JP4873812B2 - Immunodetection of RNA: DNA hybrids on microarrays - Google Patents

Description

【００８６】
【表２】

【００８７】
【表３】

【００８８】
【表４】

【００８９】
【表５】

【００９０】
【表６】

【００９１】
【表７】

【００９２】
逆転写は一般に逆転写酵素の機能する温度範囲内の任意の温度において実施してよい。好ましくは、インキュベーション温度は逆転写酵素が機能して且つプライマーが標的核酸分子にハイブリダイズしたまま残るあらゆる温度である。非好熱性逆転写酵素に関しては、好ましい温度は逆転写酵素のための最適な温度又はその付近の温度である。ほとんどの非好熱性逆転写酵素に関しては、この温度が約２５℃から４５℃の間になる。
【００９３】
好ましい態様において、好熱性逆転写酵素は選択性を増大させるために使用される。好熱性逆転写酵素が機能するもっとも高い温度は本当に高くてよい。このため、好熱性逆転写酵素を用いる場合の逆転写酵素のための好ましい温度範囲は、対象のＲＮＡ分子とプライマーの間のハイブリッドの計算された溶融温度に関してもっとも便利に記載される。そのような溶融温度は、ＲＮＡ／プライマー溶融温度（Ｒ／Ｐ Ｔｍ）と本明細書では呼ぶ。好ましい範囲は、対象のＲＮＡ分子とプライマーの間のハイブリッドの溶融温度より２０℃下であり、対象のＲＮＡ分子とプライマーの間のハイブリッドの５℃上を含む。好熱性逆転写酵素を用いる場合の他の好ましい範囲は、表２に掲載されたものを含む。
【００９４】
【表８】

【００９５】
特に注目すべきなのは、上記の列挙された範囲内の各々の特定のしかし不特定の範囲が別の好ましい範囲として意図されることである。逆転写酵素のための好ましい温度は、Ｒ／Ｐ Ｔｍより約２０℃下、Ｒ／Ｐ Ｔｍより約１５℃下、Ｒ／Ｐ Ｔｍより約１２℃下、Ｒ／Ｐ Ｔｍより約１０℃下、Ｒ／Ｐ Ｔｍより約７℃下、Ｒ／Ｐ Ｔｍより約５℃下、Ｒ／Ｐ Ｔｍより約３℃下、Ｒ／Ｐ Ｔｍより２０℃下、Ｒ／Ｐ Ｔｍより１５℃下、Ｒ／Ｐ Ｔｍより１２℃下、Ｒ／Ｐ Ｔｍより１０℃下、Ｒ／Ｐ Ｔｍより７℃下、Ｒ／Ｐ Ｔｍより５℃下、そしてＲ／Ｐ Ｔｍより３℃下を含む。通常、温度がＲ／Ｐ Ｔｍに近づけば近づくほど、ＲＮＡとプライマーの特異的ハイブリッドと非特異的ハイブリッドの間の識別の程度が高くなる。温度がＲ／Ｐ Ｔｍに近いと、しかしながら、特異的ハイブリッドの安定性の低下がプライミングの効率の低さを引き起こすかもしれない。
【００９６】
Ｒ／Ｐ Ｔｍは計算によるか又は経験的測定の何れかにより測定してよい。Ｒ／Ｐ Ｔｍを計算するためには核酸ハイブリッドの安定性を計算するためのあらゆる確立された計算式を使用してよい。Ｒ／Ｐ Ｔｍを計算するための好ましい計算式は、Ｔｍ ＝ ΔＨ／ΔＳ＋Ｒ×Ｉｎ（Ｃ／４） ＋ １６．６ｌｏｇ［Ｋ⁺］／１＋０．７［Ｋ⁺］―２３７．１５であり、完璧にマッチするＤＮＡ：ＲＮＡの安定性に基づいた研究に由来した。ＲＮＡ：ＤＮＡハイブリッドに関しては、上記式へのホルムアミド濃度の取り込みは適用できないが、何故ならば、ホルムアミド濃度とＴｍの下降の間の関係が直線ではないからである。８０％ホルムアミドにおいて、ＲＮＡ：ＤＮＡハイブリッドはＤＮＡ：ＤＮＡハイブリッドよりも安定であり、配列に依存して約１０から３０℃Ｔｍを大きくさせる（Ｈａｍｅｓ ＆ Ｈｉｇｇｉｎｓ，Ｎｕｃｌｅｉｃ Ａｃｉｄ Ｈｙｂｒｉｄｉｓａｔｉｏｎ：Ａ Ｐｒａｃｔｉｃａｌ Ａｐｐｒｏａｃｈ（ＩＲＬ Ｐｒｅｓｓ Ｌｉｍｉｔｅｄ，Ｏｘｆｏｒｄ，Ｅｎｇｌａｎｄ．１９８５））。８０％ホルムアミドにおいて反応を実施すると、ホルムアミドを従って使用することにより、ＤＮＡ：ＤＮＡ二重鎖の形成を抑制し、ＲＮＡ：ＤＮＡハイブリッドを優先的に選択し、そしてＲ／Ｐに関してのＴｍを見積もってもよい。ＲＮＡ：ハイブリッドのＴｍの見積もりのために経験的に誘導された式は、短い核酸プライマーに関して正確ではないかもしれないため、ハイブリダイゼーション温度は４０から６０℃の温度範囲において０．１−０．４Ｍの単価カチオン中でハイブリッド安定性を評価することにより好適に決定される。Ｒ／Ｐ Ｔｍは経験的に決定してもよい（Ｌｅｓｎｉｃｋ ａｎｄ Ｆｒｅｉｅｒ，Ｂｉｏｃｈｅｍｉｓｔｒｙ ３４：１０８０７−１０８１５（１９９５），ＭｃＧｒａｗ ｅｔ ａｌ．，Ｂｉｏｔｅｃｈｎｉｑｕｅｓ ８：６７４−６７８（１９９０），及びＲｙｃｈｌｉｋ ｅｔ ａｌ．，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．１８：６４０９−６４１２（１９９０））。
【００９７】
本明細書にて使用する好熱性逆転写酵素は、５０℃より上の任意の温度においてその最大活性の少なくとも５％を保持するか、又は少なくとも５０℃の最適温度を有するあらゆる逆転写酵素である。好ましい逆転写酵素は、少なくとも５０℃の最適温度を有するものである。本明細書にて使用される逆転写酵素の最大活性は、以下に記載されるアッセイにおいて測定され、与えられた逆転写酵素がその最適温度において示す、活性として定義される。本明細書にて使用される逆転写酵素の最適温度は、逆転写酵素の活性が最大になる温度において定義され、以下に記載されるアッセイにおいて測定される。与えられた逆転写酵素の最適温度は、様々な温度にて以下のアッセイにおいてその活性を測定することにより決定してよい。通常、最適温度は、アッセイが５から１０℃の間隔において実施することのみが要求される範囲内でのみ測定される必要がある。
【００９８】
固相基板への核酸配列の固定化の方法はよく確立されている。半分のプライマー及びローリングサークルの複製プライマーを含むオリゴヌクレオチドは確立されたカップリング法を使用してカップリングしてよい。例えば、付着法はＰｅａｓｅ ｅｔ ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ９１（１１）：５０２２−５０２６（１９９４）、及びＫｈｒａｐｋｏ ｅｔ ａｌ．，Ｍｏｌ．Ｂｉｏｌ（Ｍｏｓｋ）（ＵＳＳＲ）２５：７１８−７３０（１９９１）により記載される。３’アミンオリゴヌクレオチドの固定化法は、Ｓｔｉｍｐｓｏｎ ｅｔ ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ９２：６３７９−６３８３（１９９５）により記載される。オリゴヌクレオチドを固相支持体に付着させるための好ましい方法は、Ｇｕｏ ｅｔ ａｌ．，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．２２：５４５６−５４６５（１９９４）に記載される。
【００９９】
核酸又はプライマー分子の固相支持体への固定化及びアレイ化はあらゆる適切な技術を用いて達成してよい。例えば、固定化は、インサイチュ核酸合成（Ｍａｓｋｏｓ ａｎｄ Ｓｏｕｔｈｅｒｎ，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，２０：１６７９−１６８４（１９９２）；Ｐｅａｓｅ ｅｔ ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ，９１：５０２２−５０２６（１９９４））又は化学合成されたオリゴヌクレオチドの共有結合又は不動付着によるか（Ｇｕｏ ｅｔ ａｌ．，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓｅａｒｃｈ，２２：５４５６−５４６５（１９９４））又はロボットアレイ化技術と組み合わせた他の核酸、アンプリコン、ｃＤＮＡｓ等の共有結合又は不動付着の何れかにより達成してよい。他の固定化技術はＭｃＧａｌｌらへの米国特許５，４１２，０８７、Ｍａｔｓｏｎらへの米国特許５，４２９，８０７、Ｆｏｄｏｒらへの米国特許５，５１０，０８７に記載される。幾千の異なるプライマーが固相支持体上にアレイ化されて、幾千の標的核酸配列を質問してよい。核酸又はプライマーの密度はアレイ化の方法及び検出手段により適合させるべきである。
【０１００】
本発明の一つの態様は、特定の核酸プローブを含むユニバーサルアレイへの標的核酸配列のハイブリダイゼーションを含み、その際、「ユニバーサルアレイ」又は「ユニバーサルアレイ配列」は本明細書にて相互変化可能なようにあらゆる可能な塩基の組み合わせの短い核酸配列として定義される。上記ユニバーサルアレイ配列は６−１０塩基、好ましくは５−６塩基の範囲を含み、可能な組み合わせの数（即ち、固相へ結合する別々のプローブ）はそれぞれ１０２４および４０９６であり、そして核酸発現分析を可能にし、その際、結果をフィンガープリントとして使用してよく、異なる組織又はサンプルが異なるフィンガープリントを与える。
【０１０１】
第３の核酸プローブを含む態様は、「捕捉用タグ」を使用することにより固相アレイに固定化してよい。本明細書にて使用される捕捉用タグは他の化合物又はモエティに結合するかもしれないあらゆる化合物である。プライマーは即ち付着させた捕捉用タグのその結合パートナーへの結合を通して固定化される。そのような結合パートナーは本明細書にて「捕捉用ドック」と呼ぶ。捕捉用タグは化合物であり、例えば粒子又はハプテンであって、リガンド結合分子又は抗体のような他の分子に結合するか又は相互作用する化合物である。捕捉用タグと捕捉用ドックの間のそのような相互作用は特定の相互作用、例えばハプテンと抗体の間又はリガンドとリガンド結合分子の間の相互作用であることも好ましい。
【０１０２】
このアッセイのさらなる態様は、２つの隣接領域：標的核酸特異的領域及び「捕捉用配列相補体」を有する捕捉用タグを含む。本明細書にて使用される「捕捉用配列相補体」は、固相マイクロアレイに固定化された「ユニバーサル捕捉用配列」に相補な核酸配列を含み、但し、「ユニバーサル捕捉用配列」は公知であり且つ固相マイクロアレイ上のその位置が予め決定されている短い核酸配列を意味する。「捕捉用配列相補体」を含む捕捉用タグ又はプローブは、その相補体をその相補な「ユニバーサル捕捉配列」へハイブリダイズさせることにより固相マイクロアレイに固定化してよい。
【０１０３】
本発明の別の態様において、「捕捉用タグ」は固相マイクロアレイに結合したその「捕捉用ドック」にハイブリダイズする標識核酸プローブを意味し、但し、捕捉用ドックが標識核酸プローブに特異的な共通配列である。
【０１０４】
別の捕捉用タグは、オリゴヌクレオチドにカップリングするかもしれないハイブリダイゼーション又はリガンド分子を含む。核酸プローブのコンテクストにおいて記載される捕捉用タグはＳｙｖｎｅｎ ｅｔ ａｌ．，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．，１４：５０３７（１９８６）に記載された。捕捉用タグはビオチンも含み、核酸に取り込まれてよい。
【０１０５】
基板へのプライマーの接着又はカップリングは、ドックを基板に接着又はカップリングすることにより達成してよい。上記捕捉用ドックは、プライマー上の捕捉用タグに結合するか、又は相互作用することにより、プライマーの接着を媒介する。基板上に固定化された捕捉用ドックは基板上のプライマーの捕捉を可能にする。別の捕捉用ドックを別のプライマーに付着させた基板の別の領域に付着させることにより、別のプライマーに付着した別の捕捉用タグが別に、よって基板上の診断上の位置において捕捉されるかもしれない。例えば、マイクロタイタープレートの複数アッセイにおいては、９６までの異なる捕捉用タグに特異的な捕捉用ドックをマイクロタイタープレート上に、それぞれ異なる溝において固定化してよい。捕捉及び検出は、対応する核酸分子がサンプル中に存在する捕捉用タグに相当する溝においてのみ生じることになる。
【０１０６】
一つの態様において、捕捉用ドックはオリゴヌクレオチドである。基板にオリゴヌクレオチドを固定化するか又はカップリングさせる方法はよく確立されている。例えば、付着法はＰｅａｓｅ ｅｔ ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ９１（１１）：５０２２−５０２６（１９９４）、及びＫｈｒａｐｋｏ ｅｔ ａｌ．，Ｍｏｌ Ｂｉｏｌ（Ｍｏｓｋ（ＵＳＳＲ）２５：７１８−７３０（１９９１）により記載される。３’アミンオリゴヌクレオチドのカゼインコートされたスライドへの固定化の方法は、Ｓｔｉｍｐｓｏｎ ｅｔ ａｌ．，Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．ＵＳＡ ９２：６３７９−６３８３（１９９５）により記載される。オリゴヌクレオチドと固相基板に付着させるための別の方法はＧｕｏ ｅｔ ａｌ．，Ｎｕｃｌｅｉｃ Ａｃｉｄｓ Ｒｅｓ．２２：５４５６−５４６５（１９９４）に記載される。
【０１０７】
タンパク質を基板に固定化するための方法はよく確立されている。固定化は、標準固定化化学を用いた、例えば、アミン化表面、カルボキシル化表面又はヒドロキシル化表面への付着似より達成してよい。付着剤の例は、臭化シアン、スクシニミド、アルデヒド、塩化トシル、アビジン−ビオチン、光架橋剤、エポキシド、マレイミド及びグルタルアルデヒドである。これら及び他の付着剤並びにそれらの付着における使用方法は、Ｐｒｏｔｅｉｎ ｉｍｍｏｂｉｌｉｚａｔｉｏｎ：Ｆｕｎｄａｍｅｎｔａｌｓ ａｎｄ Ａｐｐｌｉｃａｔｉｏｎｓ，Ｒｉｃｈａｒｄ Ｆ．Ｔａｙｌｏｒ，ｅｄ．（Ｍ．Ｄｅｋｋｅｒ，Ｎｅｗ Ｙｏｒｋ，１９９１），Ｊｏｈｎｓｔｏｎｅ ａｎｄ Ｔｏｒｐｅ，Ｉｍｍｕｎｏｃｈｅｍｉｓｔｒｙ Ｉｎ Ｐｒａｃｔｉｃｅ（Ｂｌａｃｋｗｅｌｌ Ｓｃｉｅｎｃｔｉｆｉｃ Ｐｕｂｌｉｃａｔｉｏｎｓ，Ｏｘｆｏｒｄ，Ｅｎｇｌａｎｄ，１９８７）ページ２０９−２１６及びページ２４１−２４２、及びＩｍｍｏｂｉｌｉｚｅｄ Ａｆｆｉｎｉｔｙ Ｌｉｇａｎｄｓ，Ｃｒａｉｇ Ｔ．Ｈｅｒｍａｎｓｏｎ ｅｔ ａｌ．，ｅｄｓ．（Ａｃａｄｅｍｉｃ Ｐｒｅｓｓ，Ｎｅｗ Ｙｏｒｋ，１９９２）に記載される。タンパク質は、抗体上の遊離基を基板内に存在する反応性側鎖グループへ化学的に架橋結合することにより、基板に付着させてよい。例えば、タンパク質は架橋剤としてグルタルアルデヒド又はカルボジイミドを使用して遊離のアミノ又はカルボキシル基を含む基板に化学的に架橋結合してよい。この方法においては、遊離タンパク質を含む水性溶液をグルタルアルデヒド又はカルボジイミド存在下で固相基板とインキュベートする。標準の固定化化学は当業者に知られている。
【０１０８】
別の態様において、開示された方法の感度は、サンプル中に存在する遊離のハイブリダイズしない核酸を除去するために、ハイブリッドサンプルの繰り返しの洗浄により増加させられる。非特異的なハイブリダイズしない核酸を除去することは有用であるが、核酸中の二次構造が検出手段により認識されるかもしれず、アッセイバックグラウンドの上昇をもたらすかもしれないからである。
【０１０９】
開示された方法を用いた使用のための好ましいハイブリダイゼーションサンプル核酸検出キットは、上記方法に要求される成分のいくつか又は全てを使用して作成されてよい。上記キットは、好ましくは、対象の核酸分子上の領域に相補な固定化プライマーを含み、そしてより好ましくは対象の核酸分子上の領域に各々相補な複数の固定化プライマーを含む。
【０１１０】
好ましくは、キットが以下の成分の全て又はいくつかを含む：サンプルの安定化のためのサンプル輸送培地；検出される第２生物分子に特異的な生物分子のマイクロアレイ；ハイブリダイゼーションバッファー；ＲＮＡ：ＤＮＡハイブリッドに特異的な存在物（ｅｎｔｉｔｙ）；洗浄バッファー；増強バッファー；及びＲＮＡ：ハイブリッド特異的抗体を検出するのに必要な試薬。さらに、いくつかのキットはＲＮＡ：ＤＮＡハイブリッド依存性エキソヌクレアーゼ（ＲＮＡｓｅＨ）機能を欠く好熱性逆転写酵素を含んでよい。上記キットの別の組成物は、捕捉用配列相補領域を含む核酸プローブを含んでよい。さらに、上記キットは固相に結合した生物分子の共通配列にハイブリダイズする標識された生物分子プローブも含んでよい。キットは、限定ではないが、サンプル生物分子の検出のための生物分子のユニバーサルアレイをさらに含んでよい。キットはこれらの成分の全て又はそれらの一部を含んでよい。
【０１１１】
増幅された抗体検出のためには、上記の直接検出のためのハイブリダイゼーションキットに含まれる試薬に加えて、全て又は一部を含む以下の試薬もキットに含んでよい：検出用に標識された抗マウスＩｇＧ；ビオチン化された抗マウスＩｇＧ；標識された抗マウスストレプトアビジン；ビオチン化抗ストレプトアビジン；又はアセチル化ＢＳＡ溶液。
【０１１２】
上記キットは陰性対照及び陽性対照を含むべきである。好ましくは、陰性対照及び陽性対照のためのプローブが、核酸配列を有する固相上に含まれる。
以下の非限定実施例は本アッセイ及びキットの使用を例示する。
【０１１３】
実施例
実施例１
プローブマイクロアレイ上のＲＮＡ：ＤＮＡハイブリッドの検出
以下は、サンプル中の標的生物分子配列の検出のための開示された方法の一つの態様を実施するための好ましい方法の一例である。
【０１１４】
通常、上記アッセイは０．０５μｇの核酸のサンプルサイズを検出するために使用されるのが好ましい。もっとも好ましくは、０．０５μｇ−１０μｇの全核酸を本発明のアッセイを用いて検出する。
【０１１５】
標的核酸サンプルを、ヌクレアーゼを含まない水の中に懸濁し、そしてハイブリダイゼーションバッファーに加えた。ハイブリダイゼーション溶液を９５℃において２−５分間変性させた。標的核酸を含むハイブリダイゼーション溶液は、スポットされたオリゴヌクレオチドのマイクロアレイを有するガラススライドに加えた。６５℃において１６−２０時間ハイブリダイズさせる。直接検出するか又は増幅された検出の何れかへと続いた。
【０１１６】
直接検出に関しては、表面に結合したプライマーのマイクロアレイを有するガラススライドを３回１−２分間１ＸＰＢＳ／０．０５％Ｔｗｅｅｎ２０^TMで洗浄し、そして回転振動機上で振動させた（１１００ｒｐｍ）。ＲＮＡ：ＤＮＡ抗体染色溶液を加えたところ、全濃度は０．１４４μｇ／μｌであった。ガラススライドマイクロアレイを溶液中で１時間室温において振動させながら（１１００ｒｐｍ）インキュベートした。ガラススライドを１ＸＰＢＳ／０．０５％ Ｔｗｅｅｎ２０^TMで洗浄し、そして室温において１５分間振動させた（１１００ｒｐｍ）。次に、マイクロアレイを、マウス抗体、特にＣｙ３又はＣｙ５に対する抗体染色溶液中で室温においてインキュベートし、そして１時間１１００ｒｐｍにて振動させた。多数の蛍光染料を使用してよく、限定ではないが、例えばＣｙ３又はＣｙ５である。Ｃｙ−染料の最終濃度は１０％ヤギ血清及び１ＸＰＢＳ／０．０５ Ｔｗｅｅｎ２０^TMの溶液中で０．０４μｇ／μｌであった。わずかに厳密な手段を使用して、スライドを４回約各々１０秒間洗浄バッファー中で洗浄した。次に、スライドを増強バッファー中で５３℃において１５分間インキュベートした。次に、スライドを洗浄バッファー中で４回各々約１０秒間、ゆるい厳密性手段を用いて洗浄した。スライドガラスに結合したマイクロアレイを遠心分離により２０００ｒｐｍにて７−１０分間によるか又は乾燥するまで、乾燥させた。結果は、アレイスキャナー（Ａｆｆｙｍｅｔｒｉｘ ４１７ Ａｒｒａｙ Ｓｃａｎｎｅｒ又は均等物）中で、それぞれＣｙ３及びＣｙ５で標識された抗体により発色したスライドをスキャンするための５３２ｎｍ及び６３５ｎｍにおける光励起によりスライドを読むことにより、分析した。
【０１１７】
増幅された検出に関しては、スライドを３回１−２分間１ＸＰＢＳ／０．０５％ Ｔｗｅｅｎ２０^TM中で洗浄し、回転振動機上で１１００ｒｐｍにて振動させた。スライドをＲＮＡ：ＤＮＡハイブリッド特異的抗体染色溶液中で室温において１時間撹拌（１１００ｒｐｍ）しながらインキュベートした。スポットしたスライドはヤギ染色溶液からのビオチン化マウスＩｇＧ抗体で被覆し、そして１０分間室温においてインキュベートした。マイクロアレイを２回１−２分間それぞれ１ＸＰＢＳ／０．０５％ Ｔｗｅｅｎ２０^TM中で洗浄した。スポットしたスライドはヤギ抗マウスＲ−フィコエリトリンストレプトアビジン（０．０１μｇ／μｌ；ＳＡ−ＰＥ）染色溶液で被覆し、１０分間室温においてインキュベートした。洗浄は上記のとおり繰り返した。スポットしたスライドはストレプトアビジンに対して出現させたビオチン化ヤギ抗体（０．５ｍｇ／ｍＬ）染色溶液で被覆し、そして１０分間室温においてインキュベートした。スライドを再びヤギ抗マウスＲ−フィコエリトリンストレプトアビジン（０．０１μｇ／μｌ；ＳＡ−ＰＥ）染色溶液と共に１０分間室温においてインキュベートした。次に、スライドを３回１−２分間１ＸＰＢＳ／０．０５％ Ｔｗｅｅｎ２０^TM中で洗浄した。次に、マイクロアレイを２０００ｒｐｍにおける７−１０分間の遠心分離によるか、又は乾燥するまで、乾燥させた。結果は、アレイスキャナー（Ａｆｆｙｍｅｔｒｉｘ ４１７ Ａｒｒａｙ Ｓｃａｎｎｅｒ又は均等物）中で、それぞれＣｙ３又はＰＥ及びＣｙ５で標識された抗体により発色したスライドをスキャンするための５３２ｎｍ及び６３５ｎｍにおける光励起によりスライドを読むことにより、分析した。
【０１１８】
実施例２
サンプルハイブリダイゼーション前の
標識されたオリゴヌクレオチドのハイブリダイゼーション
Ｃｙ３又はＣｙ５で標識されたｎ−マーのオリゴヌクレオチドを、ＳＳＣ及びＳＤＳを含むハイブリダイゼーションバッファーに加え、そして９５℃にて２−５分間変性させた。多くの蛍光染料を使用してよく、限定ではないが、例えばＣｙ３及びＣｙ５である。多数の蛍光染料を使用してよく、限定ではないが、例えばＣｙ３又はＣｙ５である。スポットされたオリゴヌクレオチドを含むガラススライドをハイブリダイゼーション溶液で被覆し、そして室温において２０秒間インキュベートした。カバースリップを２ＸＳＳＣ／０．２％ＳＤＳの厳しい浸潤により除去した。如何なる残余のＳＤＳもスライドを０．０５ＸＳＳＣへ３０分間浸潤することにより洗い流された。次に、スライドを２０００ｒｐｍにて７−１０分間の遠心分離によるか又は乾燥するまで、乾燥させた。結果は、アレイスキャナー（Ａｆｆｙｍｅｔｒｉｘ ４１７ Ａｒｒａｙ Ｓｃａｎｎｅｒ又は均等物）中で、それぞれＣｙ３及びＣｙ５で標識された抗体により発色したスライドをスキャンするための５３２ｎｍ及び６３５ｎｍにおける光励起によりスライドを読むことにより、分析した。
【０１１９】
実施例３
ＲＮＡｓｅＨ機能を欠く逆転写酵素により媒介されるＲＮＡの検出
以下は、サンプル中の標的核酸配列の検出のための開示された方法のひとつの態様を実施する方法の一例である。
【０１２０】
５プライムのビオチン化された２０から３０ヌクレオチドプライマーを、ストレプトアビジンコートされた固相と混合し、そして３０から６０分間２０−２７℃において一定振動（１１００ｒｐｍ）にてインキュベートした。標的核酸のサンプルを上記固相に加えた。ハイブリダイゼーション／伸長バッファー（１００ｍＭ Ｔｒｉｓ−ＨＣｌ，ｐＨ８．３，１５０ｍＭ ＫＣｌ，６ｍＭ ＭｇＣｌ₂，２０ｍＭ ＤＴＴ，及び１ｍＭの各ｄＮＴＰ）を次に加えた。標的核酸及びプライマーは該混合物を最適なアニーリング温度まで、好ましくは６０℃にて（最適アニーリング温度は利用したプライマー及び核酸により変化する）２０−３０分間加熱することにより、アニールさせた。上記混合物を、次に、２０−２７℃に１０分間冷却した。追加のハイブリダイゼーション／伸長バッファー及び逆転写酵素、好ましくは高温安定性であってＲＮＡｓｅＨを欠く逆転写酵素を加えた。反応物を３０−６０分間４２℃においてインキュベートした。ＥＤＴＡ（０．５Ｍ）を加え、そして３７℃において３０分間インキュベートした。ＲＮＡ：ＤＮＡハイブリッド特異的アルカリホスファターゼ配合抗体混合物を加え、そして２０−２７℃において３０−６０分間インキュベートした。あらゆる未結合の抗体を洗い去り、次に化学発色基板を添加した。溶液を１５−３０分間２０−２７℃においてインキュベートした。放射されたシグナルをルミノメーターで適切な波長にて読んだ。
【０１２１】
実施例４
ＲＮＡ：ＤＮＡハイブリッド特異的抗体の結合
ハイブリダイズしたＲＮＡ：ＤＮＡサンプルを抗体と共に上記ハイブリッドの配合を許容するのに十分な時間インキュベートした。該ハイブリッドは５分から２４時間１５℃から６５℃においてプラットフォーム振動機上で０から１５００ｒｐｍのスピードにおけるインキュベーションにより抗体に結合させた。好ましくは、インキュベーション時間が３０から１２０分間であって、２０から４０℃において３００から１２００ｒｐｍのスピードであった。もっとも好ましくは、結合が約３００から１０００ｒｐｍの間の回転振動スピードにて１時間の室温における激しい振動のインキュベーションにより起こった。当業者には、インキュベーション時間、温度及び振動を変更することにより所望な別の捕捉動力学を達成してよい。
【０１２２】
実施例５
モノクローナル及びポリクローナル抗体により検出された
オリゴヌクレオチドの長さの比較
長さを変えた単一のオリゴヌクレオチドを１０の複製物にて４つの異なる濃度においてスポットした。スポットされた７２マーのオリゴヌクレオチドは、４０Ｓリボソーマルタンパク質Ｓ１１に相当するＩＭＡＧＥクローン＃２５９９８３の一部であった。より短いオリゴヌクレオチドは親の７２マーの連続的な末端除去物であった。これらのマイクロアレイを濃度を変えた相補ＲＮＡにハイブリダイズさせ、そしてモノクローナル一次ＲＮＡ：ＤＮＡハイブリッド抗体を用いて可視化させた。８００ｐＭの標的濃度において、実質的なシグナルが３０塩基のオリゴヌクレオチドにおいて観察されたが、２５塩基においてシグナルの急降下があった。図６Ａは、ＲＮＡ：ＤＮＡハイブリッド特異的モノクローナル抗体検出に際しての様々なスポットされたオリゴヌクレオチド濃度を用いた８００ｐＭのＲＮＡ濃度におけるオリゴヌクレオチドの函数としてのノイズ比に対するシグナルの結果を示す。
【０１２３】
ポリクローナル抗体検出プロトコルは、モノクローナル抗体に関しての上記プロトコルと同じであって、ウサギからのＣｙ３−標識ヤギ二次抗体を、ヤギからのマウス抗体に変えたのが例外であった。ポリクローナルを用いた結果（図６Ｂ）は、３０ 塩基未満のオリゴヌクレオチドに関するモノクローナル抗体に比較しての、顕著に改善された検出を示した。３０塩基を越えるオリゴヌクレオチドに関してはシグナルにおける顕著な差異はなかった。
【０１２４】
ポリクローナルＲＮＡ：ＤＮＡ抗体は、モノクローナル抗体を用いた検出に比較して３０塩基対未満のＲＮＡ：ＤＮＡハイブリッドを検出するのに顕著に感度の良い方法を提供した。
【図面の簡単な説明】
【図１】 図１Ａ−Ｄは、マイクロアレイ上のＲＮＡ：ＤＮＡハイブリッドの免疫抗体検出の好ましい態様ノイズ模式的代表図である。図１Ａは、図１Ｂに描かれたＲＮＡ：ＤＮＡハイブリッドを形成するマイクロアレイに付着させた相補ＤＮＡ配列へのＲＮＡサンプルのハイブリダイゼーションを示す。次に、抗体、モノクローナル又はポリクローナル、を図１Ｃに示すとおりにＲＮＡ：ＤＮＡハイブリッドに結合させる。図１Ｄは蛍光レーザースキャナーを用いた蛍光標識の検出を例示する。
【図２】 図２Ａ−Ｄは、ＲＮＡ：ＤＮＡハイブリッドの、ユニバーサル捕捉配列を含むマイクロアレイ上の免疫検出の第２の態様の模式的代表図である。図２Ａは、ユニバーサルアレイの一本鎖ＤＮＡとサンプルＲＮＡとのハイブリダイゼーションを示す。各ハイブリッドはＤＮＡ：ＤＮＡ領域及びＲＮＡ：ＤＮＡ領域を含むマイクロアレイ上に形成され、図２Ｂに描写したとおりである。抗体は図２ＣにおいてＲＮＡ：ＤＮＡハイブリッドを検出及び結合する。図２Ｄは蛍光レーザースキャナーを用いた蛍光抗体標識を含む検出の一つの手段を例示する。
【図３】 図３Ａ−Ｅは、ＲＮＡ：ＤＮＡハイブリッドの免疫検出の第３の態様の模式的代表図であって、但し、マイクロアレイは完全長配列が未知である場合のｍＲＮＡの定量のための発現された配列タグ（ＥＳＴｓ）を含む。図３Ａは、マイクロアレイに結合した短いＥＳＴｓへのサンプルＲＮＡのハイブリダイゼーションを示す。ＲＮＡと短いＤＮＡのハイブリッドの形成を図３Ｂに描写する。例えば逆転写酵素（ＲＴ）を使用して、図３Ｃに描写するように、ＤＮＡをＲＮＡの完全長まで伸長合成する。図３Ｄ及び図３Ｅは、ぞれぞれ、ＲＮＡ：ＤＮＡハイブリッドの抗体認識及びレーザースキャナーによる蛍光抗体標識の検出を例示する。
【図４】 図４Ａ−Ｄは、ＲＮＡ：ＤＮＡハイブリッドの免疫検出の第４の態様の模式的代表図であって、但し、発明は２色検出法に向けられる。図４Ａは、同一配列の領域及び可変配列の領域を含むマイクロアレイに結合した各ＤＮＡプローブを示す。標識されたＤＮＡは共通配列とハイブリダイズし、そしてＲＮＡサンプルは可変配列とハイブリダイズする。ＲＮＡ：ＤＮＡハイブリッド及びＤＮＡ：標識されたＤＮＡハイブリッドが形成され、図４Ｂに例示するとおりである。ＲＮＡ：ＤＮＡハイブリッドに対して出現させた抗体は適切な領域に結合し；マイクロアレイは２つの異なる色の蛍光レーザーによりスキャンし；そしてシグナルは図４Ｃ−４Ｄに示すとおりに標準化される。
【図５】 図５Ａ−Ｄは、ＲＮＡ：ＤＮＡハイブリッドの免疫検出の第４の態様の模式的代表図であって、但し、発明は標識された縮重ｎ−マーＤＮＡ及びサンプルＲＮＡを含む。図５Ａは、ＲＮＡサンプル及び／又は標識された縮重ＤＮＡに同時又は逐次的にハイブリダイズさせたマイクロアレイに結合させた各ＤＮＡプローブを示す。ＲＮＡ：ＤＮＡハイブリッド及び／又はＤＮＡ：標識ＤＮＡハイブリッドが形成され、図５Ｂに示されるとおりである。ＲＮＡ：ＤＮＡハイブリッドに対して出現させた抗体は適切な領域に結合し；マイクロアレイは２つの異なる色の蛍光レーザーによりスキャンし；そしてシグナルは図５Ｃ−５Ｄに示すとおりに標準化される。
【図６】 図６Ａ−Ｂは、マイクロアレイのノイズ比に対するシグナルの函数として、モノクローナル抗体（図６Ａ）及びポリクローナル抗体（図６Ｂ）による検出を用いた固相結合オリゴヌクレオチド長さの比較を表すグラフである。[0001]
Technical field
The present invention is in the general field of detection of biological molecules including DNA, RNA, proteins, etc., in particular, the field of detection of RNA: DNA hybrids on solid phases using hybridization assays.
[0002]
Background art
Many genes, including those related to disease states, and microbial and viral RNA or DNA have been isolated and sequenced. Nucleic acid probes based on such sequences are currently available for identifying a large number of genes and infections. A nucleic acid probe is a detectable nucleic acid sequence that hybridizes to complementary RNA or DNA in a test sample. Detection of the probe indicates the presence of a particular nucleic acid sequence in which the probe is specific. In addition to helping scientific research, nucleic acid probes are used to detect genetic mutations associated with viruses and bacteria, microorganisms such as yeast and protozoa, and specific disorders in patient samples.
[0003]
Grunstein. et al.Proc. Natl. Acad. Sci. USA  72: 3961 (1975) and Southern,J. et al. Mol. Biol. 98: 503 (1975) describes hybridization techniques using radiolabeled nucleic acid probes. Nucleic acid hybridization probes have the advantage of being more sensitive and specific than other detection methods and not requiring living organisms. Nucleic acid hybridization probes are often labeled with radioactive material that will be readily detected.
[0004]
Current hybridization techniques that utilize radioisotopes to label probes are expensive due to the need to monitor radioactive waste disposal and body and workplace contamination. in addition,³²Radioactive probes with short half-lives such as P require frequent production of radioactive probes. Radionucleic acid probe hybridization is therefore discouraged in commercial fields such as clinical diagnostics.
[0005]
In an attempt to avoid the problems associated with direct radioactive labeling, the probe was indirectly labeled. One common method in indirect labeling is to attach biotin (a small vitamin) to a nucleic acid probe using chemical or enzymatic techniques. Following hybridization to a specific nucleic acid, biotin is detected by reaction with streptavidin (which binds biotin tightly and is labeled with an enzyme or fluorescent dye). The bound biotin-streptavidin complex will be detected by reaction with a chromogenic substrate and the fluorescent dye will be observed when reacted with incident light of the appropriate wavelength. However, indirect labeling of hybridization probes with biotin or other haptens often increases the “hydrophobicity” of the probe. Probes tend to react non-specifically with substances other than complementary nucleic acid targets, leading to high background. Biotin labeling increases non-specific binding, which leads to high background, thereby reducing sensitivity and increasing the likelihood of false positive results. Indirect labeling is also less sensitive than direct labeling due to the limited label density; only a small fraction of bases are labeled, limiting the number of signal generating sites. Increasing the label density of the probe increases non-specific binding, increases background, and ultimately prevents the probe from hybridizing to its target due to haptene base pairing interference. Indirectly labeled probes are therefore not suitable for clinical diagnosis due to their inaccuracy and false positive results.
[0006]
Probe hybridization to specific nucleic acid sequences has been detected with intercalating agents such as acridine orange or ethidium bromide as described in US Pat. No. 4,563,417 by Albarella et al. The intercalating agent becomes inserted between the hybridized base pairs of the probe and the sample nucleic acid, and the tertiary structure of the helix is solved. Specific antibodies against the newly formed antigenic determinant created by the intercalating agent and unraveled helix are detected by conventional means. This method lacks selectivity for the target hybrid because the intercalating agent cannot recognize specific sequences. Furthermore, the antibody recognizes only the intercalating agent / nucleic acid complex and does not detect specific sequences. Thus, additional selection or purification steps are required to prevent non-specific signals, making this time consuming and laborious method unsuitable for clinical diagnosis.
[0007]
Hybridization of a probe to a specific nucleic acid sequence could also be detected with the aid of an antibody that is specific for a labeled probe as described in US Pat. No. 4,743,535 by Carrico . The probe is labeled with a detectable substance such as flavin adenine dinucleotide (FAD) or a fluorescent agent. After hybridizing to a specific nucleic acid sequence, an antibody specific for the labeled probe is detected by a biochemical reaction. This detection method also has the potential for non-specific binding and false positive results and is not well suited for clinical screening.
[0008]
Attempts have been made to increase the sensitivity of nucleic acid assays by target amplification. Methods for amplifying nucleic acid sequences are commercially available. These methods include polymerase chain reaction (PCR), ligated amplification reaction (LCR) and transcription based amplification reaction (TMA). PCR technology is described by Miehael A. et al. Innis, David H. Gelfand, John J .; Sninsky and Thomas J. et al. By WhitePCR Protocols A Guide to Methods and Applications, Pp. 39-45 and 337-385 (Academic Press, Inc., Harcourt Brace Jovanovich, Publishers, 1990). PCR technology is also described in MarxJ. L. by,Science  140: 1408-1410 (1988) and U.S. Pat. Nos. 4,683.195 and 4,683,202 to Mullis. Ligation amplification reactions are described in Wu, D. et al. Y. and Wallace, R.A. B,Genomics  4: 560-569 (1989) and Barringer, K .; J. et al. , Et al. ,Gene  89: 117-122 (1990). Transcription-based amplification reactions are described in Kwoh, D .; Y. , Et al. ,Proc. Natl. Acad. Sci. USA  86: 1173-1177 (1989). Although these methods have the advantage of high sensitivity, sample preparation is lengthy, open and expensive, tends to show false positives resulting from reaction product contaminants, and the initial amount of target nucleic acid Has the disadvantage that it cannot be accurately quantified. Amplification reaction products are most often detected by hybridization assays.
[0009]
The degree of sensitivity achieved in assays for detecting nucleic acid molecules (RNA or DNA) in a sample is generally lower for RNA than for DNA because RNA is not degraded by endogenous RNAase in the sample. This is because it is easier to receive and less RNA is available for detection. In addition, it is difficult to remove background interference caused by contaminants in the sample without causing further degradation of the target nucleic acid, such as RNA.
[0010]
  Hybridization assays for detection of nucleic acid molecules (ie RNA) have been developed. For example, hybridization protection assays for RNA are commercially available from Gen-Probe Inc. (San Diego, Calif.). Hybridization protection assays are described in Engleberg, N.C.,ASM News 57: 183-186 (1991), Amold et a1.Clin. Chem. 35: 1588-1594 (1989) and US Pat. No. 4,851,330, using single stranded nucleic acid probes linked to acridinium esters. Hybridization of the probe to the target RNA molecule protects the acridinium ester bond from thermal hydrolysis, so that the detected chemiluminescent signal is proportional to the amount of target RNA in the sample. The sensitivity of this protection assay is limited by the background luminescence caused by the unhybridized probe.
[0011]
Polyclonal and monoclonal antibodies and other similar entities are commonly used for detection purposes. In particular, polyclonal antibodies recognize multiple epitopes, whereas monoclonal antibodies recognize only one specific epitope. Monoclonal antibodies that detect RNA: DNA hybrids are currently available. Polyclonal antibodies that detect RNA: DNA hybrids have been produced, however, in general, they are not as specific as monoclonal antibodies that are designed to bind to specific epitopes.
[0012]
Monoclonal antibodies against RNA: DNA hybrids are currently available. U.S. Pat. No. 4,732,847 to Stuart and Stuart et al.Proc. Natl. Acad. Sci. USA  78: 375 1 (1981) describes a method for detecting hybridization of specific nucleic acid sequences on a solid surface containing monoclonal antibodies specific for poly (A) -poly (dT) duplexes. Is described. According to Stuart, annealing of DNA or RNA complementary to the sequence in question forms an RNA: DNA hybrid. Stuart is particularly opposed to the use of polyclonal antibodies because polyclonal antibodies cannot exclude significant binding to single or double stranded nucleic acids. Further, unlike the invention described herein, Stuart does not contemplate the advantages of polyclonal antibodies for very short oligomer arrays on glass or silicon chips. In addition, Stuart does not contemplate microarrays on glass slides or silicon chips, especially high density arrays. Stuart does not disclose binding nucleic acid probes to a solid surface. Instead, Stuart immobilizes the sample polynucleotide on the surface, while the probe (eg, a pre-determined nucleotide sequence) is in the liquid phase. In view of the foregoing, the present invention provides significant benefits and advantages in the field.
[0013]
Boguslawski et al. ,J. et al. Immunol. Methods  89: 123-130 (1986) developed a hybridization assay using isolated anti-hybrid coated polystyrene beads on filters in an attempt to reduce non-specific binding and avoid complex washing methods. A monoclonal antibody specific for the RNA: DNA hybrid secreted by the hybridoma HB8730 is disclosed by Carrico et al. In US Pat. No. 4,833,084. In the Carrico patent, RNA: DNA hybrids formed by specific reannealing of probe polynucleotides and sequences of interest can be detected sensitively and specifically by binding to monoclonal antibodies.
[0014]
By microarray is meant a regular arrangement of discrete biological molecules, including RNA, DNA, proteins, etc., arrayed or immobilized on a solid phase substrate. These microarrays of binders, such as oligonucleotides and probes, have become increasingly important tools in the biotechnology industry and related fields. Microarrays consisting of a large number of binding agents are immobilized on the surface of a solid support in a regular manner or pattern and can be used in a variety of applications including drug screening, nucleic acid screening, mutation analysis, etc. Have been found. Elements as used herein in connection with microarrays include hybridizable nucleic acid sequences, oligonucleotides, primers, probes, and the like, arranged in a distinct and identifiable manner on the surface of the substrate. And / or amino acids apply. Detection of biological molecules through the use of microarrays analyzes large numbers of samples and biological molecules, reducing the amount of sample required for analysis, reducing experimental variability, and reducing sample preparation time There are benefits to letting you verify the results and reduce the cost of such analysis.
[0015]
Currently, the primary use of microarrays is to measure gene expression in biological samples. Gene expression measurements include the detection of the presence or absence of mRNA, or the measurement of an increase or decrease in mRNA concentration. However, in order to detect hybridization in the usual way and to measure gene expression, the sample must first be purified and labeled. Common techniques for purifying and labeling samples are: 1) RNA amplification, labeling and hybridization, and 2) cDNA labeling and hybridization. The amplified portion of the first technique is described by Van Gelder et al. In US Pat. Nos. 5,716,785 and 5,891,636, published in 1998 and 1999, respectively. Highly purified total RNA or mRNA is used, which is an expensive and time consuming method. Oligo-dT primers are also used to reverse transcribe poly-A tailed mRNA into antisense single stranded cDNA. Oligo-dT further contains a sequence for T7 RNA polymerase at the 5 start end of the dT sequence. After reverse transcription, a combination of RNAase H, DNA ligase and DNA polymerase is used to generate double stranded cDNA. Since the original RT primer contained the T7 RNA polymerase promoter, the double stranded cDNA contained the complete T7 RNA promoter. The double stranded cDNA is then used as a template for T7 RNA polymerase. Approximately 100-1000 additional copies of RNA are generated from each copy of the cDNA. During the transcription process, labeled nucleotides are incorporated into the transcribed RNA. The labeled RNA is then hybridized to a DNA microarray to form a labeled RNA: DNA hybrid. The fluorescent label will be detected directly, while the indirect label will be detected after reaction with the second binding agent.
[0016]
The second sample preparation technique produces and measures labeled cDNA. In this technique, total RNA and mRNA are purified from biological samples. Oligo-dT primers are used to reverse transcribe poly-A tailed mRNA into antisense single stranded cDNA. During the reverse transcription process, labeled nucleotides are incorporated into the DNA strand to be generated. After synthesis, the RNA strand is destroyed. The labeled cDNA strand is then hybridized to the microarray. If the nucleotide is fluorescently labeled, the hybrid is directly visualized with a fluorescent array scanner. If the nucleotide is labeled with biotin, the microarray is first reacted with labeled streptavidin and then scanned.
[0017]
The disadvantages of both these techniques are several times higher. First, both require large amounts of highly purified nucleic acid (ie, RNA or DNA). Purification is time consuming and requires laborious additional steps. In addition, these techniques are inaccurate. Reverse transcription occurs at different efficiencies and kinetic rates depending on the nucleic acid sequence, artificially changing the concentration of a particular nucleic acid sequence. Prokaryotic mRNA and some eukaryotic mRNAs do not contain a poly A sequence or tail at the 3 start end, or the poly A tail is degraded during purification and thus sequences for initiating the reverse transcription step Is not present and cannot be labeled or detected with current technology. Current technology is therefore limited to sample types that can be used for detection. These methodologies also include labeled nucleotides. Incorporation of labeled nucleic acid into unlabeled nucleic acid occurs at a lower efficiency and slower rate than natural nucleotides. Once again, the label will be incorporated with different efficiencies depending on the sequence. Thus, the label density is different between different sequences and artificially changes the amount of these nucleic acids measured. Therefore, quantification is only relative. Labeled nucleic acids also exhibit hybridization kinetics that differ from natural nucleic acids and usually reduce their specificity. In addition, the method will require higher stringency hybridization conditions than unmodified nucleotides to achieve the same level of specificity. However, the use of higher stringency conditions to achieve acceptable specificity will reduce the sensitivity of detection. Thus, biological molecules (DNA) that are accurate, both time and cost efficient, and that can screen one or more sample biological molecules with higher sensitivity and minimal non-specific binding. There is a need for assays for the detection and quantitative analysis of RNA, proteins, etc.).
[0018]
Therefore, one or more biological molecules (including RNA, DNA or protein) that are easy to use for screening biological molecules, highly specific, accurate, and sensitive It would be useful to have a method for detecting and measuring (but not limited to).
[0019]
Accordingly, it is an object of the present invention to provide an assay that detects and quantifies the absence or presence of a biological molecule (including but not limited to RNA, DNA or protein).
[0020]
It is also an object of the present invention to provide a method for detecting an RNA: DNA hybrid comprising a first specific target biological molecule and a second biological probe in a sample.
[0021]
It is also an object of the present invention to provide a sensitive and quantitative assay with minimal false positives.
It is a further object of the invention to provide an assay for high volume, parallel screening.
[0022]
Summary of invention
Hybridizing a biomolecule with a complementary biomolecular probe to form a double stranded hybrid, followed by an antibody or other entity that is detectable and specifically recognizes an RNA: DNA hybrid, Assays have been disclosed for detecting and measuring biological molecules of interest, including RNA, DNA, proteins, etc., by immunological detection of these double-stranded hybrids formed on a solid phase . This method is used to detect the presence of one or more specific biological molecules present in various samples.
[0023]
The present invention provides a method for simultaneously monitoring (eg, detecting and quantifying the amount) of multiple biological molecules.
The present invention relates to assays for detecting RNA: DNA hybrids that use detectably labeled entities that are specific for recognizing RNA: DNA hybrids. Preferably, the entity is a detectably labeled RNA: DNA hybrid-specific antibody or fragment thereof. The antibody used to detect the RNA: DNA hybrid may be monoclonal or polyclonal, but is preferably polyclonal for the detection of short biological molecular probes having a length of less than 30 bases. .
[0024]
The invention also relates to assays that use the microarrays of the invention to determine physiological responses such as by gene expression, polymorphic mutation detection, or SNP analysis. The method is used to detect any and all genotypic variations, including insertion or deletion mutations.
[0025]
The present invention further relates to assays that utilize reverse transcription to extend short biological molecules, thereby facilitating detection of RNA: DNA hybrids. Preferably, the reverse transcriptase is thermostable and lacks RNAase H function.
[0026]
The invention further relates to a kit for detecting and quantifying biological molecules, wherein the kit is used to screen a sample with a number of targets described herein according to the invention.
Detailed Description of the Invention
Assays and kits for the detection and quantification of one or more target biomolecules in one or more samples are provided. In general, but not limited to, test samples consisting of biological molecules such as RNA, DNA, proteins, etc. are collected and hybridized directly or indirectly to nucleic acid probes specific to the target biological molecule bound to a solid phase. Soybeans. Non-hybridized nucleic acid sequences are preferably removed by washing. Hybridization is then detected by reaction with an RNA: DNA hybrid antibody labeled directly or indirectly with a detectable label and / or by a labeled nucleic acid sequence complementary to the bound nucleic acid probe sequence. Detected.
[0027]
In one embodiment of the present invention, preferably using a oligonucleotide or other nucleic acid spotted or bound to a solid phase, a specific nucleic acid of a sample is hybridized to a complementary nucleic acid probe to form a double-stranded RNA. : Forms a DNA hybrid. Any entity that specifically recognizes RNA: DNA hybrids, preferably antibodies or fragments thereof specific for RNA: DNA hybrids, may be used for detection and measurement.
[0028]
The present invention also utilizes short biomolecules, preferably primers or probes immobilized on a solid phase. Preferably, the primer is extended using a reverse transcriptase lacking RNAseH function, and an entity that specifically binds to an RNA: DNA hybrid specific antibody, RNA: DNA hybrid antibody fragment, or RNA: DNA hybrid is more effective. It would be desirable to be able to bind and be detected.
[0029]
A further aspect of the invention encompasses three biomolecules, all of which are preferably nucleic acids. A first sample biomolecule hybridizes to a complementary second biomolecule, preferably a probe, and simultaneously or sequentially, a second nucleic acid hybridizes to a third nucleic acid, wherein the nucleic acid One of them is immobilized on a solid phase, and the formed RNA: DNA hybrid is detected by an entity specific for the RNA: DNA hybrid.
[0030]
In a further aspect of the invention, an immobilized biomolecule, preferably consisting of a protein, binds to a sample biomolecule, preferably a nucleic acid, and is specific for an RNA: DNA hybrid if the nucleic acid is an RNA: DNA hybrid. It may be detected by a typical existence. For example, DNA may bind to the DNA binding site of the immobilized protein, where the DNA portion of the protein-DNA complex may be hybridized to RNA. In a similar manner, the immobilized protein may bind to RNA, where the RNA portion of the protein-RNA complex may bind to DNA. The resulting RNA: DNA hybrid may be detected by an entity specific for the RNA: DNA hybrid, such as an RNA: DNA hybrid specific antibody or fragment thereof.
[0031]
The present invention provides a significant effect on the art in the use of microarrays. Because either crude or purified samples may be used, the sample preparation method of the present invention is simplified and allows more accurate detection and measurement of biomolecules. Also, the biological sample need not be directly labeled for detection and measurement, avoiding the interference attributed to the label. The present invention provides a very sensitive method for detecting and measuring biomolecules, since very high label densities may be obtained by utilizing entities that bind to RNA: DNA hybrids. Such sensitive sensitivity reduces the amount of sample required for analysis. Unlike other methods, the present invention will measure prokaryotic and some eukaryotic mRNAs that lack a poly A tail or have been degraded after purification.
[0032]
Another advantage of the present invention is that reverse transcriptase is not required, but may be used if desired to increase sensitivity. One of the most effective aspects of the present invention is the direct quantification of biomolecules. Unlike other conventional techniques that quantify RNA only relatively, such as the two-color comparison method, the present invention utilizes a direct approach to interpret the results and a simplified analysis of biomolecules. In addition, the present invention allows simultaneous analysis of multiple biomolecules due to its simplified sampling method. Thus, the present invention allows for a more direct interpretation and simplification of the results.
[0033]
As used herein, a “probe” or “nucleic acid probe” as used herein is defined as a collection of one or more nucleic acids or nucleic acid-like fragments that will be detected for hybridization to a second nucleic acid. Is done. This probe will be unlabeled or labeled as described below and binding to the second nucleic acid will be detected. Probes may be made from nucleic acid sources from one or more specific parts of the genome, which may be well known or unknown, such as one or more clones, isolated whole chromosomes or chromosome fragments, polymerase chain reaction (PCR) ) A collection of amplification products, or a synthetic nucleic acid or PNA molecule. Or the probe may consist of random, semi-random, or targeted sequences. The probe may be processed in some way, for example by repeated blocking or removal of nucleic acids, or by addition of specific nucleic acids. Thus, the term “probe” as used herein refers not only to a detectable nucleic acid, but also to a detectable nucleic acid that is in a form that is applied to a target, eg, by blocking the nucleic acid. Used as a thing. This blocking nucleic acid will also be mentioned separately. What the “probe” specifically points to is clear from the context in which this word is used. In the context of use as a starting point for polymerization, ie for transcription or replication, the probe will also function as a primer.
[0034]
The probe may be an isolated nucleic acid immobilized on a solid surface. In some embodiments, the probe may be a member of a microarray of nucleic acids as described, for example, in WO 96/17958. Techniques that can produce high density microarrays are also used for this purpose (see Foder et al. Science 767-773 (1991) and US Pat. No. 5,143,854, Pirrung, M.C.). The probe may be deposited as an element on the reaction substrate to interrogate the target molecule and may be directly or indirectly labeled.
[0035]
The disclosed assay of the present invention may be used to detect and quantify any biomolecule or combination of biomolecules in a sample, where “biological molecules” used interchangeably herein. "And" biomolecule "refer to nucleic acids, amino acids, analogs, peptides, antibodies, and the like, as defined herein. “Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof, including but not limited to those derived from cells or tissues of synthesis or higher organisms such as bacteria, yeast, viruses, and plants and animals, It may be from any source and, unless otherwise limited, may include known analogs of natural nucleotides that will function in a manner similar to naturally occurring nucleotides. Peptide nucleic acids (PNAs) are also encompassed within the term nucleic acid.
[0036]
Further, herein, “nucleic acid” is defined as a single or double stranded nucleic acid ranging from 2 to about 10,000 bases in length. As used herein, “nucleic acid” refers to oligonucleotide, cDNA, mRNA, amplicon, plasmid, and the like. An “oligonucleotide” is one desired nucleic acid probe, consisting of at least 6 to about 60 bases, preferably about 15 to 30 bases, more preferably about 20 to 25 bases, and is suitable for PCR amplification, hybridization assays, and microarrays. May be used. As used herein, an oligonucleotide is substantially equivalent to the terms “amplimer” and “oligomer” as conventionally defined in the art, and as used herein “primer” and Will be used as a “probe”.
[0037]
Also, unless otherwise stated, this term includes well-known analogs of natural nucleotides that have similar binding properties as standard nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Includes nucleic acids. In addition, a particular nucleic acid sequence implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions) and complementary sequences as well as explicitly indicated sequences.
[0038]
Nucleic acid sequences for detection, referred to herein as nucleic acid molecules of interest or target nucleic acid molecules, are selected based on the need and purpose of detection. The In general, the nucleic acid molecules of interest may be selected based on well-known measures for selection of nucleic acid sequences for detection. For example, certain nucleic acid molecules may be associated with pathogens, disease states or predispositions, and detection of such nucleic acid molecules will have diagnostic value. For example, mRNA specific for cancer cells or normal cells will be detected. Further, by the methods disclosed herein, but not limited to, biological molecules consisting of proteins, peptides, primers, and DNA or RNA molecules, produced by other biological or chemical methods (CAR, NASBA It also enables detection of molecules (such as those produced by Nucleic acid detection also includes mutations, deletions, single nucleotide polymorphism insertions, and other polymorphism detections.
[0039]
“Sample” or “target sample” is used interchangeably herein, but is defined in the broadest sense and includes, but is not limited to, nucleic acids, amino acids, proteins, peptides, etc. Includes both biological material and synthetic material of biological molecules, including total genomic DNA, total RNA, eg genomic DNA or mRNA from chromosomes, or selected sequences within specific amplicons or deletions (eg specific A promoter, gene, amplification or restriction fragment, cDNA, etc.). One embodiment of the present invention is to detect either the presence or absence of a target nucleic acid sample and measure the amount of sample to be quantified. The term “target nucleic acid” refers to a specific subsequence of the larger nucleic acid to which the probe is directed, or the entire sequence (eg, gene or mRNA) at a level for detection, quantification, and presence or absence. You may point to what is needed. The difference in its use will become clear from the context.
[0040]
Biomolecular samples may be extracted from specific cells or tissues. Tissue samples from which biomolecules are prepared are typically taken from patients suspected of suffering from a disease associated with the detected amplification or deletion. In some cases, biological molecules, such as nucleic acids, may be amplified prior to hybridization using standard techniques such as PCR. Such special use of the term “nucleic acid sample” will be readily apparent to those skilled in the art from the context in which the term is used. For example, the nucleic acid sample may be a tissue extract or cell lysate sample prepared by methods well known to those skilled in the art. The sample is prepared such that the biological molecule of interest is released from the cell and is available for hybridization.
[0041]
Also, the sample for the methods disclosed in the present invention may be from any source that contains or will contain nucleic acids. The source of the nucleic acid may be in purified or unpurified form. A preferred type of sample or sample source suitable for use in the disclosed methods is a sample as known or identified as a sample suitable for use in other methods of nucleic acid detection. Many such samples are known. For example, the sample may be from an agricultural or food product, or may be a human or veterinary clinical specimen. The sample may be a biological fluid such as plasma, serum, blood, urine, sputum, cell lysate and the like. Samples will include bacteria, yeast, viruses and cells or tissues of higher organisms such as plants and animals that are likely to have the biological molecule of interest. Methods for extraction and / or purification of nucleic acids, eg RNA, are described by Maniatis et al., Molecular Cloning: A Laboratory Manual (New York, Cold Spring Harbor Laboratory, 1982).
[0042]
Since the sample may be in a crude or unpurified state, sample preparation or processing is simplified. Accurate expression detection and quantification is achieved by using a sample that is in a more natural state. Furthermore, unlike other techniques that require the presence of a poly A sequence to initiate the reverse transcriptase process to label and detect the sample, the present invention can be used to detect prokaryotic mRNA and poly A tails. 'Eukaryotic mRNA without end can be measured.
[0043]
The target biological molecule used in the disclosed methods can be of various origins, and can be either natural or synthetic. For example, various RNAs include messenger RNA, ribosomal RNA, nucleolar RNA, transfer RNA, viral RNA and heteronuclear RNA, total genomic DNA, cDNA, protein, peptide, and the like. Furthermore, the whole natural substance or a fragment thereof may be used.
[0044]
As solid phase or solid support, natural fibers such as plastic, resin, polysaccharide, silica or silica-based material, functionalized glass, modified silicon, carbon, metal, inorganic glass, membrane, nylon, silk, wool and cotton As well as polymers, but is not limited thereto. The solid phase or solid support may be either porous or nonporous. In some embodiments, the material comprising the solid support has reactive groups such as carboxy, amino, hydroxy, etc., and these reactive groups are used for covalent or non-covalent bonding of the probe. Suitable polymers include polystyrene, polyethylene glycol terephthalate, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone, polyacrylonitrile, polymethyl methacrylate, polytetrafluoroethylene, butyl rubber, styrene butadiene rubber, natural rubber, polyethylene, polypropylene, ( Examples include, but are not limited to, poly) tetrafluoroethylene, (poly) vinylidene fluoride, polycarbonate, and polymethylpentene. Suitable polymers include those outlined in US Pat. No. 5,427,779 (Elsner, H. et al., Which is hereby incorporated by reference). Solid phases and solid supports include, but are not limited to, any solid material that can bind or attach probes, primers, oligonucleotides, proteins, peptides, and the like. The solid phase and solid support can take any useful shape, such as thin films or membranes, beads, bottles, microwell plates, petri dishes, glass slides, fibers, woven fabrics, molded polymers, particles, chips and A micro particle is mentioned. Preferred substrate shapes for the solid phase are microtiter petri dishes, silicon chips, glass slides, and tagged beads.
[0045]
In normal applications where the molecule is covalently bonded to the solid substrate surface, the surface may be activated using a variety of reactive functional groups depending on the nature of the binding component and the nature of the solid substrate surface. Therefore, if necessary, a functional group may be introduced to modify the surface of the solid phase substrate, and then the functional group may be reacted with the binding component.
[0046]
A “microarray” comprises a plurality of different biological molecules such as cDNA, amplicon, plasmid, protein, peptide, etc. The plurality means at least two different biological molecules, The target molecules are immobilized on the solid phase in a matrix or structure. Theoretically, only one component may be required, but in preferred embodiments it will be at least 10, more typically at least 20, often at least 50, preferably more than 100, even more than 1,000, but generally Is about 10^FourHereinafter, more generally, there are about 100,000 or less components, and preferably about 10 to 10,000 components are immobilized on a solid phase or a solid support. Theoretically, the number of different components is 10 because it can specifically have a small amount in a limited area of designation.^FiveHowever, it is generally not necessary to exceed 100,000, and such a large number of different components only complicates the preparation of the microarray. The number of components immobilized on the solid phase is usually 10^FiveEach addressable depending on the nature of the binding component, the source of the signal, the nature of the signal to be detected, the sensitivity of the signal detection, the nature of the microarray (microarray size, etc.), the microarray manufacturing method, etc. The number of sites can increase substantially. Thus, microarrays are preferably used for “large-scale parallel screening”, as used herein to simultaneously screen for hybridization of at least 10, preferably about 1,000, more preferably about 10,000 different biological molecules. Describe.
[0047]
A preferred form of microarray includes 1-10, 10-100, most preferably over 100 different nucleic acids (preferably oligonucleotides, primers, etc.), as described herein, in small dots or elements. Arrays include deposited, spot or synthesized spot-like arrays. These nucleic acids deposited, spotted or synthesized on the solid phase are referred to as “elements” in the present invention. Typically, the element is less than about 1 mm in diameter. Usually, the size of the element is from 1 μm to about 5 mm, preferably from about 1 μm to about 1 mm. Nucleic acid primers used in the disclosed method can be synthesized using pre-made oligonucleotide synthesis methods. Such methods include standard enzymatic digestion followed by isolation of nucleotide fragments (eg, Sambrook et al., Molecular cloning: A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) (see Chapters 5 and 6)) using a Milligen or Beckman System 1 Plus DNA synthesizer (eg, model 8700 automated synthesizer or ABI model 380B from Milligen-Biosearch, Burlington, MA). There are various methods up to a simple synthesis method such as a phosphoramidite method. Synthetic methods useful for making oligonucleotides include Ikuta et al. (Ann. Rev. Biochem. 53: 323-356 (1984); phosphotriester and phosphite triester methods) and Narang et al. (Methods Enzymol , 65: 610-620 (1980); phosphotriester method).
[0048]
Another shape of the microarray is a three-dimensional array, for example, an array of color coded beads (Luminex; Austin, TX) and an array of beads with high frequency tags (PharmaSeq; Monmouth Junction, NJ). The three-dimensional microarray used in the present invention is an arbitrary solid phase having three dimensions, and each microarray has a plurality of different biological molecules (preferably nucleic acid primers) bound to the surface. Therefore, each nucleic acid primer sequence can be identified from the position of each primer on the solid phase microarray. The disclosed assay procedure may be utilized. For example, a three-dimensional microarray including a plurality of nucleic acid primers may be mixed with the target nucleic acid. If the primers are short, these molecules can be extended with a polymerase (e.g., reverse transcriptase, etc.) to produce an RNA: DNA hybrid specific material (e.g., antibodies and antibody fragments, etc.) as described herein. ) May be desirable. By capturing the antibody on the solid phase, the primer of the solid phase microarray in which the RNA: DNA hybrid is formed can be separated from the primer in which the hybrid is not formed. Subsequently, a substance specific to the RNA: DNA hybrid may be detected to identify the primer. Many other assay schemes can also be used in the disclosed methods.
[0049]
Microarrays have been proposed as a format suitable for miniaturization of assays that detect and measure RNA, DNA, proteins, etc., for example in applications to gene expression, mutation and polymorphism analysis, SNP, detection of genetic variation, etc. It is a thing. Microarrays allow levels of tens to thousands of genes or genetic variations (eg, SNPs) to be measured from a single sample on a single device. A disadvantage of conventional microarray methods is that the biological molecule to be measured (preferably a nucleic acid (RNA or DNA)) must first be labeled, and this step can be performed by one nucleic acid being another nucleic acid (e.g. This is often done by converting RNA into labeled DNA), which allows biological molecules to be detected and measured.
[0050]
Preferably, in the present invention, a “nucleic acid” in which a plurality of nucleic acid sequences such as (but not limited to) DNA, RNA, amplicon, plasmid, etc., as defined herein, are immobilized on a solid support. A “microarray” is used to hybridize complementary target nucleic acids. Microarray nucleic acids include, for example, specific genes or clone-derived sequences, probes, primers, or oligonucleotides bound to a porous or nonporous solid phase or solid support. Various sizes of nucleic acids can be used in the microarrays of the present invention.
[0051]
The nucleic acid can be bound to a solid support or substrate. Such a microarray is a solid support on which a plurality of different nucleic acids are bound or attached in an array, grid, or other organized pattern. A “nucleic acid microarray” preferably comprises an array of nucleic acid sequence strands arranged on a silicon chip, glass slide or other solid support for detection and measurement such as gene expression, mutation and polymorphism analysis. Have a wide range of uses. Several methods are available for preparing nucleic acid microarrays. The nucleic acid sequence strand can be non-covalently or covalently bound to the solid phase substrate by passive or chemical coupling methods. Another approach utilizes synthetic methods to construct nucleic acid molecules directly on the substrate surface. A simpler but more restrictive approach is to prepare a labeled nucleic acid sequence and then bind the labeled nucleic acid sequence to a substrate previously coated with a binding partner.
[0052]
Alternatively, protein and / or peptide elements can be bound to a solid support or substrate in an organized pattern. A nucleic acid, protein, peptide, and / or nucleic acid hybrid may be bound to such an immobilized protein element. Detection is performed using a substance (for example, antibody or antibody fragment) specific to RNA: DNA hybrid. When the immobilized protein or peptide binds to the RNA: DNA hybrid, the RNA: DNA hybrid portion of the protein-hybrid complex can be detected using a substance specific for the RNA: DNA hybrid. When the immobilized protein binds to DNA, the DNA portion of the protein-DNA complex can be hybridized to RNA to form an RNA: DNA hybrid. When the immobilized protein binds to RNA, the RNA portion of the protein-RNA complex may be hybridized to DNA to form an RNA: DNA hybrid. RNA: DNA hybrids can be detected using substances specific for RNA: DNA hybrids (eg, RNA: DNA hybrid specific antibodies or fragments thereof).
[0053]
A “hybrid” is a double-stranded nucleic acid consisting of RNA or DNA. The double helix is either DNA: DNA, RNA: RNA, or RNA: DNA, and may contain artificial nucleotides. An RNA homoduplex is a base-paired double-stranded RNA. An RNA: DNA heteroduplex consists of an RNA strand and a strand containing DNA nucleotide monomers. The double helix may be double-stranded as a whole or a part of it may be double-stranded. Typically, at least 10 bases of the double helix are double stranded. Expressions such as `` specifically hybridize '' or `` specific hybridization '' or `` selectively hybridize '' refer to nucleic acid molecules that are complex mixtures (e.g., under stringent conditions) Whole cell) Refers to preferential binding, duplexing, or hybridizing to a specific nucleotide sequence present in DNA or RNA.
[0054]
The nucleic acid probe immobilized on the solid phase substrate can localize the formation of RNA: DNA hybrids on the substrate. Such localization provides a convenient means of washing away reaction components that may interfere with subsequent detection steps and a convenient method of simultaneously assaying multiple different target nucleic acid sequences. RNA: DNA hybrids can be formed separately at each site where different primers are attached. To immobilize the probe to form a solid phase microarray of biological molecules, the methods described herein may be used.
[0055]
“Entity” as defined herein refers to any molecule that specifically recognizes an RNA: DNA hybrid. Examples of substances that can recognize RNA: DNA hybrids include, but are not limited to, chimeric antibodies, natural or genetically engineered proteins or nucleic acids that specifically bind to RNA: DNA hybrids.
[0056]
One preferred embodiment of the substance is an “antibody”. In the present invention, antibodies are used in the broadest sense, and whole intact antibodies, antibody fragments, recombinant antibodies, chimeric antibodies, multifunctional antibody aggregates, or antibody binding sites having the characteristics described herein In general, any antibody derivative containing at least one or other substance. Preferably, in the present invention, these substances are those that specifically detect and specifically bind RNA: DNA hybrids. Antibodies of any known class and subclass of immunoglobulins (e.g. IgG, IgM etc.) and active fragments such as IgG fragments known as Fab, F (ab ') and F (ab') 2 Is intended. Antibodies may include monoclonal antibodies (including agonists, antagonists, and neutralizing antibodies) that bind to specific epitopes as well as polyclonal antibodies with polyepitope specificity, or other substances.
[0057]
Any antibody or substance specific for double stranded RNA: DNA hybrids may be used to directly detect the hybrids of the invention. In the present invention, polyclonal antibodies are preferred in embodiments where antibodies are used to detect short nucleic acid sequences, preferably nucleic acid sequences of less than 30 bases in length.
[0058]
The antibody used to detect the RNA: DNA hybrid may be either a monoclonal antibody or a polyclonal antibody. It may be advantageous to use a mixture of monoclonal and polyclonal antibodies. Furthermore, the present invention encompasses the use of polyclonal or monoclonal antibodies that are specifically designed to have specific binding properties. For example, if a monoclonal or polyclonal antibody that specifically binds to a very short RNA chain (less than 20 base pairs) is prepared, it can be used to detect a very short RNA: DNA hybrid. In addition, monoclonal or polyclonal antibodies that are sensitive or insensitive to mismatches in RNA: DNA hybrids may be generated. Antibodies that are sensitive to mismatches in RNA: DNA hybrids have particular utility in detecting genetic variation, while antibodies that are less sensitive to mismatches in RNA: DNA hybrids are identified Used for the detection and quantification of this class of nucleic acids. Other antibodies that specifically detect nucleic acid triple helices (DNA: RNA: DNA or RNA: DNA: RNA) or DNA: PNA or RNA: PNA hybrids (where PNA is defined as peptide nucleic acid in the present invention) It can be used.
[0059]
Polyclonal antibodies to RNA: DNA hybrids can be obtained by injecting an appropriate laboratory animal with an effective amount of a peptide or antigen component, collecting serum from the animal, and isolating specific serum by any of the known immunoadsorption methods. Prepare. Animals that can be readily used to make polyclonal RNA: DNA hybrid antibodies include chickens, mice, rabbits, rats, goats, horses and the like. In a preferred embodiment of the assay of the present invention, polyclonal RNA: DNA hybrid antibodies are derived from goats immunized with RNA: DNA hybrids. Hybrid specific antibodies are purified from goat serum by affinity purification on RNA: DNA hybrids immobilized on a solid support.
[0060]
A monoclonal antibody prepared by a conventional method may be used in place of the polyclonal antibody. A variety of methods can be used to obtain appropriate antibodies specific for RNA: DNA hybrids (e.g., U.S. Pat.Nos. 4,833,084 (Carrico), 4,732,847 (Stuart et al.) And Stuart et al). , Proc. Natl. Acad. Sci. USA 78: 3751 (1981)). Monoclonal antibodies specific for RNA: DNA hybrids secreted by the hybridoma HB8730 are disclosed in US Pat. No. 4,833,084 (Carrico). Preferably, according to the present invention, monoclonal antibodies are used to detect nucleic acids longer than 30 bases.
[0061]
Isolation of anti-RNA: DNA hybridomas has improved assay development for genetic mutations associated with specific defects and detection of bacterial and viral infections. However, assays using such RNA: DNA hybrid specific monoclonal antibodies often result in high levels of non-specific binding leading to false positive results. Boguslawski et al., J. Immunol. Methods 89: 123-130 (1986) uses anti-hybrid coated polystyrene beads separated on filter paper to reduce non-specific binding and avoid cumbersome washing procedures. Hybridization assays have been developed for use.
[0062]
  Preferred antibodies to RNA: DNA hybrids were issued by the method of Kitawaga, Y. and Stollar, BD (Mol. Lmmunology 19: 413-420 (1982)) or on March 22, 1988 to Stuart et al. According to the method described in US Pat. No. 4,732,847Be prepared.
[0063]
Identification of the presence of hybrids can be accomplished by using either polyclonal or monoclonal antibodies, or using other units specific for RNA: DNA hybrid complexes. Detection can be accomplished by labeling any of the antibodies specific for the hybrid RNA: DNA complex or by using a labeled antibody that binds to the anticomplex. For example, if the antibody is derived from a mouse, any anti-complex (eg, an antibody against the murine antibody, eg, rabbit anti- (mouse IgG)) bound to the complex that binds to the solid support. anticomplex) can be combined.
[0064]
A wide variety of labels were used in other environments where they could be applied here. One of the more common labels is a radionuclide that can be used by autoradiography to visualize the binding region. Another label is a fluorescent agent such as fluorescein, mercocyanin, or rhodamine, and the presence of fluorescence can be monitored by irradiating it with excitation light. Alternatively, an enzyme can be used that results in a product that can be detected and localized in the region of the enzyme. Detection can be performed using multiple dyes or metals that can be reduced. Common enzymes include horseradish peroxidase, glucose oxidase, galactosidase, alkaline phosphatase and the like. The method of observing a particular label or detectable signal is not essential to the present invention. By using antibodies against the anticomplex, a number of labels associated with specific binding of the anticomplex to the complex can be greatly amplified.
[0065]
In order to facilitate detection of the resulting binding of antibodies to hybrids or other units specific to double-stranded hybrids to hybrids, the antibodies are usually labeled with detectable chemical groups. Can do. Examples of detectable chemical groups that can function as labels include enzymatically active groups such as coenzymes, enzyme substrates, enzyme inhibitors and enzymes themselves, fluorescent agents, chromophores, luminescers A specifically bindable ligand, such as biotin or a hapten, detectable by the binding of labeled avidin or labeled hapten, and a radioisotope.
[0066]
In order for complete hybridization to occur, optimal conditions are required to form a double-stranded hybrid. The term “stringent conditions” refers to conditions under which a probe can selectively hybridize to a complementary sequence but is weaker or not hybridize to other sequences. Complementarity between two single stranded molecules may be “partial”, in which case only some of the nucleic acids bind, or total complementarity exists between single stranded molecules In some cases it may be complete. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important during amplification reactions that rely on binding between nucleic acid strands and in the design and use of PNA molecules. For example, in the context of nucleic acid hybridization experiments such as Southern hybridization and Northern hybridization, “stringent hybridization” and “stringent hybridization wash conditions” are sequence-dependent and are subject to different environmental parameters. And different. For further guidance on nucleic acid hybridization, see Tijssen ((1993) Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acids Propbe part 1 chapter 2. “Overview of principles of hybridization and the strategy of nucleic acids propbe assays”, Elsevier , NY).
[0067]
“Substantially binding” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid, and can be applied by reducing the stringency of the hybridization medium to produce a cDNA, amplicon. Including minor mismatches that can achieve the desired detection of target polynucleotide sequences hybridized to the bound oligonucleotide sequence, including plasmids and the like.
[0068]
Hybridization of the probe nucleic acid to the nucleic acid molecule of interest can be carried out under any suitable condition, and is preferably preferred for hybridization and can be carried out under conditions that form a double-stranded hybrid. See, for example, Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989)).
[0069]
For example, in one aspect of the invention, a primer is required to initiate reverse transcription. A “primer” is defined herein as a nucleic acid molecule that can anneal to a DNA or RNA template molecule and function as a starting point for nucleic acid synthesis. Custom primers are generally synthetic oligonucleotides, including cDNAs, amplicons, plasmids, etc., but naturally occurring nucleotides also function as primers both in vitro and in vivo. In vitro use of primers includes, for example, DNA synthesis, Sanger dideoxy sequencing, and PCR.
[0070]
This particular embodiment requires a nucleic acid “template” of interest in order to identify the target nucleic acid of interest once a target nucleic acid sample has been obtained. Here, as used interchangeably herein, a “nucleic acid template” or “template” is defined as a polynucleotide sequence from which information is read to directly synthesize another macromolecule. For example, this may refer to a DNA strand that is copied during DNA synthesis or RNA transcription, or an RNA strand that is copied during reverse transcription.
[0071]
The primer of the disclosed method may be an oligonucleotide, cDNA, amplicon, plasmid, or the like, and may be either RNA or DNA having a sequence complementary to a region on the target nucleic acid molecule. As used herein, the complementary sequence of a primer refers to the “complementary portion”. As used herein, a region on a target nucleic acid molecule of interest that is complementary to a primer is referred to as a “primer complementary region”. The primer complement region of the target nucleic acid molecule of interest may be any region of the target molecule of interest. For embodiments of the assay of the invention that use reverse transcriptase, a preferred format includes a primer complement region of the target nucleic acid molecule that is somewhat away from the 5 ′ end of the template nucleic acid molecule. This obtains a longer region of the nucleic acid template between the primer hybridization site and the end of the template nucleic acid molecule, thereby amplifying the amount of RNA: DNA hybrid to be detected.
[0072]
In general, the primer complement region of the nucleic acid molecule of interest is selected based on a known classification for selecting a nucleic acid sequence for detection. For example, in order to detect a particular nucleic acid molecule from among other nucleic acid molecules, it is preferred that the primer complement region be characteristic of or unique to the target nucleic acid molecule of interest. Where it is desired that any class of RNA molecule be detected, the primer complement region is preferably selected to have a sequence that is identical or substantially identical to all of the target nucleic acid molecules of interest. Once the primer complement region is selected, the primer sequence is designed or selected to be complementary to the selected primer complement region of the molecule of interest. Any nucleic acid molecule whose sequence is known or from which the sequence can be derived can be detected using the disclosed methods.
[0073]
In the method of the present invention, the complementary portion of the primer has a length that supports specific and stable hybridization between the primer and the primer complement region. Generally, the primers of the present invention comprise 10 to 100 nucleotides, but preferably 15 to 30 nucleotides.
[0074]
The ability to characterize an individual by its genome depends on the genetic variability of the genetic information. A DNA sequence that encodes the required protein is well conserved across species, but does not have non-coding DNA regions or essential functions, and thus complete conservation of the nucleic acid sequence is not strongly selected There is a DNA region that encodes this part. These variable regions are identified by genetic markers. Typically, genetic markers are bound by probes such as oligonucleotides or amplicons that specifically bind to unique variable regions of the genome. In some cases, the presence or absence of binding to a genetic marker identifies an individual by the individual's unique nucleic acid sequence. In other cases, the marker binds to the nucleic acid sequence of all individuals, but the individual is identified by the location in the genome that is bound by the marker probe. Major causes of genetic variability are additions, deletions, or point mutations, recombination, and transposable components in the individual's genome in plant populations. The present invention can be used for detection and measurement of genotypic variation. For example, polymorphisms such as SNPs represented by different sequences can be detected.
[0075]
In general, the assay of the present invention involves the following steps:
1. By spotting or synthesizing biomolecular probes, or biomolecular probes against a solid phase via standard chemical techniques, a solid substrate (eg, plate, slide, well, dish, bead, particle, Preparing microarrays of probes that are bound to cups, strands, chips, and strips, both porous and non-porous;
2. Add a target sample containing the first biological molecule of interest to the immobilized second biomolecular probe and form an RNA: DNA hybrid;
3. adding a detectable unit specific for RNA: DNA hybrids (including RNA: DNA hybrid-specific antibodies or fragments thereof);
4. Detect units bound to immobilized RNA: DNA hybrids.
[0076]
Another aspect of the invention involves the following steps:
1. By spotting or synthesizing biomolecular probes, or biomolecular probes against a solid phase via standard chemical techniques, a solid substrate (eg, plate, slide, well, dish, bead, particle, Preparing microarrays of probes that are bound to cups, strands, chips, and strips, both porous and non-porous;
2. Add a target sample containing the first biological molecule of interest to the immobilized second biomolecular probe and form an RNA: DNA hybrid;
3. Add reverse transcriptase, preferably lacking RNAse H function and thermostability;
4. Incubate under conditions that promote reverse transcription to extend the sequence, thus forming a very long RNA: DNA hybrid and enhancing antibody detection;
5. adding a detectable unit specific for the RNA: DNA hybrid (including RNA: DNA hybrid-specific antibody or fragment thereof);
6. Detect units bound to immobilized RNA: DNA hybrids.
[0077]
A further aspect of the invention includes the following steps:
1. By spotting or synthesizing biomolecular probes, or biomolecular probes against a solid phase via standard chemical techniques, a solid substrate (eg, plate, slide, well, dish, bead, particle, Preparing microarrays of probes that are bound to cups, strands, chips, and strips, both porous and non-porous;
2. adding a target sample containing the first biological molecule of interest to the second and third unbound biomolecular probes bound to the microarray;
3. hybridizing the first target biological molecule with the complementary region of the third biomolecular probe;
4. hybridizing the immobilized second biomolecule probe to a complementary region of the third biomolecule probe that has not hybridized;
5. adding a detectable unit specific for the RNA: DNA hybrid (including RNA: DNA hybrid-specific antibody or fragment thereof);
6. Detect units bound to immobilized RNA: DNA hybrids.
[0078]
Another embodiment of the present invention includes the following steps:
1. By spotting or synthesizing biomolecular probes, or biomolecular probes against a solid phase via standard chemical techniques, a solid substrate (eg, plate, slide, well, dish, bead, particle, Preparing microarrays of probes that are bound to cups, strands, chips, and strips, both porous and non-porous;
2. A target sample containing the first biological molecule of interest is added to a second biomolecular probe bound to the microarray and a third unbound detectably labeled biomolecular probe thing;
3. Hybridizing the first target biological molecule with the complementary region of the second solid phase-bound biomolecular probe and forming an RNA: DNA hybrid;
4. hybridizing a solid phase-bound second biomolecular probe with a complementary region of a third detectably labeled biomolecular probe;
5. adding a detectable unit specific for the RNA: DNA hybrid (including RNA: DNA hybrid-specific antibody or fragment thereof);
6. Separately detect both the RNA: DNA hybrid specific unit and the detectably labeled biomolecular probe bound to the immobilized RNA: DNA hybrid.
[0079]
The disclosed assay can be used to detect a plurality of different biological molecules of interest in a sample. Preferably, the sequence present in each of the target biological molecules of interest is screened or screened using multiple probes that are entirely complementary to a region on the biological molecule of interest This is achieved by either The latter approach detects, for example, a number of different mutations for a particular gene, or a predisposition to several diseases or disorders associated with genetic mutations, including but not limited to insertion mutations or deletion mutations Preferred for use in The present invention includes, but is not limited to, gene expression, detection of biological molecules (ie, RNA, DNA, protein) on a microarray, mutation and polymorphism detection (ie, SNP), etc. Assays that can be adapted for use are also provided. In one particular embodiment, it is preferred to screen for sequences complementary to regions of the mutant nucleic acid product of these genes having the respective characteristics of the mutation. Thus, one major advantage of this assay is high throughput application, which allows large scale screening of large numbers of samples and disease potential.
[0080]
The disclosed methods can also be used to determine the ratio of expression of different biological molecular species from individual organisms or individual samples. For this purpose, this method is used to detect multiple species simultaneously. As disclosed herein, microarray detection is useful for this purpose. The disclosed methods can also be used to detect the same or related biomolecular sequences, where related biological molecules have a common sequence motif between them but are otherwise different Is. For example, a cell can contain multiple biological molecular species that have the same regulatory sequences, the same structural motifs, or other sequences in common. Such classes of nucleic acid molecules can be detected by a single probe species by designing the probes to hybridize to a common sequence.
[0081]
In the disclosed assay, RNA: DNA hybrid-specific antibodies and fragments containing RNA: DNA hybrid specific units are used to probe bioarrays that are optional but no longer require labeling of target biomolecules. A biological molecule hybridized to it is detected. In this approach, longer RNA: DNA hybrids can bind more antibodies than short RNA: DNA hybrids, so the longer the RNA: DNA hybrid, the greater the signal. Therefore, the longer the nucleic acid probe chain on the microarray, the more sensitive the detection of the target nucleic acid, or the stronger the signal intensity for a given amount of hybridized target nucleic acid. Unfortunately, in preparing these microarrays, it becomes more difficult and more expensive to synthesize, prepare, or use longer strands of probes.
[0082]
One disclosed embodiment of the assay of the present invention describes a relatively short nucleic acid probe sequence bound to a solid substrate, thereby minimizing the time, effort, and cost required to make a microarray. The target nucleic acid sequence in the sample is hybridized to these short probes to create a short RNA: DNA hybrid with a long nucleic acid tail. This short RNA: DNA hybrid will probably only bind to one or two RNA: DNA antibodies. When reverse transcriptase is added and conditions are such that reverse transcriptase occurs, the nucleic acid probe portion of the RNA: DNA hybrid is extended to the length of the target nucleic acid strand, and thus the length of the RNA: DNA hybrid. Is greatly increased. If the target nucleic acid strand is 1500 bases in length, the resulting RNA: DNA hybrid will reach 1500 base pairs. This length of RNA: DNA hybrid binds to a large number of RNA: DNA antibodies, thereby greatly increasing the intensity of the signal produced and increasing the detection sensitivity of specific target nucleic acid sequences .
[0083]
One disclosed embodiment reverse transcribes all or part of the bound nucleic acid probe sequence using a reverse transcriptase that lacks RNA: DNA hybrid-dependent exonuclease function (also commonly referred to as RNAse H function or component). Thus, a method for detecting a target nucleic acid sequence and a method for detecting the resulting RNA: DNA hybrid using an antibody specific for the RNA: DNA hybrid. The nucleic acid probe is immobilized on the solid support in order to bind the RNA: DNA hybrid to the solid support. This allows for easy separation of the hybrid from the sample solution and specific detection of nucleic acid molecules based on the position of the hybrid on the solid support.
[0084]
In one method of the invention, reverse transcription is performed using reverse transcriptase, preferably using reverse transcriptase that lacks RNAse H function. Thereafter, a reaction mixture comprising the nucleic acid molecule of interest, preferably RNA in this embodiment; a hybridized and immobilized nucleic acid primer; and a reverse transcriptase that allows reverse transcription of the RNA molecule of interest and DNA: Incubate under conditions that allow the formation of RNA hybrids. Examples of reverse transcriptases that can be used in the disclosed methods or that can be adapted for use in the disclosed methods are listed in Table 1. Preferred reverse transcriptases for use in the present invention include reverse transcriptases 18053-017, 18064-014 and 18064-071 obtained from Life Technology; reverse transcriptases M5301 and M5302 obtained from Promega; and obtained from Strategene. Reverse transcriptase 600085; each of which is disclosed in Table 1.
[0085]
[Table 1]

[0086]
[Table 2]

[0087]
[Table 3]

[0088]
[Table 4]

[0089]
[Table 5]

[0090]
[Table 6]

[0091]
[Table 7]

[0092]
Reverse transcription may generally be performed at any temperature within the temperature range in which reverse transcriptase functions. Preferably, the incubation temperature is any temperature at which the reverse transcriptase functions and the primer remains hybridized to the target nucleic acid molecule. For non-thermophilic reverse transcriptase, the preferred temperature is the temperature at or near the optimum temperature for reverse transcriptase. For most non-thermophilic reverse transcriptases, this temperature is between about 25 ° C and 45 ° C.
[0093]
In a preferred embodiment, thermophilic reverse transcriptase is used to increase selectivity. The highest temperature at which thermophilic reverse transcriptase functions can be really high. For this reason, the preferred temperature range for reverse transcriptase when using a thermophilic reverse transcriptase is most conveniently described in terms of the calculated melting temperature of the hybrid between the RNA molecule of interest and the primer. Such melting temperature is referred to herein as RNA / primer melting temperature (R / P Tm). A preferred range is 20 ° C. below the melting temperature of the hybrid between the RNA molecule of interest and the primer, and includes 5 ° C. of the hybrid between the RNA molecule of interest and the primer. Other preferred ranges when using a thermophilic reverse transcriptase include those listed in Table 2.
[0094]
[Table 8]

[0095]
Of particular note is that each specific but unspecified range within the above listed ranges is intended as another preferred range. Preferred temperatures for reverse transcriptase are about 20 ° C. below R / P Tm, about 15 ° C. below R / P Tm, about 12 ° C. below R / P Tm, about 10 ° C. below R / P Tm, R / P Tm about 7 ° C., R / P Tm about 5 ° C., R / P Tm about 3 ° C., R / P Tm 20 ° C., R / P Tm 15 ° C. Includes 12 ° C. from P Tm, 10 ° C. from R / P Tm, 7 ° C. from R / P Tm, 5 ° C. from R / P Tm, and 3 ° C. from R / P Tm. In general, the closer the temperature is to R / P Tm, the higher the degree of discrimination between RNA and primer specific hybrids and non-specific hybrids. When the temperature is close to R / P Tm, however, the reduced stability of the specific hybrid may cause inefficiency of priming.
[0096]
R / P Tm may be measured either by calculation or by empirical measurement. Any established formula for calculating the stability of a nucleic acid hybrid may be used to calculate R / P Tm. A preferred formula for calculating R / P Tm is Tm = ΔH / ΔS + R × In (C / 4) + 16.6 log [K⁺] /1+0.7 [K⁺] -237.15, derived from a study based on the perfectly matched DNA: RNA stability. For RNA: DNA hybrids, incorporation of formamide concentration into the above formula is not applicable because the relationship between formamide concentration and Tm decline is not linear. In 80% formamide, RNA: DNA hybrids are more stable than DNA: DNA hybrids, increasing the Tm by about 10 to 30 ° C. depending on the sequence (Hames & Higgins, Nucleic Acid Hybridization: A Practical Approach (IRL Press Limited). , Oxford, England. 1985)). When the reaction is carried out in 80% formamide, the use of formamide thus suppresses DNA: DNA duplex formation, preferentially selects RNA: DNA hybrids, and estimates the Tm for R / P Also good. Since the empirically derived formula for Tm estimation of RNA: hybrid may not be accurate for short nucleic acid primers, the hybridization temperature is 0.1-0.4 M in the temperature range of 40-60 ° C. It is suitably determined by evaluating the hybrid stability in the unit price cation. R / P Tm may be determined empirically (Lesnick and Freier, Biochemistry 34: 10807-10815 (1995), McGraw et al., Biotechniques 8: 674-678 (1990), and Rychlik et al., Nucleic. Acids Res.18: 6409-6412 (1990)).
[0097]
As used herein, a thermophilic reverse transcriptase is any reverse transcriptase that retains at least 5% of its maximum activity at any temperature above 50 ° C. or has an optimum temperature of at least 50 ° C. . Preferred reverse transcriptases are those having an optimum temperature of at least 50 ° C. As used herein, the maximum activity of a reverse transcriptase is measured in the assay described below and is defined as the activity that a given reverse transcriptase exhibits at its optimal temperature. As used herein, the optimal temperature for reverse transcriptase is defined at the temperature at which reverse transcriptase activity is maximal and is measured in the assay described below. The optimal temperature for a given reverse transcriptase may be determined by measuring its activity in the following assay at various temperatures. Usually the optimum temperature needs to be measured only to the extent that the assay is only required to be performed at intervals of 5 to 10 ° C.
[0098]
A method for immobilizing a nucleic acid sequence on a solid phase substrate is well established. Oligonucleotides containing half primers and rolling circle replication primers may be coupled using established coupling methods. For example, the deposition method is described by Pease et al. , Proc. Natl. Acad. Sci. USA 91 (11): 5022-5026 (1994), and Krapko et al. Mol. Biol (Mosk) (USSR) 25: 718-730 (1991). Methods for immobilizing 3 'amine oligonucleotides are described in Stimpson et al. , Proc. Natl. Acad. Sci. USA 92: 6379-6383 (1995). A preferred method for attaching oligonucleotides to a solid support is described in Guo et al. , Nucleic Acids Res. 22: 5456-5465 (1994).
[0099]
Immobilization and arraying of nucleic acid or primer molecules to a solid support may be accomplished using any suitable technique. For example, immobilization can be performed in situ nucleic acid synthesis (Maskos and Southern, Nucleic Acids Research, 20: 1679-1684 (1992); Pease et al., Proc. Natl. Acad. Sci. USA, 91: 5022-5026 (1994). ) Or by covalent bonding or immobilization of chemically synthesized oligonucleotides (Guo et al., Nucleic Acids Research, 22: 5456-5465 (1994)) or other nucleic acids, amplicons combined with robotic array technology, It may be achieved by either covalent bonding such as cDNAs or immobile attachment. Other immobilization techniques are described in US Pat. No. 5,412,087 to McGall et al., US Pat. No. 5,429,807 to Matton et al., US Pat. No. 5,510,087 to Fodor et al. Thousands of different primers can be arrayed on a solid support to interrogate thousands of target nucleic acid sequences. The density of the nucleic acid or primer should be adapted by the arraying method and detection means.
[0100]
One aspect of the present invention involves hybridization of a target nucleic acid sequence to a universal array comprising specific nucleic acid probes, wherein “universal array” or “universal array sequence” are interchangeable herein. Is defined as a short nucleic acid sequence of all possible base combinations. The universal array sequence includes a range of 6-10 bases, preferably 5-6 bases, the number of possible combinations (ie, separate probes that bind to the solid phase) are 1024 and 4096, respectively, and nucleic acid expression analysis In which case the result may be used as a fingerprint, with different tissues or samples giving different fingerprints.
[0101]
The embodiment including the third nucleic acid probe may be immobilized on the solid phase array by using a “capture tag”. As used herein, a capture tag is any compound that may bind to other compounds or moieties. The primer is thus immobilized through binding of the attached capture tag to its binding partner. Such binding partners are referred to herein as “capture docks”. A capture tag is a compound, such as a particle or hapten, which is a compound that binds to or interacts with other molecules, such as ligand-binding molecules or antibodies. It is also preferred that such interaction between a capture tag and a capture dock is a specific interaction, such as an interaction between a hapten and an antibody or between a ligand and a ligand binding molecule.
[0102]
A further aspect of this assay includes a capture tag having two flanking regions: a target nucleic acid specific region and a “capture sequence complement”. As used herein, “capture sequence complement” includes a nucleic acid sequence complementary to “universal capture sequence” immobilized on a solid phase microarray, provided that “universal capture sequence” is known. It means a short nucleic acid sequence that is present and whose position on the solid phase microarray is predetermined. A capture tag or probe comprising a “capture sequence complement” may be immobilized to a solid phase microarray by hybridizing the complement to its complementary “universal capture sequence”.
[0103]
In another embodiment of the invention, a “capture tag” means a labeled nucleic acid probe that hybridizes to its “capture dock” bound to a solid phase microarray, provided that the capture dock is specific for the labeled nucleic acid probe. It is a common sequence.
[0104]
Another capture tag includes a hybridization or ligand molecule that may couple to the oligonucleotide. The capture tags described in the context of nucleic acid probes are described in Syven et al. , Nucleic Acids Res. 14: 5037 (1986). The capture tag also contains biotin and may be incorporated into the nucleic acid.
[0105]
Adhesion or coupling of the primer to the substrate may be accomplished by adhering or coupling the dock to the substrate. The capture dock mediates primer adhesion by binding to or interacting with capture tags on the primer. A capture dock immobilized on the substrate allows capture of the primer on the substrate. By attaching another capture dock to another region of the substrate attached to another primer, another capture tag attached to another primer is captured separately, and thus at a diagnostic location on the substrate. It may be. For example, in multiple assays of microtiter plates, capture docks specific for up to 96 different capture tags may be immobilized on the microtiter plate, each in a different groove. Capture and detection will occur only in the groove corresponding to the capture tag in which the corresponding nucleic acid molecule is present in the sample.
[0106]
In one embodiment, the capture dock is an oligonucleotide. Methods for immobilizing or coupling oligonucleotides to a substrate are well established. For example, the deposition method is described by Pease et al. , Proc. Natl. Acad. Sci. USA 91 (11): 5022-5026 (1994), and Krapko et al. Mol Biol (Mosk (USSR) 25: 718-730 (1991). The method of immobilization of 3 ′ amine oligonucleotides on casein-coated slides is described by Stimpson et al., Proc. Natl. Acad. Sci.USA 92: 6379-6383 (1995) Another method for attaching oligonucleotides to solid substrates is described in Guo et al., Nucleic Acids Res.22: 5456-5465 (1994). be written.
[0107]
Methods for immobilizing proteins on a substrate are well established. Immobilization may be accomplished using standard immobilization chemistry, for example by attachment to an aminated, carboxylated or hydroxylated surface. Examples of adhesives are cyanogen bromide, succinimide, aldehyde, tosyl chloride, avidin-biotin, photocrosslinker, epoxide, maleimide and glutaraldehyde. These and other adhesives and their use in attachment are described in Protein immunization: Fundamentals and Applications, Richard F., et al. Taylor, ed. (M. Dekker, New York, 1991), Johnstone and Torpe, Immunochemistry In Practice (Blackwell Scientific Public Publications, Oxford, England, 1987) pages 209-216z, 24I, 1987, and 1924A. Hermanson et al. , Eds. (Academic Press, New York, 1992). Proteins may be attached to the substrate by chemically cross-linking free radicals on the antibody to reactive side chain groups present in the substrate. For example, the protein may be chemically cross-linked to a substrate containing free amino or carboxyl groups using glutaraldehyde or carbodiimide as a cross-linking agent. In this method, an aqueous solution containing free protein is incubated with a solid phase substrate in the presence of glutaraldehyde or carbodiimide. Standard immobilization chemistry is known to those skilled in the art.
[0108]
In another embodiment, the sensitivity of the disclosed method is increased by repeated washing of the hybrid sample to remove free unhybridized nucleic acid present in the sample. It is useful to remove non-specific non-hybridizing nucleic acids because secondary structures in the nucleic acids may be recognized by the detection means and may lead to an increase in assay background.
[0109]
Preferred hybridization sample nucleic acid detection kits for use with the disclosed methods may be made using some or all of the components required for the method. The kit preferably includes an immobilized primer that is complementary to a region on the nucleic acid molecule of interest, and more preferably includes a plurality of immobilized primers that are each complementary to a region on the nucleic acid molecule of interest.
[0110]
Preferably, the kit contains all or some of the following components: sample transport medium for sample stabilization; microarray of biomolecules specific for the second biomolecule to be detected; hybridization buffer; RNA: DNA Hybrid-specific entity; wash buffer; enhancement buffer; and RNA: reagents necessary to detect hybrid-specific antibodies. In addition, some kits may include a thermophilic reverse transcriptase that lacks RNA: DNA hybrid dependent exonuclease (RNAseH) function. Another composition of the kit may comprise a nucleic acid probe comprising a capture sequence complement region. In addition, the kit may also include a labeled biomolecule probe that hybridizes to a common sequence of biomolecules bound to the solid phase. The kit may further include, but is not limited to, a universal array of biomolecules for the detection of sample biomolecules. The kit may contain all of these components or a part thereof.
[0111]
For detection of amplified antibodies, in addition to the reagents included in the hybridization kit for direct detection described above, the kit may also include the following reagents, including all or part: labeled for detection Anti-mouse IgG; biotinylated anti-mouse IgG; labeled anti-mouse streptavidin; biotinylated anti-streptavidin; or acetylated BSA solution.
[0112]
The kit should include a negative control and a positive control. Preferably, probes for negative and positive controls are included on a solid phase having a nucleic acid sequence.
The following non-limiting examples illustrate the use of this assay and kit.
[0113]
Example
Example 1
Detection of RNA: DNA hybrids on probe microarrays
The following is an example of a preferred method for practicing one embodiment of the disclosed method for detection of a target biomolecule sequence in a sample.
[0114]
Typically, the assay is preferably used to detect a sample size of 0.05 μg nucleic acid. Most preferably, 0.05 μg-10 μg of total nucleic acid is detected using the assay of the present invention.
[0115]
The target nucleic acid sample was suspended in nuclease free water and added to the hybridization buffer. The hybridization solution was denatured at 95 ° C. for 2-5 minutes. Hybridization solution containing the target nucleic acid was added to a glass slide with a microarray of spotted oligonucleotides. Hybridize at 65 ° C. for 16-20 hours. Followed either directly detected or amplified detection.
[0116]
For direct detection, a glass slide with a microarray of primers bound to the surface was run 3 times for 1-2 minutes 1 × PBS / 0.05% Tween20^TMAnd oscillated on a rotary vibrator (1100 rpm). When the RNA: DNA antibody staining solution was added, the total concentration was 0.144 μg / μl. The glass slide microarray was incubated in the solution for 1 hour at room temperature with shaking (1100 rpm). Glass slides are 1XPBS / 0.05% Tween20^TMAnd shaken at room temperature for 15 minutes (1100 rpm). The microarray was then incubated at room temperature in an antibody staining solution for mouse antibodies, particularly Cy3 or Cy5, and shaken at 1100 rpm for 1 hour. A number of fluorescent dyes may be used, including but not limited to Cy3 or Cy5. The final concentration of Cy-dye is 10% goat serum and 1 × PBS / 0.05 Tween 20^TMIn the solution of 0.04 μg / μl. Using slightly rigorous means, the slides were washed 4 times for about 10 seconds each in wash buffer. The slides were then incubated for 15 minutes at 53 ° C. in enhancement buffer. The slides were then washed 4 times in wash buffer for about 10 seconds each using loose stringency means. The microarray bound to the glass slide was dried by centrifugation at 2000 rpm for 7-10 minutes or until dry. The results were analyzed by reading the slides in an array scanner (Affymetrix 417 Array Scanner or equivalent) with photoexcitation at 532 nm and 635 nm to scan slides developed with antibodies labeled with Cy3 and Cy5, respectively.
[0117]
For amplified detection, slides were washed 3 times for 1-2 minutes 1 × PBS / 0.05% Tween20^TMIt was washed in and vibrated at 1100 rpm on a rotary vibrator. Slides were incubated in RNA: DNA hybrid specific antibody staining solution with stirring (1100 rpm) for 1 hour at room temperature. Spotted slides were coated with biotinylated mouse IgG antibody from goat staining solution and incubated for 10 minutes at room temperature. Microarray twice for 1-2 minutes each 1XPBS / 0.05% Tween20^TMWashed in. The spotted slides were coated with goat anti-mouse R-phycoerythrin streptavidin (0.01 μg / μl; SA-PE) staining solution and incubated for 10 minutes at room temperature. Washing was repeated as described above. Spotted slides were coated with a biotinylated goat antibody (0.5 mg / mL) staining solution developed against streptavidin and incubated for 10 minutes at room temperature. The slides were again incubated with goat anti-mouse R-phycoerythrin streptavidin (0.01 μg / μl; SA-PE) staining solution for 10 minutes at room temperature. Next, slides 3 times 1-2 min 1XPBS / 0.05% Tween20^TMWashed in. The microarray was then dried by centrifugation at 2000 rpm for 7-10 minutes or until dry. The results were analyzed by reading the slides with photoexcitation at 532 nm and 635 nm to scan slides developed with antibodies labeled with Cy3 or PE and Cy5, respectively, in an array scanner (Affymetrix 417 Array Scanner or equivalent). did.
[0118]
Example 2
Before sample hybridization
Hybridization of labeled oligonucleotides
Cy3 or Cy5 labeled n-mer oligonucleotides were added to hybridization buffer containing SSC and SDS and denatured at 95 ° C. for 2-5 minutes. Many fluorescent dyes may be used, including but not limited to Cy3 and Cy5. A number of fluorescent dyes may be used, including but not limited to Cy3 or Cy5. Glass slides containing spotted oligonucleotides were coated with hybridization solution and incubated for 20 seconds at room temperature. Cover slips were removed by severe infiltration of 2XSSC / 0.2% SDS. Any remaining SDS was washed away by infiltrating the slides to 0.05XSSC for 30 minutes. The slides were then dried by centrifugation at 2000 rpm for 7-10 minutes or until dry. The results were analyzed by reading the slides in an array scanner (Affymetrix 417 Array Scanner or equivalent) with photoexcitation at 532 nm and 635 nm to scan slides developed with antibodies labeled with Cy3 and Cy5, respectively.
[0119]
Example 3
Detection of RNA mediated by reverse transcriptase lacking RNAseH function
The following is an example of a method for practicing one embodiment of the disclosed method for detection of a target nucleic acid sequence in a sample.
[0120]
Five primed biotinylated 20-30 nucleotide primers were mixed with streptavidin-coated solid phase and incubated for 30-60 minutes at 20-27 ° C. with constant vibration (1100 rpm). A sample of target nucleic acid was added to the solid phase. Hybridization / extension buffer (100 mM Tris-HCl, pH 8.3, 150 mM KCl, 6 mM MgCl₂, 20 mM DTT, and 1 mM each dNTP) were then added. Target nucleic acids and primers were annealed by heating the mixture to an optimal annealing temperature, preferably 60 ° C. (the optimal annealing temperature varies with the primer and nucleic acid utilized) for 20-30 minutes. The mixture was then cooled to 20-27 ° C. for 10 minutes. Additional hybridization / extension buffer and reverse transcriptase were added, preferably reverse transcriptase that is stable at high temperature and lacks RNAseH. The reaction was incubated for 30-60 minutes at 42 ° C. EDTA (0.5 M) was added and incubated at 37 ° C. for 30 minutes. The RNA: DNA hybrid specific alkaline phosphatase formulated antibody mixture was added and incubated at 20-27 ° C. for 30-60 minutes. Any unbound antibody was washed away and then a chemichromic substrate was added. The solution was incubated for 15-30 minutes at 20-27 ° C. The emitted signal was read with a luminometer at the appropriate wavelength.
[0121]
Example 4
RNA: DNA hybrid specific antibody binding
Hybridized RNA: DNA samples were incubated with antibodies for a time sufficient to allow the hybrid formulation. The hybrids were conjugated to the antibody by incubation at a speed of 0 to 1500 rpm on a platform shaker at 15 ° C. to 65 ° C. for 5 minutes to 24 hours. Preferably, the incubation time was 30 to 120 minutes and the speed was 300 to 1200 rpm at 20 to 40 ° C. Most preferably, the binding occurred by vigorous incubation at room temperature for 1 hour at a rotational vibration speed of between about 300 and 1000 rpm. One skilled in the art may achieve other capture kinetics as desired by varying the incubation time, temperature and vibration.
[0122]
Example 5
Detected by monoclonal and polyclonal antibodies
Comparison of oligonucleotide lengths
A single oligonucleotide of varying length was spotted at 10 different replicates at 4 different concentrations. The spotted 72-mer oligonucleotide was part of IMAGE clone # 259998 corresponding to 40S ribosomal protein S11. The shorter oligonucleotide was a continuous end remover of the parent 72mer. These microarrays were hybridized to complementary RNA of varying concentrations and visualized using monoclonal primary RNA: DNA hybrid antibodies. At a target concentration of 800 pM, a substantial signal was observed in the 30 base oligonucleotide, but there was a sharp drop in signal at 25 bases. FIG. 6A shows the signal results versus the noise ratio as a function of oligonucleotide at 800 pM RNA concentration using various spotted oligonucleotide concentrations in detecting RNA: DNA hybrid specific monoclonal antibodies.
[0123]
The polyclonal antibody detection protocol was the same as the protocol described above for the monoclonal antibody, with the exception that the Cy3-labeled goat secondary antibody from the rabbit was replaced with a mouse antibody from the goat. The results with the polyclonal (FIG. 6B) showed a significantly improved detection compared to the monoclonal antibody for oligonucleotides of less than 30 bases. There was no significant difference in signal for oligonucleotides over 30 bases.
[0124]
Polyclonal RNA: DNA antibodies provided a significantly more sensitive method for detecting RNA: DNA hybrids of less than 30 base pairs compared to detection using monoclonal antibodies.
[Brief description of the drawings]
1A-D are schematic representations of the preferred embodiment noise of immune antibody detection of RNA: DNA hybrids on microarrays. FIG. 1A shows the hybridization of an RNA sample to a complementary DNA sequence attached to a microarray forming the RNA: DNA hybrid depicted in FIG. 1B. The antibody, monoclonal or polyclonal is then bound to the RNA: DNA hybrid as shown in FIG. 1C. FIG. 1D illustrates the detection of a fluorescent label using a fluorescent laser scanner.
FIGS. 2A-D are schematic representations of a second aspect of immunodetection on a microarray containing universal capture sequences for RNA: DNA hybrids. FIGS. FIG. 2A shows hybridization of single-stranded DNA of the universal array with sample RNA. Each hybrid is formed on a microarray containing DNA: DNA regions and RNA: DNA regions, as depicted in FIG. 2B. The antibody detects and binds the RNA: DNA hybrid in FIG. 2C. FIG. 2D illustrates one means of detection involving a fluorescent antibody label using a fluorescent laser scanner.
Figures 3A-E are schematic representations of a third aspect of immunodetection of RNA: DNA hybrids, provided that the microarray is for mRNA quantification when the full-length sequence is unknown. Contains expressed sequence tags (ESTs). FIG. 3A shows hybridization of sample RNA to short ESTs bound to the microarray. The formation of RNA and short DNA hybrids is depicted in FIG. 3B. For example, reverse transcriptase (RT) is used to extend synthesize DNA to the full length of RNA, as depicted in FIG. 3C. 3D and 3E respectively illustrate antibody recognition of RNA: DNA hybrids and detection of fluorescent antibody labels with a laser scanner.
FIGS. 4A-D are schematic representations of a fourth embodiment of RNA: DNA hybrid immunodetection, provided that the invention is directed to a two-color detection method. FIG. 4A shows each DNA probe bound to a microarray containing a region of identical sequence and a region of variable sequence. The labeled DNA hybridizes with the consensus sequence and the RNA sample hybridizes with the variable sequence. RNA: DNA hybrids and DNA: labeled DNA hybrids are formed, as illustrated in FIG. 4B. Antibodies raised against the RNA: DNA hybrid bind to the appropriate region; the microarray is scanned with two different color fluorescent lasers; and the signal is normalized as shown in FIGS. 4C-4D.
FIGS. 5A-D are schematic representations of a fourth aspect of immunodetection of RNA: DNA hybrids, provided that the invention includes labeled degenerate n-mer DNA and sample RNA. FIG. 5A shows each DNA probe bound to a microarray hybridized simultaneously or sequentially to an RNA sample and / or labeled degenerate DNA. RNA: DNA hybrids and / or DNA: labeled DNA hybrids are formed, as shown in FIG. 5B. Antibodies raised against the RNA: DNA hybrid bind to the appropriate region; the microarray is scanned with two different color fluorescent lasers; and the signal is normalized as shown in FIGS. 5C-5D.
FIGS. 6A-B are graphs showing a comparison of solid phase bound oligonucleotide length using detection with monoclonal antibody (FIG. 6A) and polyclonal antibody (FIG. 6B) as a function of signal to noise ratio of the microarray. It is.

Claims (18)

A microarray method for detecting a first nucleic acid in a sample comprising the following steps:
a first nucleic acid of a) the sample and hybridized to a complementary second immobilized nucleic acid, RNA: a step of forming a DNA hybrid, step nucleic acid wherein the second comprising:
α) a common region complementary to a region of the third complementary nucleic acid that is detectably labeled; and β) a variable region complementary to the region of the first nucleic acid;
b) a step of hybridizing the second nucleic acid to the third nucleic acid to form a DNA: DNA hybrid or RNA: RNA hybrid , wherein the third nucleic acid binds to a common region of the second nucleic acid A process that is a nucleated nucleic acid ; and
c) RNA: using detectable elements which specifically bind to DNA hybrid RNA: detecting the DNA hybrid. The method of claim 1, wherein the element is an RNA: DNA hybrid specific antibody. The method of claim 1, wherein the element is a fragment of an RNA: DNA hybrid specific antibody. 4. The method according to claims 2 and 3, wherein the antibody is monoclonal. 4. The method of claims 2 and 3, wherein the antibody is polyclonal. The method of claim 1, wherein steps a) and b) occur simultaneously. The method of claim 1 wherein steps a) and b) occur sequentially. The method of claim 1, wherein a plurality of second nucleic acids are bound to a solid phase. The method of claim 1 wherein the label is a detectable chromogenic fluorescent label and the analysis utilizes a two-color detection method. A method for detecting or quantifying a target nucleic acid on a microarray having a plurality of sample detection sites comprising the following steps:
(A) a step of hybridizing a target nucleic acid to a biomolecular probe bound to a microarray to form an immobilized RNA: DNA hybrid complex;
(B) A detectably labeled biomolecule probe is hybridized to the non-hybridized portion of the biomolecule probe bound to the microarray to form an immobilized RNA: DNA hybrid complex. Process;
(C) (i) measuring the immobilized RNA: DNA hybrid complex by binding to the complex a detectable antibody that is specifically reactive to the RNA: DNA hybrid; and (ii) ) By measuring a detectably labeled biomolecular probe,
A step of detecting a target nucleic acid; and (d) a step of repeating steps (a) to (c) at each of a plurality of sample detection sites. 11. The method of claim 10 , wherein the repeating step is performed continuously. 11. The method of claim 10 , wherein the repeating steps are performed simultaneously. A method for detecting or quantifying a target nucleic acid on a microarray having a plurality of sample detection sites comprising the following steps:
(A) a step of hybridizing a part of the target nucleic acid to a biomolecular probe bound to the microarray to form an immobilized RNA: DNA hybrid;
(B) a step of hybridizing a non-hybridized portion of the target nucleic acid to a detectably labeled complementary nucleic acid probe to form an immobilized RNA: DNA hybrid complex;
(C) (i) measuring the immobilized RNA: DNA hybrid complex by binding to the complex a detectable antibody that is specifically reactive to the RNA: DNA hybrid; and (ii) ) By measuring a detectably labeled biomolecular probe,
A step of detecting a target nucleic acid; and (d) a step of repeating steps (a) to (c) at each of a plurality of sample detection sites. 14. The method of claim 13 , wherein the repeating step is performed continuously. 14. The method of claim 13 , wherein the repeating steps are performed simultaneously. A method for detecting or quantifying a target nucleic acid on a microarray having a plurality of sample detection sites comprising the following steps:
(A) a step of hybridizing a target nucleic acid to a biomolecular probe bound to a microarray to form an RNA: DNA hybrid;
(B) a step of hybridizing unhybridized biomolecules bound to the microarray to a complementary region of a detectably labeled biomolecule probe, wherein the hybridization The biomolecular probe bound to the non-microarray is different from the biomolecular probe bound to the microarray of step (a);
(C) (i) measuring the immobilized RNA: DNA hybrid complex by binding to the complex a detectable antibody that is specifically reactive to the RNA: DNA hybrid; and (ii) ) By measuring a detectably labeled biomolecular probe,
A step of detecting a target nucleic acid; and (d) a step of repeating steps (a) to (c) at each of a plurality of sample detection sites. 17. The method according to claim 16 , wherein the repeating step is performed continuously. 17. The method of claim 16 , wherein the repeating steps are performed simultaneously.

Images