an example in which the base sequence of oligonucleotide necessary as a primer for a series of reactions is set is described in the examples. This step can be achieved by denaturation by heating or DNA helicase.
[0041]
When the template nucleic acid having F2c (X2c) is RNA, the state shown in FIG. 5- (A) can also be realized by a different method. For example, if this RNA strand is degraded, R1 becomes capable of base pairing. That is, F2 is annealed to F2c of RNA, and complementary strand synthesis is performed as DNA by reverse transcriptase. Next, if the template RNA is degraded by alkali denaturation or enzymatic treatment with ribonuclease acting on DNA / RNA double-stranded RNA, the DNA synthesized from F2 becomes single-stranded. As an enzyme that selectively degrades DNA / RNA double-stranded RNA, RNaseH or the ribonuclease activity of some reverse transcriptases can be used. Thus, the reverse primer can be annealed to R1c that enables base pairing. Therefore, an outer primer for making R1c in a state capable of base bonding becomes unnecessary.
[0042]
Alternatively, the strand displacement activity using the outer primer described above can also be performed using the strand displacement activity of the reverse transcriptase. In this case, the reaction system can be composed of only reverse transcriptase. That is, using RNA as a template, synthesis of a complementary strand from F2 that anneals to F2c, and further, complementary strand synthesis and substitution using outer primer F3 that anneals to F3c located on the 3 ′ side as a starting point for synthesis are reverse transcriptase. Is possible. If the reverse transcriptase performs a complementary strand synthesis reaction using DNA as a template, complementary strand synthesis using R1c as a synthesis starting point, which anneals to the R1c using the substituted complementary strand as a template, and located on the 3 ′ side All complementary strand synthesizing reactions proceed by reverse transcriptase, including complementary strand synthesis and substitution reactions starting from R3, which anneals to R3c. Alternatively, when the reverse transcriptase cannot be expected to have a DNA / DNA strand replacement activity under given reaction conditions, a DNA polymerase having the strand replacement activity described above may be used in combination. As described above, the aspect of obtaining the first single-stranded nucleic acid using RNA as a template constitutes a desirable aspect of the present invention. Conversely, using a DNA polymerase such as Bca DNA polymerase that has strand displacement activity and also has reverse transcriptase activity, not only the synthesis of the first single-stranded nucleic acid from RNA but also the subsequent steps. Reactions using DNA as a template can also be performed with the same enzyme.
[0043]
The reaction system as described above brings about various variations unique to the present invention by using a reverse primer having a specific structure. The most effective variations are described below. That is, in the most advantageous aspect of the present invention, an oligonucleotide having the structure described in [5] is used as the reverse primer. The oligonucleotide [5] is an oligonucleotide in which an arbitrary region R2c in a complementary strand synthesized using F2 as a primer is X2c and R1c is X1c. By using such a reverse primer, a series of reactions of loop formation and complementary strand synthesis and substitution from this loop portion occurs on both the sense strand and the antisense strand (forward side and reverse side). . As a result, the synthesis efficiency of the method for synthesizing nucleic acids in which complementary base sequences are alternately linked on a single strand according to the present invention is dramatically improved, and a series of reactions can be performed isothermally. . Hereinafter, this aspect will be specifically described with reference to FIGS.
[0044]
In the following embodiment, two types of oligonucleotides based on the present invention are prepared. These are named FA and RA for explanation. The areas constituting the FA and RA are as follows.

[0045]
Here, F2 is a base sequence complementary to the nucleic acid region F2c used as a template. R2 is a base sequence complementary to an arbitrary region R2c contained in a complementary strand synthesized using F2 as a primer. F1c and R1c are arbitrary base sequences located downstream of F2c and R2c, respectively. Here, the distance between F2 and R2 may be arbitrary. Although depending on the synthesis ability of the DNA polymerase that performs complementary strand synthesis, even under a suitable condition, sufficient synthesis is possible even with a length of about 1 kbp. More specifically, when Bst DNA polymerase is used, it is surely synthesized if it has a length of 800 bp or less, preferably 500 bp or less between F2 / R2c. In PCR with a temperature cycle, the reduction in enzyme activity due to temperature change stress is said to reduce the synthesis efficiency of a long base sequence. In a desirable aspect of the present invention, since a temperature cycle in the nucleic acid amplification step is not required, synthesis and amplification can be reliably achieved even with a long base sequence.
[0046]
First, F2 of FA is annealed to the nucleic acid to be a template, and complementary strand synthesis is performed using this as a starting point for synthesis. Hereinafter, the reaction process is the same as that in the basic mode of the present invention described above (FIG. 5) until reaching (4) in FIG. The sequence annealed as F3 in (2) of FIG. 1 is the outer primer described above. When this primer is used as a starting point for synthesis, a DNA polymerase that synthesizes a strand-displacing complementary strand is used, whereby the complementary strand synthesized from FA is displaced, and base pairing is possible.
[0047]
When R2c becomes capable of base pairing in (4), RA as a reverse primer anneals with a combination of R2c / R2. Complementary strand synthesis using this as the starting point for synthesis is carried out up to the part reaching F1c, which is the 5 ′ end of FA. Subsequent to this complementary strand synthesis reaction, the outer primer R3 for replacement is also annealed, and complementary strand synthesis is performed with strand replacement, whereby the complementary strand synthesized using RA as the synthesis starting point is replaced. The complementary strand to be substituted at this time has RA on the 5 ′ side and a sequence complementary to FA located at the 3 ′ end.
[0048]
A sequence F1 complementary to F1c on the same strand is present on the 3 ′ side of the single-stranded nucleic acid thus substituted. F1 rapidly anneals to F1c arranged in the same molecule, and complementary strand synthesis begins. When the 3 ′ end (F1) anneals to F1c on the same strand, a loop containing F2c is formed. It is clear from FIG. 2- (7) that this loop portion is maintained in a state where base pairing is possible. The oligonucleotide FA of the present invention having a base sequence complementary to F2c anneals to this loop portion and becomes a starting point for complementary strand synthesis (7). Complementary strand synthesis from the loop portion proceeds while replacing the reaction product of complementary strand synthesis initiated from F1 previously initiated. As a result, the complementary strand synthesized using itself as a template becomes capable of base pairing at the 3 ′ end again. This 3 ′ end has a region R1 that can be annealed to R1c on the same strand at the 3 ′ end, and both are preferentially annealed by a rapid reaction within the same molecule. Thus, a reaction similar to the reaction from the 3 ′ end synthesized using FA as a template described above also proceeds in this region. As a result, the nucleic acid in which complementary base sequences are alternately ligated on a single strand according to the present invention continues to synthesize and replace complementary strands one after another, and continue to extend from its 3 ′ end R1. Become. Since the loop formed by annealing the 3 ′ end R1 to the same strand always contains R2c, it is always the oligonucleotide with R2 (ie, RA) that anneals to the loop portion at the 3 ′ end in subsequent reactions. )
[0049]
On the other hand, when attention is paid to a nucleic acid synthesized with a complementary strand starting from an oligonucleotide that anneals to the loop portion of a single-stranded nucleic acid that continues to elongate using itself as a template, the single-stranded nucleic acid according to the present invention is also used here. The synthesis of nucleic acids in which complementary base sequences are alternately linked is progressing. That is, the complementary strand synthesis from the loop portion is completed when RA is reached, for example, in FIG. Then, when the nucleic acid substituted by the synthesis of this nucleic acid starts complementary strand synthesis (FIG. 3- (8)), the reaction eventually reaches the loop portion that was once the starting point of synthesis, and the substitution starts again. In this way, the nucleic acid that has been synthesized from the loop portion is also replaced, and as a result, a 3 ′ end R1 that can be annealed on the same strand is obtained (FIG. 3- (10)). This 3 ′ end R1 anneals to R1c of the same strand to initiate complementary strand synthesis. Now, if F and R of this reaction are replaced, it is the same as the reaction occurring in FIG. 2- (7). Therefore, the structure shown in FIG. 3- (10) can function as a new nucleic acid that continues its extension and the generation of a new nucleic acid.
[0050]
The nucleic acid synthesis reaction starting from the nucleic acid shown in FIG. 3- (10) is always an extension starting from the 3 ′ end F1 as opposed to the synthesis reaction described so far. That is, in the present invention, along with the elongation of one nucleic acid, a reaction for continuing to supply a new nucleic acid that starts the elongation separately proceeds. Furthermore, as the strand extends, multiple loop forming sequences are provided on the same strand as well as at the ends. When these loop-forming sequences are ready for base pair formation by a strand displacement synthesis reaction, the oligonucleotide anneals and becomes a base point for a new nucleic acid generation reaction. By combining not only the terminal but also the synthesis reaction from the middle of the chain, a more efficient amplification reaction can be achieved. As described above, by combining the oligonucleotide RA according to the present invention as a reverse primer, extension and generation of a new nucleic acid associated therewith occur. Furthermore, in the present invention, the newly produced nucleic acid itself is elongated, resulting in the production of a further new nucleic acid associated therewith. The series of reactions can theoretically continue indefinitely and achieve very efficient nucleic acid amplification. Moreover, the reaction of the present invention can be carried out under isothermal conditions.
[0051]
The reaction product accumulated at this time has a structure in which the base sequence between F1 and R1 and its complementary sequence are alternately linked. However, a region composed of the base sequence of F2-F1 (F2c-F1c) or R2-R1 (R2c-R1c) is continuous at both ends of the sequence serving as a repeating unit. For example, in FIG. 3- (9), it will be in the state connected from 5 'side in the order of (R2-F2c)-(F1-R2c)-(R1-F1c)-(F2-R2c). This is because the amplification reaction according to the present invention starts with F2 (or R2) starting from the oligonucleotide as the synthesis origin, and then the complementary strand synthesis reaction from F1 (or R1) starting from its 3 ′ end as the synthesis origin. This is because the progress is based on the principle of expansion.
[0052]
Here, as the most desirable embodiment, the oligonucleotides FA and RA according to the present invention were used as oligonucleotides that anneal to the loop portion. However, the nucleic acid amplification reaction according to the present invention can be carried out by using not only oligonucleotides having these limited structures but also oligonucleotides capable of initiating complementary strand synthesis from loops. That is, as long as the 3 ′ end that continues to be extended is replaced by complementary strand synthesis from the loop, the loop portion is given again. Complementary strand synthesis starting from the loop portion always uses a nucleic acid in which complementary base sequences are alternately linked on one strand as a template, so that it is obvious that the target nucleic acid can be synthesized in the present invention. It is. However, although the nucleic acid synthesized here forms a loop after substitution and synthesizes a complementary strand, it does not have a 3 ′ end for forming a subsequent loop, and therefore cannot function as a new template. Therefore, unlike nucleic acids that have been synthesized by FA or RA, exponential amplification cannot be expected. For these reasons, oligonucleotides having structures such as FA and RA are useful for highly efficient nucleic acid synthesis based on the present invention.
[0053]
In a series of reactions, the following components can be added to a single-stranded nucleic acid that serves as a template to form a stable base-pair bond with a base sequence that is complementary to the base sequences constituting FA and RA. It is possible to proceed by simply incubating at a temperature that can be maintained and can maintain enzyme activity.
・ 4 types of oligonucleotides:
FA,
RA,
Outer primer F3,
And outer primer R3,
DNA polymerase that performs strand displacement type complementary strand synthesis,
・ Nucleotide used as substrate for DNA polymerase
[0054]
Therefore, a temperature cycle like PCR is not necessary. Here, the stable base pairing means a state in which at least a part of the oligonucleotide present in the reaction system can give a starting point for complementary strand synthesis. Desirable conditions for providing stable base pairing are, for example, setting the melting temperature (Tm) or lower. In general, the melting temperature (Tm) is a temperature at which 50% of nucleic acids having mutually complementary base sequences are in a base-paired state. Setting the temperature below the melting temperature (Tm) is not an essential condition of the present invention, but is one of the reaction conditions to be considered in order to achieve high synthesis efficiency. When the nucleic acid to be used as a template is double-stranded, at least the region where the oligonucleotide anneals needs to be in a state capable of base pairing. For this purpose, heat denaturation is generally performed, but this may be performed only once as a pretreatment before the start of the reaction.
[0055]
This reaction includes a buffer that gives a suitable pH for the enzyme reaction, salts necessary for maintaining the catalytic activity of the enzyme and annealing, an enzyme protecting agent, and a melting temperature (Tm) adjusting agent if necessary. In the coexistence of As the buffering agent, a neutral to weakly alkaline buffering agent such as Tris-HCl is used. The pH is adjusted according to the DNA polymerase used. Salts include KCl, NaCl, or (NH_Four)₂SO_FourAnd the like are appropriately added to maintain the enzyme activity and adjust the melting temperature (Tm) of the nucleic acid. As an enzyme protective agent, bovine serum albumin or saccharide is used. Furthermore, dimethyl sulfoxide (DMSO) or formamide is generally used as a regulator for the melting temperature (Tm). By using a melting temperature (Tm) adjusting agent, the annealing of the oligonucleotide can be adjusted under limited temperature conditions. Furthermore, betaine (N, N, N, -trimethylglycine) and tetraalkylammonium salts are effective in improving the strand displacement efficiency by their isostabilize action. Betaine can be expected to promote the nucleic acid amplification reaction of the present invention by adding 0.2 to 3.0 M, preferably about 0.5 to 1.5 M, in the reaction solution. These melting temperature regulators act in the direction of lowering the melting temperature, so empirically set conditions that give appropriate stringency and reactivity in consideration of other reaction conditions such as salt concentration and reaction temperature. To do.
[0056]
In the present invention, it is an important feature that a series of reactions does not proceed unless the positional relationship between a plurality of regions is always maintained. This feature can effectively prevent non-specific synthesis reaction accompanying non-specific complementary strand synthesis. That is, even if any non-specific reaction occurs, the possibility that the product becomes a starting material for the subsequent amplification step is reduced. In addition, the fact that the progress of the reaction is controlled by more regions brings about the possibility of freely configuring a detection system that enables strict identification of similar base sequences.
[0057]
This feature can be used to detect gene mutations. In the embodiment using the outer primer in the present invention, a total of four types of primers, two types of the outer primer and two types of primers composed of the oligonucleotide of the present invention, are used. That is, the synthesis reaction of the present invention does not proceed unless the 6 regions contained in the 4 types of oligonucleotides work as designed. In particular, the sequences at the 3 ′ end of each oligonucleotide that is the starting point for complementary strand synthesis and the 5 ′ end of the X1c region where the complementary sequence is the starting point for synthesis are important. Therefore, if this important sequence is designed to correspond to the mutation to be detected, by observing the synthetic reaction product according to the present invention, the presence or absence of mutation such as deletion or insertion of a base, or a gene such as SNPs. The polymorphism can be analyzed comprehensively. More specifically, the base expected to be mutated or polymorphic corresponds to the vicinity of the 3 ′ end of the oligonucleotide that is the origin of complementary strand synthesis (or the vicinity of the 5 ′ end when the complementary strand is the origin). To design. If there is a mismatch at or near the 3 ′ end, which is the origin of synthesis of the complementary strand, the nucleic acid complementary strand synthesis reaction is significantly inhibited. In the present invention, unless the terminal structure in the product at the initial stage of the reaction is repeatedly reacted, it does not lead to a high amplification reaction. Therefore, even if incorrect synthesis is performed, synthesis of complementary strands constituting the amplification reaction is always hindered at any stage, so that high-level amplification does not occur if mismatches are included. As a result, the mismatch effectively suppresses the amplification reaction and ultimately gives an accurate result. That is, it can be said that the nucleic acid amplification reaction according to the present invention has a more complete base sequence check mechanism. These characteristics are advantages that are difficult to expect in, for example, the PCR method in which amplification reaction is simply performed in two regions.
[0058]
Furthermore, the region X1c that characterizes the oligonucleotide used in the present invention becomes the starting point of synthesis only after the complementary sequence is synthesized, and this complementary sequence anneals to the newly synthesized sequence X1 in the same strand, thereby making self a template. The synthesis reaction proceeds. For this reason, even if so-called primer dimers are generated, which is often an important problem in the prior art, the oligonucleotides do not form loops. Therefore, non-specific amplification due to the primer dimer cannot occur in principle, and contributes to improving the specificity of the reaction.
[0059]
Furthermore, according to the present invention, the above-described series of reactions can be performed under isothermal conditions by combining the outer primers shown by F3 (FIG. 1- (2)) and R3 (FIG. 2- (5)). . That is, the present invention provides a method for amplifying a nucleic acid in which complementary base sequences are alternately linked on a single strand comprising the step shown in [9] above. In this method, temperature conditions are selected that cause stable annealing between F2c / F2, R2c / R2, F1c / F1, and R1c / R1, and preferably between F3c / F3 and between R3c / R3. Are set to anneal by a continuous stacking phenomenon assisted by annealing between F2c / F2 and between R2c / R2, respectively.
[0060]
In the present invention, the terms nucleic acid synthesis and amplification are used. The synthesis of nucleic acid in the present invention means the extension of nucleic acid from the oligonucleotide that is the origin of synthesis. In addition to synthesis, when the generation of another nucleic acid and the extension reaction of the generated nucleic acid occur continuously, the series of reactions are collectively called amplification.
[0061]
Now, a loop including a region F1 that can be annealed to a part F1c on the same strand at the 3 ′ end and this region F1 anneals to F1c on the same strand, thereby allowing base pairing. A single-stranded nucleic acid capable of forming is an important component of the present invention. Such a single-stranded nucleic acid can also be supplied based on the following principle. That is, complementary strand synthesis proceeds based on a primer having the following structure in advance.
5 ′-[region X1 annealed to region X1c located in the primer]-[loop forming sequence in a state capable of base pairing]-[region X1c]-[region having a sequence complementary to the template]- 3 '
[0062]
In the region having a sequence complementary to the template, two types of base sequence complementary to F1 (primer FA) and base sequence complementary to R1c (primer RA) are prepared. The base sequence constituting the nucleic acid to be synthesized at this time includes a base sequence from region F1 to region R1c and a base sequence from region R1 to region F1c having a base sequence complementary to this base sequence. . On the other hand, X1c and X1 that can be annealed inside the primer can have any sequence. However, it is desirable that the sequences of the regions X1c / X1 be different between the primers FA and RA.
[0063]
First, complementary strand synthesis is performed using the primer FA from the region F1 of the template nucleic acid. Next, the synthesized complementary region R1c is brought into a state capable of base pairing, and one primer is annealed to be a starting point for complementary strand synthesis. The 3 ′ end of the complementary strand synthesized at this time has a base sequence complementary to the primer FA constituting the 5 ′ end portion of the first synthesized strand, and therefore has a region X1 at the 3 ′ end. Annealing the region X1c on the same strand and forming a loop. Thus, the characteristic 3 ′ terminal structure according to the present invention is provided, and the subsequent reaction is the previous reaction system shown as the most desirable mode. At this time, the oligonucleotide annealed to the loop portion has a region X2 complementary to the region X2c existing in the loop at the 3 ′ end and a region X1 on the 5 ′ side. In the previous reaction system, the primer FA and RA were used to synthesize a strand complementary to the template nucleic acid, resulting in a loop structure at the 3 ′ end of the nucleic acid. This method effectively provides a terminal structure characteristic of the present invention with a short primer. On the other hand, in this embodiment, the entire base sequence constituting the loop is provided as a primer from the beginning, and synthesis of a longer primer is required.
[0064]
By using a base sequence including a restriction enzyme recognition region in the reverse primer, a different embodiment according to the present invention can be configured. Based on FIG. 6, the case where a reverse primer contains a restriction enzyme recognition sequence is demonstrated concretely. When FIG. 6- (D) is completed, a nick is generated by the restriction enzyme corresponding to the restriction enzyme recognition site in the reverse primer. Using this nick as a starting point for synthesis, a strand displacement type complementary strand synthesis reaction starts. Since the reverse primer is located at both ends of the double-stranded nucleic acid constituting (D), the complementary strand synthesis reaction is also started from both ends. Basically, it is based on the principle of the SDA method described as the prior art, but since the base sequence used as a template has a structure in which complementary base sequences are alternately linked according to the present invention, nucleic acid synthesis unique to the present invention The system is constructed. It should be designed so that the dNTP derivative is incorporated into the portion that becomes the complementary strand of the reverse primer to be nicked so as to be resistant to nuclease so as not to cause double-strand breakage by restriction enzymes.
[0065]
An RNA polymerase promoter may be inserted into the reverse primer. In this case as well, transcription is performed by RNA polymerase that recognizes this promoter from both ends of FIG. 6- (D), as in the previous embodiment applying the SDA method.
[0066]
Since the nucleic acid synthesized according to the present invention is composed of a complementary base sequence although it is a single strand, most of the nucleic acid forms a base pair bond. By utilizing this feature, it is possible to detect a synthesized product. When the nucleic acid synthesis method according to the present invention is carried out in the presence of a fluorescent dye that is a double-strand specific intercalator such as ethidium bromide, SYBR Green I, or Pico Green, the fluorescence intensity increases as the product increases. Is observed. By monitoring this, it is possible to follow the synthesis reaction in real time in a closed system. Although this type of detection system is considered to be applied to the PCR method, it is considered problematic because it cannot be distinguished from signal generation by primer dimers or the like. However, when applied to the present invention, the possibility of increasing non-specific base pair binding is very low, so high sensitivity and low noise can be expected at the same time. As with the double-strand specific intercalator, fluorescence energy transfer can be used as a method for realizing a homogeneous detection system.
[0067]
The nucleic acid synthesis method according to the present invention is supported by a DNA polymerase that catalyzes a strand displacement type complementary strand synthesis reaction. The reaction includes a reaction step that does not necessarily require a strand displacement type polymerase. However, it is advantageous to use one kind of DNA polymerase in terms of simplification of constituent reagents and economical efficiency. The following types of DNA polymerases are known. Various mutants of these enzymes can also be used in the present invention as long as they have sequence-dependent complementary strand synthesis activity and strand displacement activity. The term “mutant” as used herein refers to a product obtained by extracting only the structure that provides the catalytic activity required by the enzyme, or a product whose catalytic activity, stability, or heat resistance has been modified by amino acid mutation or the like.
Bst DNA polymerase
Bca (exo-) DNA polymerase
Klenow fragment of DNA polymerase I
Vent DNA polymerase
Vent (Exo-) DNA polymerase (excluding exonuclease activity from Vent DNA polymerase)
DeepVent DNA polymerase
DeepVent (Exo-) DNA polymerase (DeepVent DNA polymerase excluding exonuclease activity)
Φ29 phage DNA polymerase
MS-2 phage DNA polymerase
Z-Taq DNA polymerase (Takara Shuzo)
KOD DNA polymerase (Toyobo)
[0068]
Among these enzymes, Bst DNA polymerase and Bca (exo-) DNA polymerase are particularly desirable enzymes because they have a certain degree of heat resistance and high catalytic activity. Although the reaction of the present invention can be carried out isothermally in a desirable embodiment, temperature conditions suitable for enzyme stability are not always available for adjusting the melting temperature (Tm) and the like. Therefore, it is one of desirable conditions that the enzyme is thermostable. Although isothermal reaction is possible, heat denaturation may be performed to provide the initial template nucleic acid. In this respect, the use of thermostable enzymes broadens the choice of assay protocol. .
[0069]
Vent (Exo-) DNA polymerase is an enzyme with high thermostability as well as strand displacement activity. By the way, it is known that a complementary strand synthesis reaction involving strand displacement by DNA polymerase is promoted by the addition of a single strand binding protein (Paul M. Lizardi et al, Nature Genetics 19, 225). -232, July, 1998). By applying this action to the present invention and adding a single-stranded binding protein, an effect of promoting complementary strand synthesis can be expected. For example, T4 gene 32 is effective as a single-stranded binding protein for Vent (Exo-) DNA polymerase.
[0070]
For DNA polymerases that do not have 3'-5 'exonuclease activity, it is known that complementary strand synthesis does not stop at the 5' end of the template, but proceeds to a state where one base protrudes. Yes. In the present invention, such a phenomenon is undesirable because the sequence at the 3 ′ end when complementary strand synthesis reaches the end leads to the start of the next complementary strand synthesis. However, the addition of a base to the 3 ′ end by DNA polymerase is A with a high probability. Therefore, the sequence may be selected so that synthesis from the 3 ′ end starts with A so that no problem is caused even if dATP is mistakenly added with one base. In addition, even if the 3 ′ end protrudes during synthesis of the complementary strand, the 3 ′ → 5 ′ exonuclease activity can be used by digesting this to make a blunt end. For example, since natural Vent DNA polymerase has this activity, this problem can be avoided by mixing with Vent (Exo-) DNA polymerase.
[0071]
Various reagents necessary for the nucleic acid synthesis method or amplification method according to the present invention can be packaged in advance and supplied as a kit. Specifically, for the present invention, various oligonucleotides necessary as a primer for complementary strand synthesis or as an outer primer for substitution, dNTP as a substrate for complementary strand synthesis, and strand displacement type complementary strand synthesis are performed. A kit comprising a DNA polymerase, a buffer that gives conditions suitable for the enzyme reaction, and, if necessary, reagents necessary for the detection of the synthesis reaction product is provided. In particular, in a desirable embodiment of the present invention, since it is not necessary to add a reagent during the reaction, the reagent necessary for one reaction is supplied in a state of being dispensed into the reaction vessel, so that the reaction can be performed only by adding the sample. Can be started. If the system is such that the reaction product can be detected as it is using the luminescence signal or the fluorescence signal, the opening of the container after the reaction can be completely abolished. This is very desirable for preventing contamination.
[0072]
The nucleic acid synthesized by the present invention and having complementary base sequences alternately linked on a single strand has, for example, the following utility. The first is utilization of the advantages associated with a special structure having a complementary base sequence in one molecule. This feature is expected to facilitate detection. That is, a nucleic acid detection system that increases or decreases a signal accompanying base pair binding with a complementary base sequence is known. For example, a detection system that makes use of the characteristics of the synthetic product of the present invention can be realized by combining the above-described method using a double-strand specific intercalator as a detection agent. In such a detection system, once the synthetic reaction product of the present invention is heat-denatured and returned to the original temperature, intramolecular annealing occurs preferentially, so that a base pair bond is quickly formed between complementary sequences. . On the other hand, even if a non-specific reaction product is present, it does not have a complementary sequence in the molecule, so it is separated into two or more molecules by thermal denaturation, and immediately becomes the original double strand. Cannot return. Thus, noise associated with non-specific reactions can be reduced by adding a heat denaturation step before detection. When using a DNA polymerase that is not resistant to heat, the heat denaturation step also has the meaning of stopping the reaction, which is advantageous in terms of controlling the reaction time.
[0073]
The second feature is to always form a loop that is ready for base pairing. The structure of the loop in a state in which base pairing is possible is shown in FIG. As can be seen from FIG. 4, the loop is composed of a base sequence F2c (X2c) capable of annealing the primer and a base sequence interposed between F2c-F1c (X1c). The sequence between F2c and F1c (between X2c and X1c in general terms) is a base sequence derived from the template. Therefore, template-specific detection can be performed by hybridizing a probe having a complementary base sequence to this region. In addition, since this region is always in a state capable of base pairing, it is not necessary to denature by heating prior to hybridization. In addition, the base sequence which comprises the loop in the amplification reaction product of this invention can be made into arbitrary length. Therefore, for the purpose of probe hybridization, ideal reaction conditions can be constructed by separating the region to be annealed by the primer and the region to be hybridized by the probe and avoiding competition between the two. .
[0074]
A desirable embodiment of the present invention results in a number of loops capable of base pairing on a single nucleic acid strand. This means that a large number of probes can hybridize to one molecule of nucleic acid, and detection with high sensitivity is possible. In addition, not only sensitivity but also a nucleic acid detection method based on a special reaction principle such as an agglutination reaction is possible. For example, when a probe fixed to fine particles such as polystyrene latex is added to the reaction product of the present invention, aggregation of latex particles is observed with hybridization with the probe. If the intensity of aggregation is measured optically, high-sensitivity and quantitative observation is possible. Alternatively, since the agglutination reaction can be observed with the naked eye, it is possible to construct a reaction system that does not use an optical measuring device.
[0075]
Furthermore, the reaction products of the present invention that can bind many labels per nucleic acid molecule also allow for chromatographic detection. In the field of immunoassay, an analysis method (immunochromatography method) using a chromatographic medium utilizing a label that can be detected with the naked eye has been put into practical use. This method is based on the principle that an analyte is sandwiched between an antibody immobilized on a chromatographic medium and a labeled antibody, and unreacted labeled components are washed away. The reaction product of the present invention makes it possible to apply this principle to the analysis of nucleic acids. That is, by preparing a labeled probe for the loop portion and trapping it with a capture probe immobilized on the chromatographic medium, analysis in the chromatographic medium is performed. As the capture probe, a complementary sequence to the loop portion can be used. Since the reaction product of the present invention involves a large number of loop portions, it binds a large number of labeled probes, resulting in a visually recognizable signal.
[0076]
The reaction product according to the present invention which always provides a region capable of base pairing as a loop allows various other detection systems. For example, a detection system using surface plasmon resonance in which a probe for this loop portion is fixed is possible. Further, if the probe for the loop portion is labeled with a double strand specific intercalator, more sensitive fluorescence analysis can be performed. Alternatively, it can be actively utilized that the nucleic acid synthesized according to the present invention forms a loop capable of base pairing on both the 3 ′ side and the 5 ′ side. For example, one loop is designed to be a part that has a common base sequence between the normal type and the abnormal type, and the other loop is designed to be a region where a difference between the two is generated. It is possible to configure a characteristic analysis system in which the presence of a gene is confirmed with a probe for a common part and the presence or absence of an abnormality is confirmed in the other region. Since the nucleic acid synthesis reaction according to the present invention can be carried out isothermally, it is also a remarkable advantage that real-time analysis can be performed with a general fluorometer. Nucleic acid structures that anneal on the same strand have been known so far. However, nucleic acids in which complementary base sequences are alternately linked on a single strand that can be obtained by the present invention are novel in that they contain a large number of loop portions that allow other oligonucleotides to base pair. .
[0077]
On the other hand, it is also possible to use a large number of loop portions provided by the reaction product according to the present invention as probes. For example, in a DNA chip, it is necessary to accumulate probes at a high density in a limited area. However, current technology limits the number of oligonucleotides that can be immobilized on a certain area. Therefore, if the reaction product of the present invention is used, a large number of probes that can be annealed can be immobilized at a high density. That is, the reaction product according to the present invention may be immobilized on a DNA chip as a probe. The reaction product can be immobilized by a known technique after amplification, or the immobilized oligonucleotide is used as an oligonucleotide in the amplification reaction of the present invention, resulting in an immobilized reaction product. You can also. If the probe immobilized in this way is used, many sample DNAs can be hybridized in a limited area, and as a result, a high signal can be expected.
[0078]
【Example】
[Example 1] Amplification of region in M13mp18
Using M13mp18 as a template, an attempt was made to synthesize nucleic acids in which complementary base sequences were alternately linked on a single strand according to the present invention. There are four types of primers used in the experiment: M13FA, M13RA, M13F3, and M13R3. M13F3 and M13R3 are outer primers for substituting the first nucleic acid obtained using M13FA and M13RA as starting points for synthesis, respectively. Since the outer primer is a primer that should be the starting point for complementary strand synthesis after M13FA (or M13RA), it was designed to anneal to the region adjacent to M13FA (or M13RA) using the contiguous stacking phenomenon. Moreover, these primer concentrations were set high so that annealing of M13FA (or M13RA) occurred preferentially.
[0079]
The base sequences constituting each primer are as shown in the sequence listing. The structural features of the primers are summarized below. FIG. 7 shows the positional relationship of each region with respect to the target base sequence (target).

[0080]
With such a primer, a nucleic acid is synthesized in which the region from M13mp18 region F1c to R1c and its complementary base sequence are alternately linked on a single strand across a loop-forming sequence containing F2c. The reaction solution composition for the nucleic acid synthesis method according to the present invention using these primers is shown below.
[0081]
Reaction solution composition (in 25 μL)
20 mM Tris-HCl pH8.8
10 mM KCl
10mM (NH_Four)₂SO_Four
6 mM MgSO_Four
0.1% Triton X-100
5% dimethyl sulfoxide (DMSO)
0.4mM dNTP
Primer:
800 nM M13FA / SEQ ID NO: 1
800nM M13RA / SEQ ID NO: 2
200 nM M13F3 / SEQ ID NO: 3
200 nM M13R3 / SEQ ID NO: 4
Target: M13mp18 dsDNA / SEQ ID NO: 5
Reaction: The reaction solution was heated at 95 ° C. for 5 minutes to denature the target to form a single strand. The reaction solution was transferred to ice water, 4 U of Bst DNA polymerase (NEW ENGLAND BioLabs) was added, and the mixture was reacted at 65 ° C. for 1 hour. After the reaction, the reaction was stopped at 80 ° C. for 10 minutes and transferred again to ice water.
Confirmation of reaction: 1 μL of loading buffer was added to 5 μL of the above reaction solution, and electrophoresed at 80 mV for 1 hour using 2% agarose gel (0.5% TBE). XIV (100 bp ladder, manufactured by Boehringer Mannheim) was used as a molecular size marker. The gel after electrophoresis was stained with SYBR Green I (Molecular Probes, Inc.) to confirm the nucleic acid. The results are as shown in FIG. Each lane corresponds to the next sample.
1. XIV size marker
2. 1fmol M13mp18 dsDNA
3. No target
[0082]
In lane 3, no band was confirmed except that the unreacted primer was stained. In the case of lane 2, when the target was present, the product was confirmed as a ladder of a low-size band and a smear stain at a high size, and a band hardly migrated in the gel. Among the low-size bands, the bands near 290 bp and 450 bp are products in which SEQ ID NO: 11 and SEQ ID NO: 12 are double strands as predicted by the synthesis reaction of the present invention (FIG. 2- (7) and FIG. 2- (10) correspond to the double strand) and SEQ ID NO: 13 (corresponding to the long single strand in FIG. 3- (9)), the size matches. It was confirmed that the reaction was proceeding as expected. The high-size smear pattern and the non-migrated band indicate that the reaction is basically a continuous reaction, so that the reaction product does not have a constant size, and a partial single strand, or Since it has a complex structure in which a double-stranded complex is formed, it was considered that the result of such migration was given as a result.
[0083]
[Example 2] Confirmation of restriction enzyme digestion of reaction product
For the purpose of clarifying the structure of a nucleic acid in which complementary base sequences were alternately linked on the single strand according to the present invention obtained in Example 1, digestion with a restriction enzyme was performed. While digestion with a restriction enzyme produces a fragment as expected, if the high-size smear pattern or non-migrated band observed in Example 1 disappears, these are all single-stranded synthesized according to the present invention. It can be presumed that the nucleic acid is composed of alternately linked complementary base sequences.
[0084]
The reaction solution of Example 1 was pooled for 8 bottles (200 μL), treated with phenol, and purified by ethanol precipitation. This precipitate was collected and redissolved with 200 μL of TE buffer, and 10 μL was digested with restriction enzymes BamHI, PvuII, and HindIII at 37 ° C. for 2 hours, respectively. The digest was electrophoresed at 80 mV for 1 hour using a 2% agarose gel (0.5% TBE). SUPER LADDER-LOW (100 bp ladder) (Gensura Laboratories, Inc.) was used as a molecular size marker. The gel after electrophoresis was stained with SYBR Green I (Molecular Probes, Inc.) to confirm the nucleic acid. The results are as shown in FIG. Each lane corresponds to the next sample.
1. XIV size marker
2. BamHI digest of purified product
3. PvuII digest of purified product
4. HindIII digest of purified product
[0085]
Here, the base sequence constituting the amplification product having a relatively short chain length is presumed to be SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and the like. Table 1 shows the size of each restriction enzyme-digested fragment estimated from these base sequences. L in the table indicates that the migration position is uncertain because it is a fragment containing a loop (single strand).
[0086]
[Table 1]
Restriction enzyme digestion fragment of the amplification product according to the invention

[0087]
Since most of the undigested band changed to a band of estimated size after digestion, it was confirmed that the target reaction product was amplified. It was also shown that there were almost no non-specific products.
[0088]
[Example 3] Promotion of amplification reaction by addition of betaine
Experiments were conducted to examine the effects of betaine (betaine: N, N, N, -trimethylglycine, SIGMA) on the amplification reaction of nucleic acids. In the same manner as in Example 1, a method for synthesizing nucleic acids in which relative base sequences were alternately linked on a single strand according to the present invention using M13mp18 as a template was carried out in the presence of various concentrations of betaine. The primers used in the experiment are the same as those used in Example 1. The amount of template DNA is 10^{-twenty one} mol (M13mp18) and water was used as a negative control. The betaine to be added was added to the reaction solution so as to have a concentration of 0, 0.5, 1, 2M. The reaction solution composition is shown below.
Reaction solution composition (in 25 μL)
20 mM Tris-HCl pH8.8
4 mM MgSO_Four
0.4mM dNTPs
10 mM KCl
10mM (NH_Four)₂SO_Four
0.1% TritonX-100
Primer:
800 nM M13FA / SEQ ID NO: 1
800nM M13RA / SEQ ID NO: 2
200 nM M13F3 / SEQ ID NO: 3
200 nM M13R3 / SEQ ID NO: 4
Target: M13mp18 dsDNA / SEQ ID NO: 5
The polymerase used, the reaction conditions, and the electrophoresis conditions after the reaction are the same as those described in Example 1.
[0089]
The results are shown in FIG. In the reaction in the presence of betaine concentrations of 0.5 and 1.0 M, the amount of amplified product increased. On the other hand, when it was increased to 2.0M, no amplification product was confirmed. Thus, it was shown that the amplification reaction was promoted in the presence of moderate betaine. The decrease in amplification product at a betaine concentration of 2M was thought to be due to an excessive decrease in Tm.
[0090]
[Example 4] Amplification of HBV gene sequence
The nucleic acid synthesis method according to the present invention was attempted using M13mp18 dsDNA incorporating a partial sequence of the HBV gene as a template. The primers used in the experiment are HB65FA (SEQ ID NO: 6), HB65RA (SEQ ID NO: 7), HBF3 (SEQ ID NO: 8), and HBR3 (SEQ ID NO: 9). HBF3 and HBR3 are outer primers for substituting the first nucleic acid obtained using HB65FA and HB65RA as the synthesis starting points, respectively. Since the outer primer is a primer that should be the starting point for complementary strand synthesis after HB65FA (or HB65RA), it was designed to anneal to the region adjacent to HB65FA (or HB65RA) using the contiguous stacking phenomenon. Moreover, these primer concentrations were set high so that annealing of HB65FA (or HB65RA) preferentially occurred. The target sequence (430 bp) of this example derived from HBV incorporated into M13mp18 is shown in Sequence 10.
[0091]
The base sequences constituting each primer are as shown in the sequence listing. The structural features of the primers are summarized below. Further, the positional relationship of each region with respect to the target base sequence (target) is shown in FIG.

[0092]
With such a primer, the region from F1c to R1c of M13mp18 (HBV-M13mp18) incorporating the partial sequence of the HBV gene and its complementary base sequence are single-stranded across the loop-forming sequence containing F2c. Nucleic acids linked alternately above are synthesized. The reaction was carried out under the same conditions as in Example 1 except that the above primers were used, and the reaction solution was analyzed by agarose electrophoresis. The results are as shown in FIG. Each lane corresponds to the next sample.
1. XIV size marker
2. 1fmol HBV-M13mp18 dsDNA
3. No target
[0093]
Similar to Example 1, only when target was present, the product was confirmed as a low-size band ladder and a high-size smear stain and a band that was hardly migrated in the gel (lane 2). ). Among the low-size bands, the bands around 310 bp and 480 bp are the products predicted by this reaction, and the size matches the double strands of SEQ ID NO: 17 and SEQ ID NO: 18. It was confirmed that it was progressing as expected. The high-size smear pattern and the non-migrated band were presumed to be caused by the structure of the synthetic product characteristic of the present invention as described in the results of Example 1. From this experiment, it was confirmed that the present invention can be implemented even if the target sequence to be amplified is different.
[0094]
[Example 5] Confirmation of size of synthesis reaction product
In order to confirm the structure of the nucleic acid synthesized according to the present invention, its length was analyzed by electrophoresis under alkaline denaturing conditions. Add 1 μL of alkaline loading buffer to 5 μL of the reaction solution in the presence of the target of Example 1 and Example 4 and use a 0.7% agarose gel (50 mM NaOH, 1 mM EDTA) for 14 hours at 50 mA. Electrophoresed. As a molecular size marker, a HindIII digested fragment of lambda phage was used. The gel after electrophoresis was neutralized with 1M Tris pH 8, and then stained with SYBR Green I (Molecular Probes, Inc.) to confirm the nucleic acid. The results are shown in FIG. Each lane corresponds to the following sample.
1. HindIII digestion fragment of lambda phage
2. Reaction product of Example 1
3. Reaction product of Example 4
[0095]
When the reaction product is run under alkaline denaturing conditions, the size can be confirmed in a single-stranded state. In both Example 1 (lane 2) and Example 4 (lane 3), the main product was confirmed to be within 2 kbase. It was also found that the product according to the present invention extends to at least 6 kbase or more within the range that can be confirmed by this analysis. In addition, it was reconfirmed that the bands that were not migrated under the undenatured conditions of Example 1 and Example 4 were separated into individual single strands in the denatured state and the size was reduced.
[0096]
[Example 6] Confirmation of target concentration-dependent amplification in amplification of the region in M-13mp13
The influence of the target concentration change on the nucleic acid synthesis method according to the present invention was observed. The nucleic acid synthesis method according to the present invention was carried out under the same conditions as in Example 1 except that the target M13mp18 dsDNA was 0 to 1 fmol and the reaction times were 1 hour and 3 hours. In the same manner as in Example 1, electrophoresis was performed on a 2% agarose gel (0.5% TBE), and nucleic acids were confirmed by SYBR Green I (Molecular Probes, Inc.) staining. XIV (100 bp ladder, Boehringer Mannheim) was used as a molecular size marker. The results are shown in FIG. 14 (top: reaction time 1 hour, bottom: reaction time 3 hours). Each lane corresponds to the next sample.
1. M13mp18 dsDNA 1x10^-15mol / tube
2. M13mp18 dsDNA 1x10^-16mol / tube
3. M13mp18 dsDNA 1x10^-17mol / tube
4. M13mp18 dsDNA 1x10^-18mol / tube
5. M13mp18 dsDNA 1x10^-19mol / tube
6. M13mp18 dsDNA 1x10^-20mol / tube
7. M13mp18 dsDNA 1x10^{-twenty one}mol / tube
8. M13mp18 dsDNA 1x10^{-twenty two}mol / tube
9. No target
10. XIV size marker
[0097]
A band common to each lane seen in the lower part of the electrophoresis image is a stained unreacted primer. Regardless of the reaction time, no amplification product is observed when no target is present. Only in the presence of the target, a staining pattern of the amplification product was obtained depending on the concentration of the target. Moreover, the amplification product could be confirmed to a lower concentration by extending the reaction time.
[0098]
[Example 7] Detection of point mutation (point mutation)
(1) Production of M13mp18FM (mutant type)
M13mp18 (wild type) and M13mp18FM (mutant type) were used as target DNA. Mutant M13mp18FM is produced by LA PCR^TM One base substitution was introduced using in vitro Mutagenesis Kit (Takara Shuzo). Thereafter, the sequence was confirmed by sequencing. The sequence in the F1 region is shown below.
Wild type: CCGGGGATCCTCTAGAGTCG (SEQ ID NO: 19)
Mutant: CCGGGGATCCTCTAGAGTCA (SEQ ID NO: 20)
[0099]
(2) Primer design
Primers to be used were such that the bases of the FA-primer F1c region at the 5 ′ end were wild-type and mutant-types with different sequences. FIG. 15 shows the position of the mutation and the positional relationship of each region with respect to the target base sequence (target).
[0100]
(3) Amplification reaction
Using M13mp18 (wild type) and M13mp18FM (mutant type) as templates, experiments were conducted to determine whether template-specific amplification reactions would occur with specific primer combinations shown below.
Wild type amplification primer set: FAd4, RAd4, F3, R3
Mutant amplification primer set: FAMd4, RAd4, F3, R3
The base sequence of each primer is as follows.
FAd4: CGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (SEQ ID NO: 21)
FAMd4: TGACTCTAGAGGATCCCCGGTTTTTGTTGTGTGGAATTGTGAGCGGAT (SEQ ID NO: 22)
RAd4: CGTCGTGACTGGGAAAACCCTTTTTGTGCGGGCCTCTTCGCTATTAC (SEQ ID NO: 23)
F3: ACTTTATGCTTCCGGCTCGTA (SEQ ID NO: 24)
R3: GTTGGGAAGGGCGATCG (SEQ ID NO: 25)
[0101]
(4) M13mp18 point mutation detection
The composition of the reaction solution is as follows.

[0102]
Target M13mp18 or M13mp18FM 1 fmol (2 μl) was added to the reaction solution, heated at 95 ° C. for 5 minutes, and the target was denatured to form a single strand. The reaction solution was transferred to ice water, 1 μL (8 U) of Bst DNA polymerase (NEW ENGLAND BioLabs) was added, and the mixture was reacted at 68 ° C. or 68.5 ° C. for 1 hour. After the reaction, the reaction was stopped at 80 ° C. for 10 minutes and transferred again to ice water.
As shown in FIG. 16, when wild type FAd4 was used as the FA primer, amplification was effectively observed only in the presence of the wild type template. On the other hand, when the mutant FAMd4 was used as the FA primer, amplification was effectively observed only in the presence of the wild type template.
From the above results, it was shown that point mutations can be detected efficiently by using the amplification reaction of the present invention.
[0103]
[Example 8] Amplification reaction targeting mRNA
The method for synthesizing nucleic acids according to the present invention was attempted using mRNA as a target nucleic acid. Target mRNAs include prostate cancer cell line LNCaP cell (ATCC No. CRL-1740), which is a cell expressing prostate specific antigen (PSA), and chronic myeloid leukemia cell line K562, which is a non-expressing cell. cell (ATCC No. CCL-243), 1:10⁶~ 100: 10⁶The total RNA was extracted using the RNeasy Mini kit from Qiagen (Germany). There are four types of primers used in the experiment: PSAFA, PSARA, PSAF3, and PSAR3. PSAF3 and PSAR3 are outer primers for substituting the first nucleic acid obtained from PSAFA and PSARA, respectively. In addition, these primer concentrations were set high so that annealing of PSAFA (or PSARA) occurred preferentially. The base sequences constituting each primer are as follows.
Primer:
PSAFA: TGTTCCTGATGCAGTGGGCAGCTTTAGTCTGCGGCGGTGTTCTG (SEQ ID NO: 26)
PSARA: TGCTGGGTCGGCACAGCCTGAAGCTGACCTGAAATACCTGGCCTG (SEQ ID NO: 27)
PSAF3: TGCTTGTGGCCTCTCGTG (SEQ ID NO: 28)
PSAR3: GGGTGTGGGAAGCTGTG (SEQ ID NO: 29)
[0104]
The structural features of the primers are summarized below. Moreover, the positional relationship of each primer with respect to the DNA base sequence which codes target mRNA, and the recognition site | part of the restriction enzyme Sau3A I are shown in FIG.

[0105]
The composition of the reaction solution for the nucleic acid synthesis method according to the present invention is shown below.
Reaction solution composition (in 25 μL)
20 mM Tris-HCl pH8.8
4 mM MgSO_Four
0.4mM dNTPs
10 mM KCl
10mM (NH_Four)₂SO_Four
0.1% TritonX-100
0.8M betaine
5mM DTT
1600nM PSAFA & PSARA primer
200nM PSAF3 & PSAR3 primer
8U Bst DNA polymerase
100U ReverTra Ace (TOYOBO, Japan)
5 μg total RNA
[0106]
All ingredients were mixed on ice. In this experiment, mRNA (single strand) is the target, so the step of making single strand by heat denaturation is unnecessary. The reaction was carried out at 65 ° C. for 45 minutes and stopped at 85 ° C. for 5 minutes. After completion of the reaction, 5 μL of the reaction solution was electrophoresed using 2% agarose and detected with SYBR Green I.
The results are shown in FIG. Each lane corresponds to the following sample.
[0107]
Lane Bst RT LNCaP cell count / 10⁶K562
1-+ 0
2 + 10
3 + − 0
4 + -10
5 + + 0
6 + +1
7 + +10
8 1 μL of the reaction solution in lane 6 digested with Sau3A I
9 1 μL of the reaction solution in lane 7 digested with Sau3A I
10 Size marker 100bp ladder (New England Biolabs)
[0108]
Without either Bst DNA polymerase or ReverTra Ace, no amplification product was obtained (lanes 1 to 4). In the presence of both enzymes, amplification products were detected in the presence of LNCaP-derived RNA (lanes 5-7). Detection was possible even with RNA extracted from one LNCaP per million K562 cells (lane 6). When the amplified product was digested with the restriction enzyme site Sau3A I in the sequence inside the target, it was digested into a fragment of the expected size (lanes 8 and 9).
From the above results, it was confirmed that the target reaction product was obtained even when RNA was used as a target in the nucleic acid synthesis method of the present invention.
[0109]
【The invention's effect】
The novel oligonucleotide according to the present invention and a method for synthesizing a nucleic acid using the same provide a method for synthesizing a nucleic acid in which complementary base sequences are alternately linked on a single strand that does not require complicated temperature control. The complementary strand synthesized using the oligonucleotide according to the present invention as a primer is always the synthesis starting point for the synthesis of a new complementary strand using the 3 ′ end as a template. At this time, accompanied by the formation of a loop that causes annealing of a new primer, the product of the complementary strand synthesis reaction using itself synthesized as a template by synthesizing the complementary strand from this portion is replaced again to allow base pairing. It becomes a state. The nucleic acid synthesized using itself as a template that can be obtained in this way contributes to the improvement of the efficiency of nucleic acid synthesis by combining with a known nucleic acid synthesis method such as SDA.
[0110]
Furthermore, according to a desirable embodiment of the present invention, not only the efficiency improvement of a known nucleic acid synthesis method is achieved, but also a complicated temperature control is not required, a high amplification efficiency can be expected, and a high specificity is achieved. A novel nucleic acid synthesis method that can be achieved is provided. That is, by applying the oligonucleotide according to the present invention to a template strand and its complementary strand, nucleic acids in which complementary base sequences are alternately linked on one strand are synthesized continuously. . This reaction continues in principle until the starting material required for synthesis is depleted, during which time new nucleic acids starting synthesis from the loop portion continue to be generated. Thus, extension from oligonucleotides annealed to the loop performs strand displacement that provides 3'-OH for the extension of long single-stranded nucleic acids (ie, multiple sets of complementary base sequences linked together). . On the other hand, a long single-stranded 3′-OH achieves its own elongation by performing a complementary strand synthesis reaction using itself as a template, and at the same time, replaces a new complementary strand that has been synthesized from a loop. Such an amplification reaction process proceeds under isothermal conditions while maintaining high specificity.
[0111]
The oligonucleotide in the present invention functions as a primer for the nucleic acid synthesis reaction according to the present invention only when two consecutive regions are arranged as designed. This greatly contributes to maintaining specificity. For example, in PCR, high specificity can be expected in the present invention compared to the case where nonspecific amplification reaction is initiated by nonspecific misannealing regardless of the intended positional relationship between two primers. Can be easily explained. Using this feature, SNPs can be accurately detected with high sensitivity.
[0112]
A feature of the present invention is that such a reaction can be easily achieved with a very simple reagent configuration. For example, although the oligonucleotide according to the present invention has a special structure, it is a matter of selection of a base sequence, and is merely an oligonucleotide as a substance. In a desirable embodiment, the reaction can proceed only with a DNA polymerase that catalyzes a strand displacement type complementary strand synthesis reaction. Furthermore, when the present invention is carried out using RNA as a template, all enzyme reactions can be performed in a single manner by using a DNA polymerase having both reverse transcriptase activity and strand displacement type DNA polymerase activity, such as Bca DNA polymerase. This can be done with any enzyme. The reaction principle that realizes an advanced nucleic acid amplification reaction by such a simple enzyme reaction has not been known so far. Alternatively, even when applied to a known nucleic acid synthesis reaction such as SDA, a new enzyme is not required in combination with the present invention, and various reaction systems can be obtained simply by combining oligonucleotides based on the present invention. Can be adapted. Therefore, the nucleic acid synthesis method according to the present invention can be said to be advantageous in terms of cost.
As described above, the nucleic acid synthesis method of the present invention and the oligonucleotides therefor simultaneously solve a plurality of difficult problems such as operability (temperature control unnecessary), improvement in synthesis efficiency, economy, and high specificity. To provide a new principle.
[0113]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing a part (1)-(4) of the reaction principle of a preferred embodiment of the present invention.
FIG. 2 is a schematic diagram showing a part (5)-(7) of the reaction principle of a preferred embodiment of the present invention.
FIG. 3 is a schematic diagram showing a part (8)-(10) of the reaction principle of a preferred embodiment of the present invention.
FIG. 4 is a schematic diagram showing the structure of a loop formed by a single-stranded nucleic acid according to the present invention.
FIG. 5 is a schematic diagram showing a part (A)-(B) of the basic embodiment according to the present invention.
FIG. 6 is a schematic diagram showing a part (C)-(D) of the basic embodiment according to the present invention.
FIG. 7 is a diagram showing the positional relationship of each base sequence constituting an oligonucleotide in the target base sequence of M13mp18.
FIG. 8 is a photograph showing the result of agarose electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid according to the present invention using M13mp18 as a template.
Lane 1: XIV size marker
Lane 2: 1 fmol M13mp18 dsDNA
Lane 3: No target
FIG. 9 is a photograph showing the result of digesting the product of the nucleic acid synthesis reaction according to the present invention obtained in Example 1 with a restriction enzyme and performing agarose electrophoresis.
Lane 1: XIV size marker
Lane 2: BamHI digest of purified product
Lane 3: PvuII digest of purified product
Lane 4: HindIII digest of purified product
FIG. 10 is a photograph showing the result of agarose electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid of the present invention by adding betaine using M13mp18 as a template. 0, 0.5, 1, and 2 represent betaine concentrations (M) added to the reaction solution. N is a negative control, -21 is a template DNA concentration of 10^{-twenty one}Represents mol.
FIG. 11 is a diagram showing the positional relationship of each base sequence constituting an oligonucleotide in a target base sequence derived from HBV.
FIG. 12 is a photograph showing the result of agarose electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid according to the present invention using HBV-M13mp18 incorporated in M13mp18 as a template.
Lane 1: XIV size marker
Lane 2: 1 fmol HBV-M13mp18 dsDNA
Lane 3: No target
FIG. 13 is a photograph showing a result of alkali denaturing gel electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid according to the present invention.
Lane 1: lambda phage digested HindIII
Lane 2: reaction product of Example 1
Lane 3: reaction product of Example 3
FIG. 14 is a photograph showing the result of agarose electrophoresis of the product obtained by the method for synthesizing a single-stranded nucleic acid according to the present invention when the concentration of M13mp18 as a target was changed. The upper result is a reaction time of 1 hour, and the lower result is a reaction time of 3 hours.
Lane 1: M13mp18 dsDNA 1x10^-15mol / tube
Lane 2: M13mp18 dsDNA 1x10^-16mol / tube
Lane 3: M13mp18 dsDNA 1x10^-17mol / tube
Lane 4: M13mp18 dsDNA 1x10^-18mol / tube
Lane 5: M13mp18 dsDNA 1x10^-19mol / tube
Lane 6: M13mp18 dsDNA 1x10^-20mol / tube
Lane 7: M13mp18 dsDNA 1x10^{-twenty one}mol / tube
Lane 8: M13mp18 dsDNA 1x10^{-twenty two}mol / tube
Lane 9: No target
Lane 10: XIV size marker
FIG. 15 is a diagram showing the position of mutation and the positional relationship of each region with respect to the target base sequence (target). The underlined guanine is replaced with adenine in the mutant form.
FIG. 16 is a photograph showing the result of agarose electrophoresis of a product obtained by the amplification reaction of the present invention.
M: 100bp ladder (New England Biolabs)
N: No mold (purified water)
WT: Wild type template M13mp18 1 fmol
MT: Mutant template M13mp18FM 1 fmol
FIG. 17 is a diagram showing the positional relationship of each base sequence constituting an oligonucleotide in a base sequence encoding a target mRNA.
FIG. 18 is a photograph showing the result of agarose electrophoresis of a product obtained by the method for synthesizing a single-stranded nucleic acid according to the present invention using mRNA as a target.

Claims (10)

A method for synthesizing a nucleic acid comprising complementary base sequences alternately linked on a single strand, comprising the following steps.
a) A region F1 that can be annealed to a part F1c on the same strand is provided at the 3 ′ end, and this region F1 anneals to F1c, thereby forming a loop including a region F2c capable of base pairing. Providing a nucleic acid capable of
b) Complementary strand synthesis starting from the 3 ′ end of F1 annealed to F1c,
c) An oligonucleotide containing F2 having a sequence complementary to the region F2c at its 3 ′ end is annealed, and this is used as a starting point for synthesis to carry out complementary strand synthesis using a polymerase that catalyzes a strand displacement complementary strand synthesis reaction, and step b) Substituting the complementary strand synthesized in
d) Annealing a polynucleotide containing a sequence complementary to an arbitrary region in the complementary strand that has been replaced in step c) and capable of base pairing, and complementing the strand using the 3 'end as a synthesis origin Performing complementary strand synthesis with a polymerase that catalyzes the strand synthesis reaction, and replacing the complementary strand synthesized in step c)   In step d), the synthesis origin is the region R1 existing at the 3 ′ end on the same strand that can be annealed to the region R1c, and the loop including the region R2c capable of base pairing by annealing to R1c The method of claim 1, wherein: The method according to claim 1, wherein the nucleic acid in step a) is a second nucleic acid provided by the following steps.
i) An oligonucleotide region F2 including a region having the same base sequence as F1c linked to the 5 ′ side of a region having a base sequence F2 complementary to F2c is annealed to a nucleic acid region F2c as a template. Process,
ii) synthesizing a first nucleic acid having a base sequence complementary to the template using the oligonucleotide F2 as a synthesis origin;
iii) An arbitrary region of the first nucleic acid synthesized in step ii) by performing strand displacement complementary strand synthesis starting with the oligonucleotide F3 that anneals to an arbitrary region F3c further 3 ′ of F2c , In a state capable of base pairing,
iv) annealing an oligonucleotide having a base sequence complementary to the region capable of base pairing of the first nucleic acid in step iii), and synthesizing the second nucleic acid using it as a synthesis origin; Further , a step of synthesizing a strand-displacement complementary strand starting from the synthesis of the oligonucleotide R3 that anneals to an arbitrary region R3c on the 3 ′ side to make the F1 at the 3 ′ end ready for base pairing. When the region capable of base pairing in step iii) is R2c, and the region present on the 5 ′ side of R2c is R1c, the oligonucleotide in step iv) is a base sequence R2 complementary to R2c. The method according to claim 3 , wherein the oligonucleotide comprises a region having the same base sequence as that of R1c linked to the 5 ′ side of the region having R. The method according to claim 4 , wherein the melting temperature of each oligonucleotide used in the reaction and its complementary region in the template has the following relationship under the same stringency.
(F3c / F3 and R3c / R3) ≦ (F2c / F2 and R2c / R2) ≦ (F1c / F1 and R1c / R1) The method according to any one of claims 3 to 5 , wherein the template nucleic acid is RNA, and the complementary strand synthesis in step ii) is performed with an enzyme having reverse transcriptase activity. A method for amplifying a nucleic acid in which complementary base sequences are alternately linked on a single strand by repeating the following steps.
A) When the 3 ′ end and the 5 ′ end are provided with regions each consisting of a base sequence complementary to the end region on the same strand by the method according to claim 4 , and the complementary base sequences are annealed. Providing a template in which a loop capable of base pairing is formed between the two,
B) Complementary strand synthesis using the 3 ′ end of the template annealed to the same strand as a synthesis origin,
C) An oligonucleotide containing a complementary base sequence at the 3 ′ end in the loop located on the 3 ′ end side of the loop is annealed to the loop portion, and this is used as a starting point for synthesis to catalyze the strand displacement complementary strand synthesis reaction. A complementary strand synthesis using a polymerase that replaces the complementary strand synthesized in step B) so that the 3 ′ end is capable of base pairing, and D) the 3 ′ end is replaced in step C). A step in which a strand that is capable of base pairing is used as a new template in step A) A kit for synthesizing a nucleic acid comprising complementary base sequences alternately linked on a single strand, comprising the following elements.
For a template nucleic acid containing region F3c, region F2c, and region F1c in this order from the 3 ′ side,
i) an oligonucleotide comprising a region having the same base sequence as F1c linked to the 5 ′ side of the region having a base sequence F2 complementary to F2c ;
ii) an oligonucleotide comprising a base sequence complementary to any region R2c in a complementary strand synthesized using the oligonucleotide of i) as a primer;
iii) an oligonucleotide having a nucleotide sequence complementary to the F3c,
iv) an oligonucleotide having a base sequence complementary to the region R3c located 3 ′ of any region R2c in the complementary strand synthesized using the oligonucleotide i) as a starting point ;
v) a DNA polymerase that catalyzes a strand displacement type complementary strand synthesis reaction; and
vi) Nucleotides that serve as substrates for element v) The oligonucleotide of ii) has a base sequence R2 complementary to R2c with respect to an arbitrary region R2c and a region R1c located on the 5 ′ side of the complementary strand synthesized from the oligonucleotide of i). The kit according to claim 8 , which is an oligonucleotide containing a region having the same base sequence as that of R1c on the 5 ' side of the region . A kit for detecting a target base sequence, further comprising a detection agent for detecting a product of a nucleic acid synthesis reaction in the kit according to claim 8 or 9 .

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