JP2004147597A - Probe for detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a probe compound for detecting a target nucleic acid, capable of functionally detecting the nucleic acid in high sensitivity while hardly causing cross hybridization. <P>SOLUTION: The probe has a nucleic acid having not only a specific complementary sequence complementary to a specified sequence but also a common complementary sequence complementary to the whole or a part of a common sequence, and shorter than the chain length of the specific complementary sequence. The method comprises measuring an amount of the probes immobilized on a carrier for immobilization. The method is the one for estimating the amounts of two or more kinds of target nucleic acids present in a sample respectively. The measurement of a fixed amount of the probes is facilitated by the microarray. The following problems (1) and (2) when detecting the two or more target nucleic acids having the common sequences at the both terminals of the specific sequences are solved: (1) the hybridization between the target nucleic acid and the probe is hardly formed by forming an intramolecular secondary structure because of the long target nucleic acid to cause false negative data sometimes; and (2) the stability of the hybridization is low to require the cleaning under a mild condition because the specific sequence is short compared to the length of the target nucleic acid and to cause the false negative data sometimes. <P>COPYRIGHT: (C)2004,JPO

Description

（１）リンカー配列が同一塩基の連続であるプローブ
プローブ例１（ｐｒｏｂｅ １）：

プローブ例２（ｐｒｏｂｅ ２）：

リンカー配列に同一の塩基配列を選んだ例。リンカー配列にＣやＧでなくＡやＴを選ぶのは、Ｇ，Ｃ塩基同士のミスマッチ塩基Ｇ−Ｇ， Ｃ−ＣなどよりＡ， Ｔ塩基同士のミスマッチのＡ−Ａ， Ｔ−Ｔの安定性の方が低いからである。また、Ａ、Ｔを選択することによりプローブが分子内構造をとりにくいようにすることができる。
【００５３】
（２）リンカー配列がすべてミスマッチになるようにしたプローブ
プローブ例３（ｐｒｏｂｅ ３）：

プローブ例４（ｐｒｏｂｅ ４）：

プローブ例５（ｐｒｏｂｅ ５）：

例３， ４は（１）のリンカー配列で、Ａ−Ｔマッチになっている部分をＡ−ＡミスマッチかＴ−Ｔミスマッチに変えたプローブの例。例５はさらに別のミスマッチにしたもの。
ミスマッチの塩基には次の８通りの組み合わせがある。
Ａ−Ａ， Ａ−Ｃ， Ａ−Ｇ， Ｃ−Ｃ， Ｃ−Ｔ， Ｇ−Ｇ， Ｇ−Ｔ， Ｔ−Ｔ
これらの中のいずれかのミスマッチになるようにリンカー配列を設計することにより、ミスマッチのリンカー配列が得られる。
以上はプローブの３’端側に共通配列に相補な配列をつけた場合であるが、これが５’端にあってもかまわない。また、３’端側、５’端側両方に存在しても良い。
【００５４】
本発明において、「マッチ」という用語は、生体内の核酸において通常認められるワトソン・クリック塩基対が特定の塩基対において形成された状況を意味する用語である。即ち、一般的な塩基対であるＡ−Ｔ、Ｇ−Ｃ、Ａ−Ｕのうちの何れかの対で相補鎖形成する状況を意味する。しかしながら、本用語は対応する塩基間で水素結合を形成する塩基対を広く意味しており、Ａ、Ｔ、Ｇ、Ｃ以外の塩基において塩基対が形成される場合などにおいても本用語が使用される。
【００５５】
本発明において、「パーフェクトマッチ（ＰＭ）」という用語は、形成される塩基対が前記「マッチ」する組み合わせのみからなる塩基対の状況を意味する用語である。即ち、ある鎖長の異なる２配列が「パーフェクトマッチ（ＰＭ）」である場合、２配列のうち短い配列の全体にわたり上記の「マッチ」の定義を満たす塩基対が形成されることを意味する。例えば、特定配列に対して相補となる配列（両者の鎖長は一致）、ターゲット核酸の部分配列に対して全塩基が相補であるプローブの配列（両者の鎖長は不一致）などは、互いに「パーフェクトマッチ（ＰＭ）」する配列である。
【００５６】
また本発明において、「ミスマッチ（ＭＭ）」という用語は、配列中に本来は塩基対を形成しない塩基対を有することを広く意味する用語である。「ミスマッチ（ＭＭ）」な塩基対とは、一般的にＤＮＡにおいてはＡ−Ａ、Ａ−Ｃ、Ａ−Ｇ、Ｃ−Ｃ、Ｃ−Ｔ、Ｇ−Ｇ、Ｇ−Ｔ、Ｔ−Ｔのうちの何れかの組み合わせとなる塩基対である。しかしながら、本用語は対応する塩基間で水素結合を形成しない塩基対を広く意味しており、Ａ、Ｔ、Ｇ、Ｃ以外の塩基において塩基対が形成されない場合などにおいても本用語が使用される。
【００５７】
前記固定化用支持体は、ガラス、プラスチックなど材料を問わず、平板、ビーズ状など形態も問わない。また、磁気ビーズを使用すれば液中のビーズを容易に沈降させることができ洗浄操作などを簡便に行うことができる。前記マイクロアレイの場合には、スライドガラスなどの平板上の支持体が好適に使用される。
【００５８】
前記固定化用リンカーは、本発明のプローブを前記支持体上に固相化できるものであれば特に限定されず、当業者において通常使用されている公知のものが使用できるが、一般的には長鎖アルキル、アミノアルキルなどが使用できる。
【００５９】
本発明のプローブは、何れも当業者に公知の手法を用いて化学合成できる、または、合成の受託業者に委託して合成することができる。
前記プローブを使用したマイクロアレイの作成も当業者に公知の方法で作成できる。例えば、前記プローブが核酸のみからなる場合にはスライドガラス上で固相合成する方法、または合成した前記プローブをピンにより支持体上に印刷する方法などにより作成できる。
【００６０】
［本発明で提供される第一の方法］
本発明の第一の方法は、本発明のプローブを、前記共通配列に相補的な配列を含み且つ標識物質で標識した既知量の固定量測定用核酸を使用して測定する方法であって、
１）前記プローブと、前記固定量測定用核酸とを接触させ、両者を特異的にハイブリダイズさせる工程と、
２）工程１におけるハイブリッドの生成を、前記標識物質に基づく測定手段により測定する工程と、
３）工程２で得られる測定結果に基づき、前記支持体上の前記プローブの量を計算する工程と、
を具備することを特徴とするものである。
【００６１】
前記標識物質は、実験の目的に応じて任意の標識物質を使用できる。マイクロアレイを実施する場合、前記標識物質は蛍光色素であり、好ましくはシアニン ３（Ｃｙ３；Ｃｙａｎｉｎｅ３）もしくはシアニン ５（Ｃｙ５；Ｃｙａｎｉｎｅ５）などのシアニン色素が使用される。
【００６２】
前記固定量測定用核酸は、本発明のプローブの前記共通相補配列を特異的に測定できる核酸であり、前記共通相補配列の全体もしくは一部と相補的な配列を具備し、前記標識物質で標識された核酸である。
【００６３】
前記測定手段は、前記標識物質の存在を検出し、その存在量を測定できる機能を備える分析機器が使用される。前記標識物質が蛍光物質であれば光学的な検出装置を備えた機器が使用される。
【００６４】
前記「計算」は、既知量の前記固定量測定用核酸を使用することにより前記支持体上に固相化されたプローブの量を計算することである。１分子の前記プローブは１分子の前記固定量測定用核酸とハイブリダイズして二重鎖を形成する。従って、コントロールとして測定する既知量の前記固定量測定用核酸の値を測定し、前記プローブとハイブリダイズした前記固定量測定用核酸の値を測定し、両測定値から支持体上の前記プローブの固相化量が計算される。
【００６５】
［本発明で提供される第二の方法］
更に、本発明の第二の方法は、マイクロアレイ上に固相化された前記プローブと、前記共通配列に相補的な配列を含み且つ標識物質Ａで標識した既知量の固定量測定用核酸とを使用して、前記マイクロアレイ上で前記ターゲット核酸の存在量を測定する方法であって、
１）前記標識物質Ａとは異なる標識物質Ｂで標識した前記ターゲット核酸を含む試料を作成する工程と、
２）前記プローブと、工程１の試料と、前記固定量測定用核酸とを接触させ、特異的にハイブリダイズさせる工程と、
３）工程２のハイブリッドの生成を、使用する標識物質に基づく測定手段により測定する工程と、
４）工程３で得られる測定結果に基づき、該試料中の前記ターゲット核酸の存在量を計算する工程と、
を具備し、
マイクロアレイ上の各々のスポット間の前記プローブの固相化量比が、標識物質Ａの測定結果から計算され（計算値Ａ）、
前記ターゲット核酸として使用される既知量のターゲット核酸の前記プローブに対するハイブリダイズ量が、標識物質Ｂの測定結果から計算され（計算値Ｂ）、
該試料中に存在する複数種類の前記ターゲット核酸の各々の存在量を計算値ＡおよびＢから外挿して推定することを特徴とするものである。
【００６６】
前記固定量測定用核酸は、上記の第一の方法で定義した固定量測定用核酸と同義である。
前記マイクロアレイは、当業者に公知の方法で本発明のプローブをマイクロアレイ上に固相化して作成できる。しかし、前記方法は、１支持体上に多種類のプローブが固相化される様式であればマイクロアレイ以外の方式であっても実施できる。従って、各種マイクロプレートに前記プローブを固相化して測定することもできる。また、例えば前記ビーズに多種類のプローブが固相化されている場合には、各々のプローブをマイクロプレートに播種して本方法を実施することもできる。
【００６７】
前記試料は、一般的な適用においては各種の核酸（例えば、生体から抽出したｍＲＮＡ若しくはゲノムのＤＮＡなど）を前記ターゲット核酸に転換したものを含む混合物である。しかし、生体の核酸に由来する核酸だけでなく、人工的に設計された配列を有しても、前記ターゲット核酸の定義を満たす核酸であれば前記試料として使用できる。本発明で好適に使用される前記ターゲット核酸の態様を以下の［本発明の態様］において説明する。
【００６８】
標識物質Ａおよび標識物質Ｂは互いに異なる標識物質であり、互いに影響せず各々の存在量を別個に測定し得るものであればよい。好ましくは、双方にはＣｙ３もしくはＣｙ５のどちらかが使用される。即ち、標識物質ＡがＣｙ５の場合には標識物質ＢはＣｙ３が使用され、その逆の組み合わせであってもいい。
【００６９】
前記計算値Ａは、標識物質Ａの測定結果から計算される計算値である。この計算値から、マイクロアレイ上の各々のスポット間の前記プローブの固相化量比が算出される。計算値Ａは標識物質Ａの測定値から第一の方法の「計算」について説明した通りに計算できる。
【００７０】
前記計算値Ｂは、標識物質Ｂの測定結果から計算される計算値である。この計算値から、前記ターゲット核酸として使用される既知量のターゲット核酸の前記プローブ（ここで使用する既知量のターゲット核酸に対応する前記プローブである、従って該ターゲットに対する特異的相補配列を有するプローブである）に対するハイブリダイズ量が算出される。
【００７１】
計算値Ｂは、例えば下記のように測定できる。
１）同一マイクロアレイ上に内部標準物質として既知量のターゲット核酸をスポットして測定する。
２）同時に対応する前記プローブへの既知量のターゲット核酸のハイブリダイズした量を測定する。
３）１）の測定値を基に内部標準物質の量と測定値との関係から検量線を作成し、２）の測定値と前記検量線から前記プローブに結合したターゲット核酸量が算出される。
【００７２】
同一スポット上の前記計算値ＡおよびＢを算出して計算値Ｂをスポット毎に固相化量比を表す計算値Ａにより補正して別種類の前記プローブが固相化されたスポットの測定値に外挿することにより試料中の全てのターゲット核酸の絶対量が測定できる。
【００７３】
以下、本発明の代表的な実施態様について本願に添付される図面を参照して説明する。但し、本発明の技術的範囲はこれら具体的な態様に限定されるものではない。
［本発明の態様］
＜本発明のターゲット核酸：図１〜２＞
以下に本発明が主に対象とするターゲット核酸について説明する。
図１〜２は共通プライマー配列をそなえたターゲット核酸の遺伝子検出への使用例を説明する図である。これらの図により、本発明のプローブが対象とするターゲット核酸の作成の手順が例示される。
【００７４】
核酸の構造（図１、Ａ）は、ターゲット核酸を作成する際に使用される共通プライマー配列（共通配列１および２）を具備する核酸である（右側が３’末端）。
このような組を検出遺伝子に応じて多種類合成する。共通配列１， ２は検出対象にかかわらず同一の配列を有するものである（図１、Ｂ）。
検出対象の一本鎖ＤＮＡの多数を含有する試料中でハイブリダイゼーションし、リガーゼ反応する（図１、Ｃ）。
検出対象が前記試料中に存在していた場合、連結される（図１、Ｃ）が、検出対象が存在しなかった場合、連結されずそのままになる（図１、Ｄ）。
【００７５】
図２において、連結されたターゲット核酸（図１、Ｃ）を処理する手順を説明する。
ターゲット核酸の配列構成は図２の（Ａ）に示すようなものであり、中央部に連結された特異的配列を有している（この鎖は検出プローブに相補な側を示す）。
【００７６】
以下に説明する工程（図２Ｂの１〜３）で図２の（Ａ）の核酸が増幅され且つ同時に標識付けされ、単鎖抽出される。即ち、
（１）ターゲット核酸ライブラリを標識した共通プライマーでＰＣＲ増幅する工程。
（２）工程（１）のＰＣＲ産物をストレプトアビジンのついた磁気ビーズに捕獲する工程。
（３）捕獲したターゲット核酸から蛍光標識した単鎖ＤＮＡを抽出する工程。
このようにして本発明のターゲット核酸を作成できる。
【００７７】
以下に従来型プローブと比較した本発明のプローブの特徴を態様ごとに例示する。
＜従来型プローブの説明：図３＞
図３にターゲット核酸検出のためのプローブの従来例を説明する。
ターゲット核酸の特異的配列に特異的にハイブリッドを形成する配列を具備する単鎖のＲＮＡ、ＤＮＡまたはＰＮＡなどの化合物をプローブに用いる。たとえば図２の（３）のターゲット核酸の特異的配列に相補的なプローブで検出する場合、図３のようなハイブリッドを形成することになる。
【００７８】
＜本発明のプローブの実施態様：図４〜５＞
《プローブ態様１（延長型プローブ）》
図４に本発明の一態様である、延長型プローブを用いた際のハイブリッド形成の例を説明する。
（１）にターゲット核酸の特異的配列に相補な配列（特異的相補配列） ＋ ターゲット核酸５’側共通プライマー配列の３’側の一部に相補な配列（共通相補配列）を備えたプローブの例を示す。
（２）にターゲット核酸の特異的配列に相補な配列（特異的相補配列） ＋ ターゲット核酸３’側共通プライマー配列の５’側の一部に相補な配列（共通相補配列）を備えたプローブの例を示す。
（３）ターゲット核酸の特異的配列に相補な配列（特異的相補配列） ＋ ターゲット核酸５’側共通プライマー配列の３’側の一部に相補な配列（共通相補配列） ＋ ターゲット核酸３’側共通プライマー配列の５’側の一部に相補な配列（共通相補配列）を備えたプローブの例を示す。
以上のようなプローブを使用することによってプローブ長を長く設定することができる。
【００７９】
《プローブ態様２（末端捕捉型プローブ）》
図５に本発明の一態様である、末端捕捉型プローブを用いた際のハイブリッド形成の例を説明する。
（１）にターゲット核酸の特異的配列に相補な配列（特異的相補配列） ＋ リンカー配列 ＋ ターゲット核酸５’側共通プライマー配列の５’末端側の一部に相補な配列（共通相補配列）を備えたプローブの例を示す。
（２）にターゲット核酸の特異的配列に相補な配列（特異的相補配列） ＋ リンカー配列 ＋ ターゲット核酸３’側共通プライマー配列の３’末端側の一部に相補な配列（共通相補配列）を備えたプローブの例を示す。
（３）にターゲット核酸の特異的配列の両側にリンカー配列をもつプローブの例を示す。
上記で説明した各態様のように、本発明のプローブはターゲット核酸に対して様々に設定できるが、好ましくはプローブの末端でターゲット核酸の末端を捕捉するように設定することが好ましい。このように設定することにより、ハイブリ効率を上げることができる。
【００８０】
この理由を図７を参照して以下に説明する。
図７は矢印で示された鎖状分子のそれぞれの部位が持つ局在化した運動エネルギを「横への広がり」として表現している。
図７の右側のプローブ分子では５’が固相化されているため運動量は小さく，３次元的に自由な３’末端に向けて運動量が大きくなる。もっとも大きな運動量を持つのは３’末端である。
左側のターゲット核酸分子については，対称中心となる鎖状分子中央で運動量が最も小さい。分子中央が運動する場合，両末端を引っ張らなくてはならないため抵抗が大きく，周囲の分子（溶媒など）も２次元的な衝突をするに過ぎない。運動量がもっとも大きいのは５’及び３’両末端である。末端では運動する際の分子内での抵抗が小さい上に，周囲の分子が３次元的に衝突する。
【００８１】
化学反応は正しい方向から，ある一定以上のエネルギをもって，衝突することによって生じる。ハイブリダイゼーション反応についても例外ではなく，まずは分子同士が衝突しなければならない。つまりは衝突頻度が重要となる。
【００８２】
上記のような反応系の場合，局所的な分子間衝突確率がもっとも高い部分はプローブの３’末端とターゲット核酸の５’末端，３’末端である。
【００８３】
ただし，プローブの３’末端とターゲット核酸の３’末端が衝突した場合ではハイブリダイゼーションは行なわれない。したがって，もっとも反応効率が高いのはプローブの３’端でターゲット核酸の５’端を捕捉する形となる。
【００８４】
＜本発明のプローブを使用する核酸の測定方法：図６＞
図６において本発明のプローブ固定量の測定方法を説明する。
マイクロアレイに本発明のプローブを用いる場合、各プローブの固定量の測定ができ、各スポットに捕獲されたターゲット核酸の絶対量測定に使える。
たとえば図６（１）のように本発明のプローブが固定されている場合。
図６（２）のように、Ｃｙ３で蛍光標識したターゲット核酸をハイブリダイズさせる。このハイブリダイゼーションを阻害しない程度の量のＣｙ５で蛍光標識した固定量測定用核酸も同時にハイブリダイズさせる。
【００８５】
次にハイブリダイゼーションの済んだマイクロアレイを蛍光スキャナで読み取る。Ｃｙ５の蛍光波長とＣｙ３の蛍光波長の２つの画像が得られるが、Ｃｙ５画像から各プローブの固定量比を反映した蛍光強度が分かり、Ｃｙ３画像は各ターゲット核酸の量比に応じた蛍光強度が得られる（３）。ここで量が既知のＣｙ３で標識したターゲット核酸を混ぜておくと、すべてのターゲット核酸の絶対量が測定できる。
【００８６】
以上、本発明のプローブの５’末端側が支持体上に固相化されている態様について説明した。しかしながら、本発明のプローブの３’末端側が支持体上に固相化される別態様についても同様に実施でき、且つ同様の効果を奏することは当業者に容易に理解されるであろう。従って、３’末端側が支持体上に固相化されるプローブおよび該プローブを使用する方法も、同様に本発明に含まれるものである。
【００８７】
以下、実施例により本発明を更に具体的に説明する。但し、本発明はこれら実施例にその技術的範囲が限定されるものではない。
【００８８】
【実施例】
［実施例１］
《ターゲット核酸中央でのハイブリダイゼーションとターゲット核酸 ５’末端でのハイブリダイゼーションとの比較》
＜実験方法＞
▲１▼ターゲット核酸として、５’末端をＣｙ３標識したｒＥ２４３配列７５ ｍｅｒを使用した。ｒＥ２４３配列は、それぞれ２５ ｍｅｒの３’部、中央部、５’部の３つの部分配列からなる全長７５ ｍｅｒの配列である。
▲２▼前記▲１▼のターゲット核酸に対するプローブとして、ｒＥ２４３の中央部２５ ｍｅｒに相補な ＣＤ２４３プローブ，ｒＥ２４３の５’部２５ ｍｅｒに相補なＣＤ１４７プローブを使用した。
【００８９】
前記ターゲット核酸及び前記プローブが固相化されたマイクロアレイを用いたハイブリダイゼーションにより該プローブを評価した。実験に使用した装置、器具、試薬の詳細を実験結果の後に記載する。
【００９０】
まず，５’末端をＣｙ３標識したｒＥ２４３が２．５ｐｍｏｌ含まれるように１ ｘ ＳＳＣ（０．２％ＳＤＳ含有） １００μｌ中に調製し，これをターゲット核酸 溶液とした。このターゲット核酸 溶液をハイブリ前に９４℃で５分間加熱し，前処理を行なった。
【００９１】
ＣＤ２４３プローブ，ＣＤ１４７プローブを固相化した検出用マイクロアレイにハイブリウェル（後述）を装着した。ハイブリ直前に前処理したターゲット核酸 溶液 １００μｌをハイブリウェルにインジェクトし，このマイクロアレイを４０℃のインキュベータ中でハイブリダイゼーション反応を６０分間行なった。
【００９２】
次にハイブリ後のマイクロアレイをインキュベータから取り出し，ハイブリウェルを剥離した後すみやかに１ ｘ ＳＳＣで満たした染色バットに投入し，スライドラックとスライドウオッシャーを使用して１０分間揺動洗浄した。つづいて同様に０．１ ｘ ＳＳＣで５分間揺動洗浄した後，フロンガス（ＨＦＣ１３４ａ）を使用したブロアーで乾燥させた。これをＳｃａｎＡｒｒａｙ４０００を用いて観察し，スキャン画像を市販の定量化ソフトウェアをもちいて定量した。
【００９３】
＜実験結果＞
以下に示す結果は各プローブ，４スポットの定量値を平均化した値である。
【００９４】
【表１】

【００９５】
以上の結果から７５ｍｅｒのターゲット核酸中央部２５ｍｅｒに相補なＣＤ２４３プローブよりも、ターゲット核酸の５’末端から２５ｍｅｒに相補なＣＤ１４７プローブを使用した場合にシグナル強度が高いことが明らかになった。つまり，ターゲット核酸の５’末端に相補的な配列をプローブとして使用してハイブリさせると効率が高いと言える。
【００９６】
＜実験条件の詳細＞
・検出用チップ：
アミノ基標識した合成オリゴＤＮＡを市販の白板スライドガラス基板と共有結合させる当業者に通常用いられる方法により作成した。スポッティングは１５０μｍのピンヘッドを持つ市販のスポッタ（ピンピッチ４．５ｍｍ，スポットピッチ０．５ｍｍ）をもちいて，各プローブを４スポットずつ点着した。
【００９７】
・検出ターゲット核酸：
検出する配列と同じ配列をもつ合成オリゴＤＮＡをテンプレートとして，ビオチン化したｆｗｄプライマ及びＣｙ３標識したｒｅｖプライマを使用してＰＣＲ反応をおこなった。そのＰＣＲ産物をストレプトアビジン磁気ビーズにより精製した。
【００９８】
ストレプトアビジン磁気ビーズは高温度条件下においてもビオチンと比較的安定した結合を形成することを利用してＣｙ３化単鎖ＤＮＡ（ｓｓ−ＤＮＡ）を単離する。即ち、磁気ビーズにＰＣＲ産物を捕獲し、それを熱水中でデハイブリダイゼーションして、Ｃｙ３化されたｓｓ−ＤＮＡ ターゲット核酸のみを単離した（図２を参照）。
【００９９】
・ハイブリダイゼーション条件：
ターゲット核酸 ： Ｃｙ３標識ｒＥ２４３ ２．５ ｐｍｏｌ
反応溶液 ： ０．２％ ＳＤＳ １ ｘ ＳＳＣ １００μｌ
ハイブリウェル ： Ｇｒａｃｅ ＢＩＯ−ＬＡＢＳ製Ｈｙｂｒｉｗｅｌｌ（ＨＢＷ７５）
反応温度 ： ４０℃
前処理 ： ９４℃ ５分間
・洗浄後処理：
スライドウオッシャー（十条フィールド株式会社ＳＷ−４）
・観察条件：
ＧＳＩ ｌｕｍｏｎｉｃｓ社ｓｃａｎａｒｒａｙ４０００を使用し，ＬＡＳＥＲ ／ ＰＭＴの設定はそれぞれ１００％ ／１００％とした。解像度は５μｍに設定している。
・定量方法：
市販のマイクロアレイ定量ソフトウェア（ＱｕａｎｔＡｒｒａｙ）にて定量した。
【０１００】
＜配列の詳細情報＞
以下に使用した配列について記載する。配列設計にはクロスハイブリ特性，Ｔｍ値を考慮して人工的に設計した配列を使用している。
（表記は５’→３’）
▲１▼ターゲット核酸配列
・５’末端Ｃｙ３標識ｒＥ２４３ ７５ｍｅｒ
ＴｇＡＡｇＴＴＣＣｇＣＴＴＴｇｇＡｇＴｇｇＣＴＡＡｇｇｇＡｇＣＡＡＡＴＡＴＡＡＡＴｇＣｇＡＴｇｇＣＴＴＴｇｇＴＴＡＡＣＴｇＣＣＴＴＣＣＣＣＴＣＡＣＡＡ（配列番号７）
▲２▼プローブ配列
・５’末端アミノ基標識ＣＤ１４７ プローブ（ターゲット核酸の５’端側２５ｍｅｒに相補）
ＴＴＡｇＣＣＡＣＴＣＣＡＡＡｇＣｇｇＡＡＣＴＴＣＡ（配列番号８）
・５’末端アミノ基標識ＣＤ２４３ プローブ（ターゲット核酸の中央２５ｍｅｒに相補）
ＡｇＣＣＡＴＣｇＣＡＴＴＴＡＴＡＴＴＴｇＣＴＣＣＣ（配列番号９）
［実施例２］
《プローブの反応特異性と共通相補配列の延長方向に関する実施例》
＜実験方法＞
▲１▼ターゲット核酸として、５’末端をＣｙ３標識したｒＣ２４３配列７５ ｍｅｒを使用した。ｒＣ２４３配列は、それぞれ２５ ｍｅｒの３’部、中央部、５’部の３つの部分配列からなる全長７５ ｍｅｒの配列である。
【０１０１】
▲２▼前記▲１▼のターゲット核酸に対するプローブとして、ｒＣ２４３にパーフェクトマッチするＣ２４３ プローブ ７５ ｍｅｒ，ｒＣ２４３の両末端から２５ ｍｅｒはマッチするが中央部２５ ｍｅｒがミスマッチとなっているＣ０３７ プローブ ７５ ｍｅｒ，ｒＣ２４３の５’末端から５０ｍｅｒまでに相補な２４３＋ＣＥＤ プローブ ５０ ｍｅｒ，ｒＣ２４３の５’末端から２５ｍｅｒまで相補であるがｒＣ２４３の中央部２５ ｍｅｒとはミスマッチな０３７＋ＣＥＤ プローブ ５０ ｍｅｒ，ｒＣ２４３の３’末端から５０ｍｅｒに相補である０１４ＳＤ＋２４３ プローブ ５０ｍｅｒおよびｒＣ２４３の中央部２５ ｍｅｒに相補であるＣＤ２４３ プローブを使用した。
【０１０２】
前記ターゲット核酸及び前記プローブが固相化されたマイクロアレイを用いたハイブリダイゼーションにより該プローブを評価した。実験に使用した装置、器具、試薬の詳細を実験結果の後に記載する。
【０１０３】
まず，５’末端をＣｙ３標識したｒＣ２４３が５ｐｍｏｌ含まれるように１ ｘ ＳＳＣ（０．２％ ＳＤＳ含有） １００μｌ中に調製し，これをターゲット核酸 溶液とした。このターゲット核酸 溶液をハイブリ前に９４℃で５分間加熱し，前処理を行なった。
【０１０４】
プローブを固相化した検出用マイクロアレイにハイブリウェルを装着した。直前に前処理したターゲット核酸 溶液 １００μｌをハイブリウェルにインジェクトし，このマイクロアレイを４０℃のインキュベータ中で６０分間のハイブリダイゼーション反応を行なった。
【０１０５】
次にハイブリ後のマイクロアレイをインキュベータから取り出し，ハイブリウェルを剥離した後すばやく０．１ ｘ ＳＳＣで満たした染色バットに投入し，ラックとスライドウオッシャーを使用して５分間揺動洗浄した後，フロンガス（ＨＦＣ１３４ａ）を使用したブロアーで乾燥させた。これをＳｃａｎＡｒｒａｙ４０００を用いて観察し，ｓｃａｎ画像を市販の定量化ソフトウェアをもちいて定量した。
【０１０６】
＜実験結果＞
以下に示す結果は各プローブ，４スポットの定量値を平均化した値である。
【０１０７】
【表２】

【０１０８】
以上の結果から５’末端固相化プローブ（ターゲット核酸の５’末端との相補性を利用してハイブリするプローブ）は、ターゲット核酸に対してマッチする配列の鎖長にかかわらずハイブリ効率が高いことが明らかになった。しかし， ５’末端部分２５ｍｅｒのみがマッチすれば、中央２５ｍｅｒ部分での反応特異性がなくとも反応することが明らかとなった（０３７＋ＣＥＤ プローブ）。つまりＰＭ／ＭＭ比（パーフェクトマッチ／ミスマッチ比）が小さいため、中央２５ｍｅｒ部分での反応特異性を判別出来ないことが明らかとなった。
【０１０９】
また，３’末端捕捉型プローブ（同じターゲット核酸末端を利用する５０ｍｅｒのプローブであるが、ターゲット核酸の３’末端を捕捉する形のプローブ；０１４ＳＤ＋２４３ プローブ）のハイブリ効率が格段に低いことが明らかになった。中央２５ｍｅｒ部分のみと相補なＣＤ２４３プローブのハイブリ効率は更に低いものであった。
【０１１０】
＜実験条件の詳細＞
・検出用チップ：
アミノ基標識した合成オリゴＤＮＡを市販のスライドガラス基板と共有結合させる当業者に通常用いられる方法により作成した。基材は通常の白板のスライドガラスを使用した。スポッティングは１５０μｍのピンヘッドを持つ市販のスポッタ（ピンピッチ４．５ｍｍ，スポットピッチ０．５ｍｍ）をもちいて，各プローブを前記基材上に４スポットずつ点着した。
【０１１１】
・検出ターゲット核酸：
検出する配列と同じ配列をもつ合成オリゴＤＮＡをテンプレートとして，ビオチン化したｆｗｄプライマ及びＣｙ３標識したｒｅｖプライマを使用してＰＣＲ反応をおこなった。そのＰＣＲ産物をストレプトアビジン磁気ビーズにより精製した。
【０１１２】
ストレプトアビジン磁気ビーズは高温度条件下においてもビオチンと比較的安定した結合を形成することを利用してＣｙ３化単鎖ＤＮＡ（ｓｓ−ＤＮＡ）を単離する。即ち、磁気ビーズにＰＣＲ産物を捕獲し、それを熱水中でデハイブリダイゼーションして、Ｃｙ３化されたｓｓ−ＤＮＡ ターゲット核酸のみを単離した（図２を参照）。
【０１１３】
・ハイブリダイゼーション条件：
ターゲット核酸 ： Ｃｙ３標識ｒＣ２４３ ５ ｐｍｏｌ
反応溶液 ： ０．２％ ＳＤＳ １ ｘ ＳＳＣ １００μｌ
ハイブリウェル ： Ｇｒａｃｅ ＢＩＯ−ＬＡＢＳ製Ｈｙｂｒｉｗｅｌｌ（ＨＢＷ７５）
反応温度 ： ４０℃
前処理 ： ９４℃ ５分間
・洗浄後処理：
スライドウオッシャー（十条フィールド株式会社ＳＷ−４）
・観察条件：
ＧＳＩ ｌｕｍｏｎｉｃｓ社ｓｃａｎａｒｒａｙ４０００を使用し，ＬＡＳＥＲ ／ ＰＭＴの設定はそれぞれ１００％ ／１００％とした。解像度は５μｍに設定している。
・定量方法：
市販のマイクロアレイ定量ソフトウェア（ＱｕａｎｔＡｒｒａｙ）にて定量した。
【０１１４】
＜配列情報の詳細＞
以下に使用した配列について記載する。配列設計にはクロスハイブリ，Ｔｍ値を考慮して人工的に設計した配列を使用している。
（表記は５’→３’）
▲１▼ターゲット核酸配列
・５’末端Ｃｙ３標識ｒＣ２４３ ７５ｍｅｒ
ｇＡＴＴＣｇＴＣｇＴｇＴｇＣＣＴＴＴｇＡＣＴｇＴＴｇｇｇＡｇＣＡＡＡＴＡＴＡＡＡＴｇＣｇＡＴｇｇＣＴＴＴｇｇＴＴＡＡＣＴｇＣＣＴＴＣＣＣＣＴＣＡＣＡＡ（配列番号１０）
▲２▼プローブ配列
・５’末端アミノ基標識Ｃ２４３ プローブ ７５ ｍｅｒ （ターゲット核酸にパーフェクトマッチ）
ＴＴｇＴｇＡｇｇｇｇＡＡｇｇｃＡｇＴＴＡＡｃｃＡＡＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ（配列番号１１）
・５’末端アミノ基標識ＣＤ０３７ プローブ ７５ ｍｅｒ（ターゲット核酸の中央部２５ｍｅｒのみミスマッチ）
ＴＴｇＴｇＡｇｇｇｇＡＡｇｇｃＡｇＴＴＡＡｃｃＡＡＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ（配列番号１２）
・５’末端アミノ基標識 ２４３＋ＣＥＤ プローブ ５０ ｍｅｒ（ターゲット核酸５’末端から５０ｍｅｒに相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ（配列番号１３）
・５’末端アミノ基標識 ０３７＋ＣＥＤ プローブ ５０ ｍｅｒ（ターゲット核酸５’末端から２５ｍｅｒにマッチ，ターゲット核酸の中央部２５ｍｅｒにはミスマッチ）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＡＡｃｃＴｃｃｃＡＴＴｃＡｃｃＡＴＴｇＴｃＡｇＡｇ（配列番号１４）
・５’末端アミノ基標識 ０１４ＳＤ＋２４３ プローブ ５０ ｍｅｒ（ターゲット核酸３’末端から５０ｍｅｒに相補）
ＴＴｇＴｇＡｇｇｇｇＡＡｇｇｃＡｇＴＴＡＡｃｃＡＡＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃ（配列番号１５）
・５’末端アミノ基標識 ＣＤ２４３ プローブ ２５ ｍｅｒ（ターゲット核酸の中央部２５ｍｅｒに相補）
ＡｇＣＣＡＴＣｇＣＡＴＴＴＡＴＡＴＴＴｇＣＴＣＣＣ（配列番号１６）
［実施例３］
《ＥＤ延長型プローブとターゲット核酸５’末端捕捉型プローブ》
＜実験方法＞
▲１▼ターゲット核酸として、５’末端をＣｙ３標識したｒＣ２１９配列７５ｍｅｒを使用した。ｒＣ２１９配列は、それぞれ２５ ｍｅｒの３’部、中央部、５’部の３つの部分配列からなる全長７５ ｍｅｒの配列である。
【０１１５】
▲２▼前記▲１▼のターゲット核酸に対するプローブとして、以下のＡ〜Ｈにグループ分けされるプローブ、即ち、
（Ａ） ：コントロールプローブ；
ターゲット核酸の中央部２５ｍｅｒに対応する配列を具備するプローブ。
（Ｂ〜Ｅ）：ＥＤ延長型プローブ；
ターゲット核酸中央部２５ｍｅｒに対応する配列に加え、該配列の３’末端に連続的に、対応するターゲット核酸の配列に相補となるように１０， １５， １８， ２１ ｍｅｒと延長した配列を更に具備する、それぞれ３５， ４０， ４３， ４６ ｍｅｒのプローブ，
（Ｆ〜Ｈ）：ターゲット核酸５’末端捕捉型プローブ；
ターゲット核酸中央部２５ｍｅｒに対応する配列に加え、該配列の３’末端側にミスマッチ配列をはさみ、更にターゲット核酸の５’末端から５， １０， １５ ｍｅｒの配列に相補な配列を具備する５０ ｍｅｒのプローブ，
を使用した。
【０１１６】
上述のＡ〜Ｈの各々のプローブ群は、前記「ターゲット核酸の中央部２５ｍｅｒに対応する配列」が、ターゲット核酸中央部と相補になるよう設計された「中央部ＰＭプローブ」、及びターゲット核酸中央部とミスマッチになるよう設計された「中央部ＭＭプローブ」とに更に分けられる。このような２種類のプローブの反応性を比較検討することによって、上述した各種プローブのターゲット核酸中央部２５ｍｅｒへの反応特異性を評価することができる。
【０１１７】
上記の（１）のターゲット核酸及び（２）のプローブが固相化されたマイクロアレイを用いたハイブリダイゼーションにより該プローブを評価した。実験に使用した装置、器具、試薬の詳細を実験結果の後に記載する。
【０１１８】
まず，市販のハイブリダイゼーションバッファ１００μｌ中に５’末端をＣｙ３標識したｒＣ２１９が５ｐｍｏｌ含まれるように調製し，これをターゲット核酸 溶液とした。
【０１１９】
前記プローブが固相化された検出用マイクロアレイにハイブリウェルを装着した。ハイブリ直前に前処理したターゲット核酸 溶液 １００μｌをハイブリウェルにインジェクトし，このマイクロアレイを５０℃のインキュベータ中でハイブリダイゼーション反応を３時間行なった。
【０１２０】
次にハイブリ後のマイクロアレイをインキュベータから取り出し，ハイブリウェルを剥離した後すばやく１ ｘ ＳＳＣで満たした染色バットに投入し，ラックとスライドウオッシャーを使用して１０ｍｉｎ揺動洗浄した。つづいて同様に０．１ ｘ ＳＳＣで５ｍｉｎ揺動洗浄した後，ＨＦＣ１３４ａを使用したブロアーで乾燥させた。
【０１２１】
これをＳｃａｎＡｒｒａｙ４０００を用いて観察し，スキャン画像を市販の定量化ソフトウェアをもちいて定量した。
【０１２２】
＜実験結果＞
以下に示す結果は各プローブ，４スポットの定量値を平均化した値である。
【０１２３】
【表３】

【０１２４】
【表４】

【０１２５】
表３及び表４中のＣＤはターゲット核酸 ７５ ｍｅｒ中央部２５ｍｅｒの配列名を示し，該ＣＤは本実施例で使用したターゲット核酸であるｒＣ２１９配列中の２１９ プローブに対して相補な配列を意味する。
ターゲット核酸５’端方向延長型プローブでは，プローブ長が各プローブで異なり，＋ １０ ｍｅｒ，＋ １５ｍｅｒ，＋ １８ ｍｅｒ，＋ ２１ ｍｅｒはそれぞれ全長が３５ ｍｅｒ， ４０ ｍｅｒ， ４３ ｍｅｒ， ４６ ｍｅｒとなる。
【０１２６】
ターゲット核酸５’末端捕捉型プローブについてはコントロール ２５ ｍｅｒはＣＤ（ターゲット核酸の中央部２５ ｍｅｒのみ），＋ ５ ｍｅｒ， ＋ １０ ｍｅｒ， ＋ １５ ｍｅｒはそれぞれＣＤ ２５ ｍｅｒに加え２０ ｍｅｒ， １５ ｍｅｒ， １０ ｍｅｒのミスマッチ領域をはさみターゲット核酸の５’端から相補な配列を５ ｍｅｒ， １０ ｍｅｒ， １５ ｍｅｒもつ５０ ｍｅｒのプローブとなっている。
また，表３及び４は同一のマイクロアレイ上で得た結果であり，相互間で直接的な比較が可能である。
【０１２７】
結果については，以下のようにまとめられる。
（１）ターゲット核酸 ５’端方向延長型プローブにおいては＋ １０ ｍｅｒでＰＭのプローブはシグナルが約２倍に上昇したのに対しＭＭのプローブではほとんどシグナルの上昇が見られなかった。
【０１２８】
（２）ターゲット核酸 ５’端方向延長型プローブにおいては＋ １５ ｍｅｒ以上でＰＭのシグナル上昇とともにＭ Ｍのシグナル上昇も見られ始め配列特異性が失われ始めた。
【０１２９】
（３）ターゲット核酸 ５’末端捕捉型プローブにおいてはターゲット核酸 ５’末端捕捉長５ ｍｅｒにおいて既にシグナル増強が観察され，１０ ｍｅｒまではＰＭのシグナルのみが上昇しＭＭのシグナルはほとんど変化しない。
【０１３０】
（４）ターゲット核酸 ５’末端捕捉型プローブにおいてはターゲット核酸 ５’末端捕捉長１５ ｍｅｒにおいてＭＭのシグナル増強が見られる。
【０１３１】
（５）ターゲット核酸 ５’端方向延長型プローブとターゲット核酸 ５’末端捕捉型プローブを比較した場合，ターゲット核酸 ５’端方向延長型プローブの ＋１０ ｍｅｒ（マッチ長３５ ｍｅｒ）とターゲット核酸 ５’末端捕捉型プローブの ＋ ５ｍｅｒ（マッチ長３０ ｍｅｒ）でほぼ同等のハイブリ強度を示し，ターゲット核酸 ５’末端捕捉型プローブ ＋ １０ ｍｅｒ（マッチ長３５ ｍｅｒ）ではマッチ長はターゲット核酸 ５’端方向延長型プローブの ＋ １０ ｍｅｒと変わらないが，ターゲット核酸 ５’末端捕捉型の方がシグナルが強い（該条件における末端捕捉型の測定値は飽和しているので実際には更に強いシグナル強度である）。
【０１３２】
・検出用チップ：
アミノ基標識した合成オリゴＤＮＡを市販のスライドガラス基板と共有結合させる当業者に通常用いられる方法により作成した。基材は通常の白板のスライドガラスを使用した。スポッティングは１５０μｍのピンヘッドを持つ市販のスポッタ（ピンピッチ４．５ｍｍ，スポットピッチ０．５ｍｍ）をもちいて，各プローブを前記基材上に４スポットずつ点着した。
【０１３３】
・検出ターゲット核酸：
検出する配列と同じ配列をもつ合成オリゴＤＮＡをテンプレートとして，ビオチン化したｆｗｄプライマ及びＣｙ３標識したｒｅｖプライマを使用してＰＣＲ反応をおこなった。そのＰＣＲ産物をストレプトアビジン磁気ビーズにより精製した。
【０１３４】
ストレプトアビジン磁気ビーズは高温度条件下においてもビオチンと比較的安定した結合を形成することを利用してＣｙ３化単鎖ＤＮＡ（ｓｓ−ＤＮＡ）を単離する。即ち、磁気ビーズにＰＣＲ産物を捕獲し、それを熱水中でデハイブリダイゼーションして、Ｃｙ３化されたｓｓ−ＤＮＡ ターゲット核酸のみを単離した（図２を参照）。
【０１３５】
・ハイブリダイゼーション条件：
ターゲット核酸 ： Ｃｙ３標識ｒＣ２１９ ５ ｐｍｏｌ
反応溶液 ： ＧｌａｓｓＨｙｂ ハイブリダイゼーション 溶液 （ＣＬＯＮＴＥＣＨ）
ハイブリウェル ： Ｇｒａｃｅ ＢＩＯ−ＬＡＢＳ製Ｈｙｂｒｉｗｅｌｌ（ＨＢＷ７５）
反応温度 ： ５０℃
・洗浄後処理：
スライドウオッシャー（十条フィールド株式会社ＳＷ−４）
・観察条件：
ＧＳＩ ｌｕｍｏｎｉｃｓ社ｓｃａｎａｒｒａｙ４０００を使用し，ＬＡＳＥＲ ／ ＰＭＴの設定はそれぞれ１００％ ／１００％とした。解像度は５μｍに設定した。
・定量方法：
市販のマイクロアレイ定量ソフトウェア（ＱｕａｎｔＡｒｒａｙ）により定量を行った。
【０１３６】
＜配列情報の詳細＞
以下に使用した配列について記載する。配列設計にはクロスハイブリ，Ｔｍ値を考慮して人工的に設計した配列を使用している。
（表記は５’→３’）
▲１▼ターゲット核酸配列
・５’末端Ｃｙ３標識ｒＣ２１９ ７５ｍｅｒ
ｇＡＴＴＣｇＴＣｇＴｇＴｇＣＣＴＴＴｇＡＣＴｇＴＴＡＣＴｇＡＡＣＴＴＴＴＴＣＣＡＴｇＣＣｇｇＴＣＡＡＴＴｇｇＴＴＡＡＣＴｇＣＣＴＴＣＣＣＣＴＣＡＣＡＡ（配列番号１７）
▲２▼プローブ配列
コントロールプローブ；
（Ａ）ターゲット核酸中央部２５ｍｅｒに対応するプローブ
・５’末端アミノ基標識ＣＤ０３２ プローブ ２５ ｍｅｒ （ミスマッチプローブ）
ＡＴＴＡＣｇＣＡＣｇＡＡＡＴＴＡＣＡＣＣｇｇＴＡＣ（配列番号１８）
・５’末端アミノ基標識ＣＤ０３７ プローブ ２５ ｍｅｒ （ミスマッチプローブ）
ＴＴＡＡＴＣＡＣＣｇＡＣＴｇＴＣＴＴＣｇＴＴｇＣＣ（配列番号１９）
・５’末端アミノ基標識ＣＤ２１９ プローブ ２５ ｍｅｒ （ターゲット核酸の中央部２５ｍｅｒに相補）
ＴＴｇＡＣＣｇｇＣＡＴｇｇＡＡＡＡＡｇＴＴＣＡｇＴ（配列番号２０）
・５’末端アミノ基標識ＣＤ２４３ プローブ ２５ ｍｅｒ （ミスマッチプローブ）
ＡｇＣＣＡＴＣｇＣＡＴＴＴＡＴＡＴＴＴｇＣＴＣＣＣ（配列番号２１）
ＥＤ延長型プローブ；
（Ｂ） ターゲット核酸中央部２５ｍｅｒに加えターゲット核酸 ５’末端方向に１０ ｍｅｒ延長した配列に対応するプローブ
・５’末端アミノ基標識０３２ ＋ １０ ｍｅｒ プローブ ３５ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１０ｍｅｒ相補）
ＡＴＴＡｃｇｃＡｃｇＡＡＡＴＴＡｃＡｃｃｇｇＴＡｃＡＡｃＡｇＴｃＡＡＡ（配列番号２２）
・５’末端アミノ基標識０３７ ＋ １０ ｍｅｒ プローブ ３５ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１０ｍｅｒ相補）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＡＡｃＡｇＴｃＡＡＡ（配列番号２３）
・５’末端アミノ基標識２１９ ＋ １０ ｍｅｒ プローブ ３５ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒに相補，ターゲット核酸 ５’側１０ｍｅｒ相補）
ＴＴｇＡｃｃｇｇｃＡＴｇｇＡＡＡＡＡｇＴＴｃＡｇＴＡＡｃＡｇＴｃＡＡＡ（配列番号２４）
・５’末端アミノ基標識２４３ ＋ １０ ｍｅｒ プローブ ３５ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１０ｍｅｒ相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＡＡｃＡｇＴｃＡＡＡ（配列番号２５）
（Ｃ）ターゲット核酸中央部２５ｍｅｒに加えターゲット核酸 ５’末端方向に１５ ｍｅｒ延長した配列に対応するプローブ
・５’末端アミノ基標識０３２ ＋ １５ ｍｅｒ プローブ ４０ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１５ｍｅｒ相補）
ＡＴＴＡｃｇｃＡｃｇＡＡＡＴＴＡｃＡｃｃｇｇＴＡｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃ（配列番号２６）
・５’末端アミノ基標識０３７ ＋ １５ ｍｅｒ プローブ ４０ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１５ｍｅｒ相補）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃ（配列番号２７）
・５’末端アミノ基標識２１９ ＋ １５ ｍｅｒ プローブ ４０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒに相補，ターゲット核酸 ５’側１５ｍｅｒ相補）
ＴＴｇＡｃｃｇｇｃＡＴｇｇＡＡＡＡＡｇＴＴｃＡｇＴＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃ（配列番号２８）
・５’末端アミノ基標識２４３ ＋ １５ ｍｅｒ プローブ ４０ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１５ｍｅｒ相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃ（配列番号２９）
（Ｄ） ターゲット核酸中央部２５ｍｅｒに加えターゲット核酸 ５’末端方向に１８ ｍｅｒ延長した配列に対応するプローブ
・５’末端アミノ基標識０３２ ＋ １８ ｍｅｒ プローブ ４３ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１８ｍｅｒ相補）
ＡＴＴＡｃｇｃＡｃｇＡＡＡＴＴＡｃＡｃｃｇｇＴＡｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇ（配列番号３０）
・５’末端アミノ基標識０３７ ＋ １８ ｍｅｒ プローブ ４３ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１８ｍｅｒ相補）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇ（配列番号３１）
・５’末端アミノ基標識２１９ ＋ １８ ｍｅｒ プローブ ４３ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒに相補，ターゲット核酸 ５’ 側１８ｍｅｒに相補）
ＴＴｇＡｃｃｇｇｃＡＴｇｇＡＡＡＡＡｇＴＴｃＡｇＴＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇ（配列番号３２）
・５’末端アミノ基標識２４３ ＋ １８ ｍｅｒ プローブ ４３ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側１８ｍｅｒ相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇ（配列番号３３）
（Ｅ） ターゲット核酸中央部２５ｍｅｒに加えターゲット核酸 ５’末端方向に２１ ｍｅｒ延長した配列に対応するプローブ
・５’末端アミノ基標識０３２ ＋ ２１ ｍｅｒ プローブ ４６ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側２１ ｍｅｒ相補）
ＡＴＴＡｃｇｃＡｃｇＡＡＡＴＴＡｃＡｃｃｇｇＴＡｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇ（配列番号３４）
・５’末端アミノ基標識０３７ ＋ ２１ ｍｅｒ プローブ ４６ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側２１ ｍｅｒ相補）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇ（配列番号３５）
・５’末端アミノ基標識２１９ ＋ ２１ ｍｅｒ プローブ ４６ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒに相補，ターゲット核酸 ５’側２１ ｍｅｒ相補）
ＴＴｇＡｃｃｇｇｃＡＴｇｇＡＡＡＡＡｇＴＴｃＡｇＴＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇ（配列番号３６）
・５’末端アミノ基標識２４３ ＋ ２１ ｍｅｒ プローブ ４６ ｍｅｒ （ターゲット核酸中央部２５ｍｅｒミスマッチ，ターゲット核酸５’側２１ ｍｅｒ相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇ（配列番号３７）
ターゲット核酸５’末端捕捉型プローブ；
（Ｆ）ターゲット核酸中央部２５ｍｅｒに対応する配列と、ターゲット核酸 ５’末端から５ｍｅｒに相補的な配列とが、ミスマッチ領域２０ｍｅｒを介して連結されているプローブ
このミスマッチを意図した部分には，この領域に含まれる４種類の塩基のうちで最も含有量の少ないＴで置換することにした。
【０１３７】
＊参考 ＣＥＤのプローブ配列： ＡＡｃＡｇＴｃＡＡＡｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ （配列番号３８）の左から２０塩基の部分において
Ａ：９ ４５％
Ｔ：１ ５％
ｃ：６ ３０％
ｇ：４ ２０％
・５’末端アミノ基標識０３２ ＋ ５’ ５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ ２０のミスマッチ領域を介して ターゲット核酸５’末端から５ ｍｅｒ相補）
ＡＴＴＡｃｇｃＡｃｇＡＡＡＴＴＡｃＡｃｃｇｇＴＡｃＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴｇＡＡＴｃ（配列番号３９）
・５’末端アミノ基標識０３７ ＋ ５’ ５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ ２０のミスマッチ領域を介してターゲット核酸５’末端から５ ｍｅｒ相補）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴｇＡＡＴｃ（配列番号４０）
・５’末端アミノ基標識２１９ ＋ ５’ ５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒに相補，Ｔ ｘ ２０のミスマッチ領域を介してターゲット核酸５’末端から５ ｍｅｒ相補）
ＴＴｇＡｃｃｇｇｃＡＴｇｇＡＡＡＡＡｇＴＴｃＡｇＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴｇＡＡＴｃ（配列番号４１）
・５’末端アミノ基標識２４３ ＋ ５’ ５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ ２０のミスマッチ領域を介してターゲット核酸５’末端から５ ｍｅｒ相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴｇＡＡＴｃ（配列番号４２）
（Ｇ）． ターゲット核酸中央部２５ｍｅｒに対応する配列と、ターゲット核酸５’末端から１０ ｍｅｒに相補な配列とが、とミスマッチ領域１５ ｍｅｒを介して連結されているプローブ
・５’末端アミノ基標識０３２ ＋ ５’ １０ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ １５のミスマッチ領域を介して ターゲット核酸５’末端から１０ ｍｅｒ相補）
ＡＴＴＡｃｇｃＡｃｇＡＡＡＴＴＡｃＡｃｃｇｇＴＡｃＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＡｃｇＡｃｇＡＡＴｃ（配列番号４３）
・５’末端アミノ基標識０３７ ＋ ５’ １０ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ １５のミスマッチ領域を介してターゲット核酸５’末端から１０ ｍｅｒ相補）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＡｃｇＡｃｇＡＡＴｃ（配列番号４４）
・５’末端アミノ基標識２１９ ＋ ５’ １０ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒに相補，Ｔ ｘ １５のミスマッチ領域を介してターゲット核酸５’末端から１０ ｍｅｒ相補）
ＴＴｇＡｃｃｇｇｃＡＴｇｇＡＡＡＡＡｇＴＴｃＡｇＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＡｃｇＡｃｇＡＡＴｃ（配列番号４５）
・５’末端アミノ基標識２４３ ＋ ５’ １０ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ １５のミスマッチ領域を介してターゲット核酸５’末端から１０ ｍｅｒ相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＴＴＴＴＴＴＴＴＴＴＴＴＴＴＴＡｃｇＡｃｇＡＡＴｃ（配列番号４６）
（Ｈ）ターゲット核酸中央部２５ｍｅｒに対応する配列と、ターゲット核酸 ５’末端から１５ ｍｅｒに相補な配列とが、ミスマッチ領域１０ ｍｅｒを介して連結されているプローブ
・５’末端アミノ基標識０３２ ＋ ５’ １５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ １０のミスマッチ領域を介して ターゲット核酸５’末端から１５ ｍｅｒ相補）
ＡＴＴＡｃｇｃＡｃｇＡＡＡＴＴＡｃＡｃｃｇｇＴＡｃＴＴＴＴＴＴＴＴＴＴｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ（配列番号４７）
・５’末端アミノ基標識０３７ ＋ ５’ １５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ １０のミスマッチ領域を介してターゲット核酸５’末端から１５ ｍｅｒ相補）
ＴＴＡＡＴｃＡｃｃｇＡｃＴｇＴｃＴＴｃｇＴＴｇｃｃＴＴＴＴＴＴＴＴＴＴｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ（配列番号４８）
・５’末端アミノ基標識２１９ ＋ ５’ １５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒに相補，Ｔ ｘ １０のミスマッチ領域を介してターゲット核酸５’末端から１５ ｍｅｒ相補）
ＴＴｇＡｃｃｇｇｃＡＴｇｇＡＡＡＡＡｇＴＴｃＡｇＴＴＴＴＴＴＴＴＴＴＴｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ（配列番号４９）
・５’末端アミノ基標識２４３ ＋ ５’ １５ ｍｅｒ プローブ ５０ ｍｅｒ （ターゲット核酸中央部２５ ｍｅｒミスマッチ，Ｔ ｘ １０のミスマッチ領域を介してターゲット核酸５’末端から１５ ｍｅｒ相補）
ＡｇｃｃＡＴｃｇｃＡＴＴＴＡＴＡＴＴＴｇｃＴｃｃｃＴＴＴＴＴＴＴＴＴＴｇｇｃＡｃＡｃｇＡｃｇＡＡＴｃ（配列番号５０）
【０１３８】
【発明の効果】
プローブがターゲット核酸に特異的な配列以外に、共通配列に相補的な配列を備えるので、
１）プローブ長を長く設定できる。このことからより高温でハイブリダイゼーション反応を実施できる。従って、ハイブリダイゼーション反応中にターゲット核酸が安定な構造をとりにくくなり、ターゲット核酸の反応性が高まる。
【０１３９】
２）すべての検出用プローブが共通な配列をそなえることになる。これに対してプローブの共通相補配列部分にハイブリダイズする検出用の標識をした核酸を準備する。この標識をした核酸をハイブリダイズさせ、プローブの固定量を計測できる。
【０１４０】
３）各プローブの固定化量の比を計測することができ、さらに絶対量を決めた標準検体を用いればすべてのプローブで検出しているターゲット核酸の量を計測することができる。
【０１４１】
また、前記プローブに更にリンカー配列を備えたプローブを使用することにより、よりハイブリダイゼーションがおきやすく、反応速度が上がる。長さの割に、分子内構造をとりにくい、低温で反応できるプローブが得られる。また、高感度な核酸検出ができる。
【０１４２】
【配列表】

【図面の簡単な説明】
【図１】共通プライマー配列をそなえたターゲット核酸の遺伝子検出への使用例を示す図である。
【図２】ターゲット核酸の配列構成を示す図である。
【図３】ターゲット核酸検出のための従来のプローブの例を示す図である。
【図４】延長型プローブによるハイブリッド形成の形態を説明する図である。
【図５】末端捕捉型プローブによるハイブリッド形成の形態を説明する図である。
【図６】プローブ固定量の測定方法を説明する図である。
【図７】ターゲット核酸の末端を捕捉するとハイブリ効率が向上する理由を説明する図である。
【図８】ターゲット核酸の中央部でのハイブリと５’末端部でのハイブリの比較実験の概略を説明する図である。
【図９】プローブの反応特異性と共通相補配列の延長方向に関する比較実験の概略を説明する図である。
【図１０】ＥＤ延長型プローブと末端捕捉型プローブに関する比較実験の概略を説明する図である。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a probe technology for detecting a nucleic acid.
[0002]
[Prior art]
DNA microarrays have a structural feature in that many types of probes are immobilized at high density on an inert support, and can simultaneously detect and measure many types of nucleic acid samples such as genes present in the same sample. This is a method for analyzing nucleic acids having functional characteristics. Therefore, in the art, it is assumed that many types of probes are reacted under the same conditions, and it is generally considered that each probe preferably has a uniform reactivity.
[0003]
DNA microarrays include a method in which cDNA derived from a living body is fixed on a support and a method in which synthetic oligo DNA probes are fixed. In the latter method, the DNA probe is prepared by chemically synthesizing the DNA probe, so that the probe sequence can be arbitrarily set. Therefore, it is relatively easy to prepare various kinds of probe groups having the above-mentioned uniform reactivity.
[0004]
In designing a probe group having uniform reactivity, preferable conditions that the DNA sequence of each probe should have are (1) that it is complementary only to the gene of the detection target (target nucleic acid), (2) the melting temperature ( Tm) in a certain range, and (3) an array that is difficult to take a secondary structure. Appropriate sequences are selected according to such criteria so that the detection results for each gene do not greatly differ.
[0005]
However, whether the probe DNA of the microarray is solid-phase synthesized (Patent Literature 1) or fixed by printing with a pin (Patent Literature 2), the amount of probe fixed between spots is always uneven. For this reason, even if the reactivity is homogenized using a probe whose appropriate sequence has been selected, the method of comparing the measured values between spots cannot quantify the abundance of each gene in the sample. Until now it was impossible.
[0006]
Next, the target nucleic acid detected by the above-described probe will be described. In the prior art, the target nucleic acid to be detected is obtained by converting mRNA extracted and purified from a living body into cDNA. However, inventions called tags (Patent Literature 3, Patent Literature 4), zip codes (Patent Literature 5) or orthonormalized arrays (Patent Literature 6) have recently been filed. These techniques are methods of converting each gene sequence into a format containing an oligo DNA called a tag designed to have a uniform Tm and to reduce cross-hybridization, and to detect the sequence. That is, each gene sequence is converted into a nucleic acid (tagged target nucleic acid) having both the tag sequence and the gene-specific gene sequence by this conversion. By this processing, a plurality of types of tag sequences can be added to a specific nucleic acid, and a nucleic acid having information of various tags can be prepared for comparative analysis.
[0007]
Also, a method of amplifying a tagged target nucleic acid (Patent Document 7) has been reported. In this method, each gene sequence is first converted into a tagged target nucleic acid having appropriate primer sequences at both ends. Next, the tagged target nucleic acid is amplified by polymerase chain reaction (PCR) using a primer set complementary to the primer sequence. In this way, the tagged target nucleic acid can be amplified. To detect the tagged target nucleic acid amplified in this way, magnetic microbeads or fluorescently labeled microbeads having a probe complementary to the sequence to be detected immobilized thereon (Patent Document 8), or a microarray in which probes are spotted on a slide glass, etc. Used.
[0008]
Further, if the primer is a common primer common to each tagged target nucleic acid, each tagged target nucleic acid can be amplified simultaneously using the common primer.
Generally, a sequence of about 20 to 30 bases suitable for PCR is selected as these common primer sequences.
[0009]
As described above, in the technique using a tag, a sequence of at least about 20 bases is selected for each of the tag sequence and the common primer sequence in addition to the gene sequence. Therefore, the length of the tagged target nucleic acid, which is the nucleic acid to be detected, must be a relatively long nucleic acid of about 60 bases at least.
[0010]
However, sequences common to each tagged target nucleic acid are not used due to cross-reactivity (cross-hybridization) issues. Thus, in contrast, the chain length that can be used substantially as a probe is limited to a short one. That is, for a typical purpose of obtaining information on each gene, only a sequence specific to each gene in the tagged target nucleic acid is used as a probe sequence. Similarly, only a tag sequence specific to a tag is used as a probe sequence for the purpose of acquiring information on each tag. Thus, in the prior art, only the sequence complementary to the tag sequence or gene sequence in the tagged target nucleic acid is used as the probe sequence.
[0011]
It is generally known that DNA having a long chain length of about 60 bases easily takes an intramolecular structure. For this reason, a tagged target nucleic acid having a long chain length has low reactivity with a probe and cannot be said to be optimal as a nucleic acid to be detected. One of the problems with using tag technology is such low reactivity. As a means for improving the reactivity with the probe, it is conceivable to set the probe length to be longer and hybridize at a higher temperature. However, it has never been performed because of the limitation of the probe length described above.
[0012]
[Patent Document 1]
Japanese Patent Publication No. 9-501561
[0013]
[Patent Document 2]
Japanese Patent No. 3272365
[0014]
[Patent Document 3]
Japanese Patent Publication No. 10-507357
[0015]
[Patent Document 4]
JP-T-2002-518060
[0016]
[Patent Document 5]
JP 2001-519648 A
[0017]
[Patent Document 6]
JP-A-2002-181813
[0018]
[Patent Document 7]
JP 2001-514483 A
[0019]
[Patent Document 8]
Japanese Patent Publication No. 10-507357
[0020]
[Problems to be solved by the invention]
The conventional microarray has the following problems.
-It was difficult to measure the amount of probe fixed on each spot where the probe was fixed.
In addition, when a target nucleic acid having a common sequence such as a primer sequence common to a plurality of target nucleic acids at both ends is detected, there are the following problems.
-If the common sequence exists in the probe sequence, the target nucleic acid will hybridize to all the probes, and the specific target nucleic acid cannot be specifically detected.
-The target nucleic acid forms a relatively stable intramolecular secondary structure because it is relatively long. Therefore, hybridization was hardly formed between the target nucleic acid and the probe, that is, false negative data was easily generated.
-Although the target nucleic acid is relatively long and easy to take a structure, the length of the specific sequence at the center of the target nucleic acid is short and the hybridization stability is low, so that the detection sensitivity cannot be increased. Due to this low stability, the washing step of the non-specific adsorbate had to be performed under mild conditions, and false positive data was likely to be generated.
The present invention focuses on these points, and an object of the present invention is to provide a probe compound for more functional detection and detection of a target nucleic acid with high sensitivity and low cross-hybridization.
[0021]
[Means for Solving the Problems]
The present invention solves the problem of the present invention by providing a probe and a method described below. That is,
A probe for measuring a target nucleic acid having a common sequence at both ends of a specific sequence,
The probe includes, in addition to the specific complementary sequence complementary to the specific sequence, a common complementary sequence that is complementary to all or a part of the common sequence and shorter than the specific complementary sequence. Having a nucleic acid;
The common complementary sequence is linked directly or via a linker to the 3 ′ end and / or 5 ′ end of the specific complementary sequence; and
One end of the probe is immobilized directly on a support for immobilization or via a linker for immobilization,
A probe is provided.
[0022]
Further, a method for measuring the probe using a known amount of a fixed amount measurement nucleic acid containing a sequence complementary to the common sequence and labeled with a labeling substance,
1) a step of bringing the probe into contact with the fixed amount measurement nucleic acid and specifically hybridizing the two;
2) measuring the production of the hybrid in step 1 by a measuring means based on the labeling substance;
3) calculating the amount of the probe on the support based on the measurement result obtained in the step 2,
There is provided a method comprising:
[0023]
Furthermore, using the probe immobilized on a microarray and a known amount of a fixed amount measurement nucleic acid containing a sequence complementary to the common sequence and labeled with a labeling substance A, A method for measuring the abundance of a target nucleic acid,
1) preparing a sample containing the target nucleic acid labeled with a labeling substance B different from the labeling substance A;
2) a step of bringing the probe, the sample of step 1, and the nucleic acid for measuring an immobilized amount into contact with each other, and specifically hybridizing;
3) measuring the production of the hybrid in step 2 by a measuring means based on the labeling substance used;
4) calculating the abundance of the target nucleic acid in the sample based on the measurement result obtained in step 3;
With
The immobilization ratio of the probe at each spot on the microarray is calculated from the measurement result of the labeling substance A (calculated value A),
A hybridization amount of the known amount of the target nucleic acid used as the target nucleic acid to the probe is calculated from the measurement result of the labeling substance B (calculated value B),
A method is provided, wherein the abundance of each of a plurality of types of the target nucleic acids present in the sample is extrapolated from calculated values A and B and estimated.
[0024]
BEST MODE FOR CARRYING OUT THE INVENTION
For the purpose of specifically detecting only a specific target nucleic acid, it is common technical knowledge in the technical field to use a sequence specifically existing only in the target nucleic acid as a probe. Therefore, it is understood in the prior art that a sequence complementary to a sequence common to a plurality of target nucleic acids should be excluded from the probe.
The present invention has advantageous effects which cannot be expected from the related art by adopting a configuration different from the recognition of the related art.
That is, the present invention is to solve the problem of the present invention by providing a probe and a method described below.
[0025]
[Probe provided by the present invention]
According to the present invention, a probe for measuring a target nucleic acid having a common sequence at both ends of a specific sequence,
The probe includes, in addition to the specific complementary sequence complementary to the specific sequence, a common complementary sequence that is complementary to all or a part of the common sequence and shorter than the specific complementary sequence. Having a nucleic acid;
The common complementary sequence is linked directly or via a linker to the 3 ′ end and / or 5 ′ end of the specific complementary sequence; and
One end of the probe is immobilized directly on a support for immobilization or via a linker for immobilization,
A probe is provided.
[0026]
Hereinafter, the target nucleic acid targeted by the present invention will be described.
The target nucleic acid is an object (detected nucleic acid) to be detected and measured. The target nucleic acid is generally a nucleic acid having the common sequence at both ends of a nucleic acid having the specific sequence, the tag sequence and the like, but may be a nucleic acid having the common sequence at one end.
[0027]
The specific sequence is a partial sequence consisting of a part of a sequence limitedly included in a specific nucleic acid molecule (group) among nucleic acid molecules present in a sample to be detected.
In a general application, the specific sequence is a sequence derived from a specific gene or gene group (such as a gene family), and is a sequence that is specifically distinguished from a sequence derived from another gene or gene group. .
[0028]
The sequence information of the gene to be used as the specific sequence can be obtained from a public data bank or the like, and the information is appropriately selected from the sequence information of the gene to be actually tested according to a method generally used in a microarray or the like. be able to.
[0029]
The common sequence is a sequence provided at an end of a nucleic acid having the specific sequence, and is a sequence common to many nucleic acids. The consensus sequence can be designed using any specific sequence other than the specific sequence, but is preferably a primer sequence that can be used for PCR in general applications.
[0030]
The sequence used for the common sequence can be appropriately determined by those skilled in the art by comprehensively determining cross-hybridization, Tm value and the like according to the purpose to be used.
Representative examples of target nucleic acids targeted by the present invention are described in [Embodiments of the present invention] below.
[0031]
Hereinafter, components of the probe of the present invention will be described.
The specific complementary sequence is a sequence complementary to the specific sequence.
Therefore, the specific complementary sequence is appropriately selected from the whole or a part of the specific sequence according to a technique usually used in a microarray or the like depending on the purpose of the experiment.
[0032]
The general conditions that the specific complementary sequence should have are (1) that it is complementary only to the target nucleic acid to be detected, (2) that the melting temperature (Tm) is in a certain range, 3) It is difficult to take a secondary structure.
The number of bases of the specific complementary sequence is in the range of about 10 to 100 or about 10 to 50, more preferably 15 to 80, still more preferably 20 to 60, still more preferably 25 to 40, and most preferably 25 bases. Degree is used.
[0033]
The common complementary sequence is a sequence complementary to the common sequence.
Accordingly, the common complementary sequence is similarly selected from the whole or a part of the common sequence, but the chain length is selected to be shorter than the chain length of the specific complementary sequence.
[0034]
The chain length of the specific complementary sequence and the common complementary sequence can be appropriately set in consideration of various “chain length setting conditions” depending on the experiment to be used. The “chain length setting conditions” include “sequence conditions” such as GC content and Tm determined specifically for the sequence of the probe, and “hybridizing conditions” such as salt concentration and temperature of a buffer solution during hybridization. included.
[0035]
Those skilled in the art can implement the present invention by appropriately setting “chain length setting conditions”. A general procedure for determining the chain length of the specific complementary sequence and the common complementary sequence of the probe of the present invention will be described below.
[0036]
Procedure for probe length determination:
In general, the target nucleic acid is designed first, that is, the sequence composition and chain length of the consensus sequence on the target nucleic acid, and the sequence composition and chain length of the specific sequence. In a general application, the consensus sequence is a primer sequence for a polymerase chain reaction, and is set at both ends of the specific sequence.
Subsequently, a probe of the present invention capable of appropriately reacting with the target nucleic acid thus designed is secondly designed. That is, according to the sequence of the target nucleic acid, a specific complementary sequence having an “appropriate chain length” is added with a common complementary sequence having an “appropriate chain length” for design.
[0037]
The “appropriate chain length” of the specific complementary sequence is determined in consideration of the above “general conditions that the specific complementary sequence should have” in a plurality of probes used simultaneously.
[0038]
“Appropriate chain length” in the common complementary sequence can be set as follows.
That is, after a specific sequence to be used is determined and its chain length is determined, a specific complementary sequence and a mismatch sequence having the same chain length as the specific complementary sequence are designed.
Next, a common complementary sequence having several kinds of chain lengths is added to the same side end of each nucleic acid comprising the specific complementary sequence or the mismatch sequence. In this way, several pairs of positive and negative control probes, each having the same common complementary sequence, are created (ie, different pairs will have common complementary sequences of different chain lengths).
[0039]
Next, using the positive and negative control probes, the above-mentioned hybridization conditions which allow only the positive control to hybridize to the target nucleic acid, that is, depend on the specific complementary sequence, are set. Such conditions depending on the specific complementary sequence can be achieved by optimizing the conditions in a manner well known to those skilled in the art, such as setting the temperature and salt concentration at the time of hybridization to be high. Thus, experimental conditions such as “hybridization conditions” can be optimized.
[0040]
The “chain length setting condition” can be set by the above procedure.
However, in general applications, the number of bases of the common complementary sequence ranges from 1 to 5, 1 to 10, 1 to 15, 1 to 18, 1 to 21 or 1 to 30, preferably 5 to 10, 5 to 5. A range of 15, 5 to 18, 5 to 21, or 5 to 30, more preferably a range of 10 to 15, 10 to 18, or 10 to 21, most preferably about 10 bases is used.
[0041]
In the extended probe, the number of bases of the nucleic acid of the common complementary sequence is 1 to 5, 1 to 10, 1 to 15, 1 to 18 or 1 to 21, preferably 5 to 10, 5 to 15 or 5 to 5. 18, more preferably in the range of 10 to 15.
[0042]
The extended probe is an embodiment of the probe of the present invention, and is a probe in which the common complementary sequence is provided continuously at one end (both ends or one end) of the specific complementary sequence. Examples of this type of probe are illustrated in the following [Embodiments of the present invention].
[0043]
In the end capture probe, the number of bases of the nucleic acid of the common complementary sequence is 1 to 5, 1 to 10 or 1 to 15, preferably 5 to 10 or 5 to 15, more preferably 10 to 15. Ranges are used.
[0044]
The end capture probe is one embodiment of the probe of the present invention, and is a probe in which the common complementary sequence is provided indirectly via a linker at both ends (both ends or one end) of the specific complementary sequence. is there. An example of this type of probe is also exemplified in the following [Embodiments of the present invention].
[0045]
The subject of the present invention can be achieved as long as the nucleic acid in the probe of the present invention hybridizes with the target nucleic acid (forms a complementary strand) with at least a specific complementary sequence portion and a common complementary sequence. Accordingly, the probes of the present invention may be nucleic acids that are rare or non-natural in nature, such as modified nucleic acids. However, generally, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), etc., which are usually found in living organisms, are preferably used.
[0046]
The linker in the probe of the present invention connects the common complementary sequence and the specific complementary sequence constituting the probe of the present invention at both ends of the linker.
It is preferable that the orientation of both complementary sequences in the probe is designed to be constant throughout the probe. However, even if the directions are reversed in the probe, it is sufficient that the common complementary sequence and the specific complementary sequence can specifically form a hybrid with the target nucleic acid, and the length of the linker is sufficiently long to increase the mobility. Such a configuration can be adopted as long as the member is provided.
[0047]
The linker is not limited as long as the probe to which the linker is linked can be used substantially in experiments, and may be any as long as it can serve as a spacer. The linker length (linker length), low reactivity, It is selected in consideration of mobility and the like.
The linker length may be any length as long as the common complementary sequence and the specific complementary sequence to the target nucleic acid can form a complementary hybrid. In general, when a common complementary sequence and a specific complementary sequence are individually hybridized to a target nucleic acid, the distance may be approximately the same as the distance that can be taken between the two sequences.
The low reactivity means that the linker portion of the probe of the present invention does not interact with other substances at the time of hybridization or the degree thereof is small.
The mobility means that the linker portion has high mobility in a liquid and can be freely bent.
Specific examples of the linker include a linear carbon (alkyl chain), preferably a nucleic acid composed of an adenine base, a nucleic acid composed of a thymine base, and a polypeptide chain. When the linker is a nucleic acid and the entire probe is composed of only the nucleic acid, the entire probe can be synthesized. However, when the members constituting the probe cannot be synthesized by the same synthetic system, the probe is prepared by covalently bonding an appropriate functional group of each member in an appropriate manner.
[0048]
The linker does not contribute to the stabilization of the hybrid, but by appropriately setting the linker, the common complementary sequence at the probe end can be set to form a hybrid with the most reactive end of the target nucleic acid. When a nucleic acid is used as the linker, the sequence is set to mismatch, so that the binding of the target nucleic acid can be strengthened without particularly increasing the hybridization temperature. In particular, if a relatively mismatched base such as A or T is used as the linker sequence, the probe length can be increased while the probe hardly takes an intramolecular structure.
[0049]
The case where a nucleic acid having a DNA sequence is used as the “linker” will be specifically described below.
General precautions when selecting a linker sequence (nucleic acid sequence used for the linker) include the following 1) to 2).
[0050]
1) It is preferable not to use relatively stable bases, such as G and C, even for a very complicated sequence or mismatch, because it may cause cross-hybridization to form a hybrid with an unintended target nucleic acid. When such a sequence is adopted, a specific target nucleic acid is easily attached to all probes because the linker sequence is present in all probes. Also,
2) When the linker sequence is made a perfect match sequence, the probe length becomes longer, so that the hybridization temperature must be higher, and the experiment becomes difficult from the aspects of evaporation of the liquid, temperature control, and the like.
[0051]
The linker sequence is selected in consideration of the above-mentioned precautions, and will be described in more detail below with reference to an actual DNA sequence as an example when selecting the linker sequence.
For example, when the following 45-mer target nucleic acid (target nucleic acid 1) is detected.
[0052]

(1) Probe whose linker sequence is a sequence of identical bases
Probe example 1 (probe 1):

Probe example 2 (probe 2):

Example in which the same base sequence was selected for the linker sequence. The reason for choosing A or T instead of C or G for the linker sequence is that the mismatched bases A and T of the A and T bases are more stable than the mismatched bases G and G of the G and C bases. Because sex is lower. Further, by selecting A and T, it is possible to make it difficult for the probe to take an intramolecular structure.
[0053]
(2) Probe in which all linker sequences are mismatched
Probe example 3 (probe 3):

Probe example 4 (probe 4):

Probe example 5 (probe 5):

Examples 3 and 4 are examples of probes in which the linker sequence of (1) is replaced with an AA mismatch or an TT mismatch in the AT match. Example 5 is yet another mismatch.
There are the following eight combinations of mismatched bases.
AA, AC, AG, CC, CT, GG, GT, TT
By designing a linker sequence so as to cause any of these mismatches, a mismatched linker sequence can be obtained.
The above is the case where a sequence complementary to the common sequence is provided at the 3 ′ end of the probe, but this may be at the 5 ′ end. Also, it may be present on both the 3 ′ end side and the 5 ′ end side.
[0054]
In the present invention, the term "match" is a term that refers to a situation where a Watson-Crick base pair normally found in a nucleic acid in a living body is formed at a specific base pair. That is, it means a situation where a complementary strand is formed by any of AT, GC, and AU, which are general base pairs. However, this term broadly refers to a base pair that forms a hydrogen bond between the corresponding bases, and this term is also used when a base pair is formed at a base other than A, T, G, and C. You.
[0055]
In the present invention, the term “perfect match (PM)” is a term that means the state of base pairs in which the formed base pairs consist only of the above “matched” combinations. That is, when two sequences having different chain lengths are “perfect match (PM)”, it means that a base pair satisfying the above definition of “match” is formed over the shorter of the two sequences. For example, a sequence complementary to a specific sequence (both have the same chain length) and a probe sequence in which all bases are complementary to the partial sequence of the target nucleic acid (both have different chain lengths) are mutually " The sequence is a "perfect match (PM)".
[0056]
Further, in the present invention, the term "mismatch (MM)" is a term broadly meaning that the sequence has base pairs that do not originally form base pairs. "Mismatch (MM)" base pairs are generally defined as AA, AC, AG, CC, CT, GG, GT, TT in DNA. It is a base pair that is any combination of the above. However, the term broadly refers to a base pair that does not form a hydrogen bond between the corresponding bases, and is used even when a base pair other than A, T, G, and C does not form a base pair. .
[0057]
The support for immobilization is not limited to materials such as glass and plastic, and may be in any form such as a flat plate or a bead. In addition, if magnetic beads are used, beads in the liquid can be easily settled, and a washing operation or the like can be easily performed. In the case of the microarray, a support on a flat plate such as a slide glass is suitably used.
[0058]
The immobilization linker is not particularly limited as long as the probe of the present invention can be immobilized on the support, and known linkers commonly used by those skilled in the art can be used. Long chain alkyl, aminoalkyl and the like can be used.
[0059]
Any of the probes of the present invention can be chemically synthesized using a method known to those skilled in the art, or can be synthesized by outsourcing a synthesis contractor.
Preparation of a microarray using the probe can also be performed by a method known to those skilled in the art. For example, when the probe comprises only nucleic acid, it can be prepared by a method of solid phase synthesis on a slide glass, or a method of printing the synthesized probe on a support with a pin.
[0060]
[First method provided by the present invention]
The first method of the present invention is a method of measuring the probe of the present invention using a known amount of a fixed amount measurement nucleic acid containing a sequence complementary to the common sequence and labeled with a labeling substance,
1) a step of bringing the probe into contact with the fixed amount measurement nucleic acid and specifically hybridizing the two;
2) measuring the production of the hybrid in step 1 by a measuring means based on the labeling substance;
3) calculating the amount of the probe on the support based on the measurement result obtained in the step 2,
It is characterized by having.
[0061]
As the labeling substance, any labeling substance can be used according to the purpose of the experiment. When performing microarray, the labeling substance is a fluorescent dye, and a cyanine dye such as cyanine 3 (Cy3; Cyanine3) or cyanine 5 (Cy5; Cyanine5) is preferably used.
[0062]
The fixed amount measurement nucleic acid is a nucleic acid capable of specifically measuring the common complementary sequence of the probe of the present invention, and has a sequence complementary to all or a part of the common complementary sequence, and is labeled with the labeling substance. Nucleic acid.
[0063]
As the measuring means, an analytical instrument having a function of detecting the presence of the labeling substance and measuring the amount of the labeling substance is used. If the labeling substance is a fluorescent substance, a device equipped with an optical detection device is used.
[0064]
The “calculation” is to calculate the amount of the probe immobilized on the support by using a known amount of the nucleic acid for measuring the fixed amount. One molecule of the probe hybridizes with one molecule of the fixed amount measurement nucleic acid to form a double strand. Therefore, the value of the fixed amount measurement nucleic acid of a known amount to be measured as a control is measured, the value of the fixed amount measurement nucleic acid hybridized with the probe is measured, and the value of the probe on the support is measured from both the measured values. The amount of solid phase is calculated.
[0065]
[Second method provided by the present invention]
Further, in the second method of the present invention, the probe immobilized on a microarray and a known amount of a fixed amount measurement nucleic acid containing a sequence complementary to the common sequence and labeled with a labeling substance A are used. Use, a method for measuring the abundance of the target nucleic acid on the microarray,
1) preparing a sample containing the target nucleic acid labeled with a labeling substance B different from the labeling substance A;
2) a step of bringing the probe, the sample of step 1, and the nucleic acid for measuring an immobilized amount into contact with each other, and specifically hybridizing;
3) measuring the production of the hybrid in step 2 by a measuring means based on the labeling substance used;
4) calculating the abundance of the target nucleic acid in the sample based on the measurement result obtained in step 3;
With
The immobilization ratio of the probe between each spot on the microarray is calculated from the measurement result of the labeling substance A (calculated value A),
A hybridization amount of the known amount of the target nucleic acid used as the target nucleic acid to the probe is calculated from the measurement result of the labeling substance B (calculated value B),
The abundance of each of the plurality of types of target nucleic acids present in the sample is extrapolated from the calculated values A and B and estimated.
[0066]
The fixed amount measuring nucleic acid has the same meaning as the fixed amount measuring nucleic acid defined in the first method.
The microarray can be prepared by immobilizing the probe of the present invention on a microarray by a method known to those skilled in the art. However, the above method can be carried out by a method other than the microarray as long as various kinds of probes are immobilized on one support. Therefore, the probe can be immobilized on various microplates for measurement. Further, for example, when many kinds of probes are immobilized on the beads, each probe can be seeded on a microplate to carry out the present method.
[0067]
The sample is a mixture containing various nucleic acids (for example, mRNA or genomic DNA extracted from a living body) converted into the target nucleic acid in a general application. However, not only a nucleic acid derived from a nucleic acid of a living body but also a nucleic acid satisfying the definition of the target nucleic acid, even if it has an artificially designed sequence, can be used as the sample. Embodiments of the target nucleic acid suitably used in the present invention will be described in the following [Embodiments of the present invention].
[0068]
The labeling substance A and the labeling substance B are labeling substances different from each other, and may be any as long as they can measure the abundance of each without affecting each other. Preferably, either Cy3 or Cy5 is used for both. That is, when the labeling substance A is Cy5, Cy3 is used as the labeling substance B, and the reverse combination may be used.
[0069]
The calculated value A is a calculated value calculated from the measurement result of the labeling substance A. From the calculated value, the immobilization ratio of the probe between each spot on the microarray is calculated. The calculated value A can be calculated from the measured value of the labeling substance A as described in the “calculation” of the first method.
[0070]
The calculated value B is a calculated value calculated from the measurement result of the labeling substance B. From this calculated value, the probe of a known amount of the target nucleic acid to be used as the target nucleic acid (the probe corresponding to the known amount of the target nucleic acid used herein, and thus the probe having a specific complementary sequence to the target) ) Is calculated.
[0071]
The calculated value B can be measured, for example, as follows.
1) A known amount of target nucleic acid is spotted as an internal standard substance on the same microarray and measured.
2) Simultaneously measure the amount of hybridization of a known amount of the target nucleic acid to the corresponding probe.
3) A calibration curve is created from the relationship between the amount of the internal standard substance and the measured value based on the measured value in 1), and the amount of the target nucleic acid bound to the probe is calculated from the measured value in 2) and the calibration curve. .
[0072]
The calculated values A and B on the same spot are calculated, and the calculated value B is corrected for each spot by the calculated value A representing the immobilization ratio, and the measured value of the spot on which another type of the probe is immobilized The absolute amounts of all target nucleic acids in the sample can be measured by extrapolating the target nucleic acids.
[0073]
Hereinafter, typical embodiments of the present invention will be described with reference to the accompanying drawings. However, the technical scope of the present invention is not limited to these specific embodiments.
[Aspect of the present invention]
<Target nucleic acid of the present invention: FIGS.
Hereinafter, the target nucleic acid mainly targeted by the present invention will be described.
1 and 2 are diagrams illustrating an example of use of a target nucleic acid having a common primer sequence for gene detection. These figures illustrate the procedure for preparing a target nucleic acid targeted by the probe of the present invention.
[0074]
The structure of the nucleic acid (FIG. 1, A) is a nucleic acid having a common primer sequence (common sequences 1 and 2) used for preparing a target nucleic acid (3 'end on the right).
Many such sets are synthesized according to the detection gene. The common sequences 1 and 2 have the same sequence regardless of the detection target (FIG. 1, B).
Hybridization occurs in a sample containing a large number of single-stranded DNAs to be detected, and a ligase reaction occurs (FIG. 1, C).
If the detection target is present in the sample, it is linked (FIG. 1, C), but if the detection target does not exist, it is left unlinked (FIG. 1, D).
[0075]
In FIG. 2, a procedure for processing the linked target nucleic acid (FIG. 1, C) will be described.
The sequence configuration of the target nucleic acid is as shown in FIG. 2 (A), and has a specific sequence linked at the center (this strand shows the side complementary to the detection probe).
[0076]
In the steps described below (1-3 in FIG. 2B), the nucleic acid of FIG. 2 (A) is amplified and simultaneously labeled and single-stranded extracted. That is,
(1) A step of PCR-amplifying a target nucleic acid library with a labeled common primer.
(2) a step of capturing the PCR product of step (1) on magnetic beads having streptavidin.
(3) a step of extracting a fluorescently labeled single-stranded DNA from the captured target nucleic acid.
Thus, the target nucleic acid of the present invention can be prepared.
[0077]
The characteristics of the probe of the present invention as compared with the conventional probe will be exemplified for each embodiment.
<Description of conventional probe: Fig. 3>
FIG. 3 illustrates a conventional example of a probe for detecting a target nucleic acid.
A compound such as single-stranded RNA, DNA, or PNA having a sequence that specifically hybridizes to a specific sequence of the target nucleic acid is used as a probe. For example, when detection is performed with a probe complementary to the specific sequence of the target nucleic acid in (3) of FIG. 2, a hybrid as shown in FIG. 3 is formed.
[0078]
<Embodiment of Probe of the Present Invention: FIGS.
<< Probe mode 1 (extended probe) >>
FIG. 4 illustrates an example of hybridization according to one embodiment of the present invention using an extended probe.
(1) a probe having a sequence complementary to the specific sequence of the target nucleic acid (specific complementary sequence) + a sequence complementary to a part of the 3 ′ side of the common primer sequence on the 5 ′ side of the target nucleic acid (common complementary sequence) Here is an example.
(2) a probe having a sequence complementary to the specific sequence of the target nucleic acid (specific complementary sequence) + a probe having a sequence complementary to a part of the 5 ′ side of the 3 ′ common primer sequence of the target nucleic acid (common complementary sequence) Here is an example.
(3) a sequence complementary to a specific sequence of the target nucleic acid (specific complementary sequence) + a sequence complementary to a part of the 3 'side of the common primer sequence at the 5' side of the target nucleic acid (a common complementary sequence) + a 3 'side of the target nucleic acid An example of a probe having a sequence complementary to a part of the common primer sequence at the 5 ′ side (common complementary sequence) is shown.
By using such a probe, the probe length can be set long.
[0079]
<< Probe mode 2 (end capture probe) >>
FIG. 5 illustrates an example of hybridization using an end-capture probe according to one embodiment of the present invention.
In (1), a sequence complementary to the specific sequence of the target nucleic acid (specific complementary sequence) + a linker sequence + a sequence complementary to a part of the 5 'end of the target primer 5' common primer sequence (common complementary sequence) The example of the equipped probe is shown.
In (2), a sequence complementary to the specific sequence of the target nucleic acid (specific complementary sequence) + a linker sequence + a sequence complementary to a part of the 3 ′ end of the target nucleic acid 3′-side common primer sequence (common complementary sequence) The example of the equipped probe is shown.
(3) shows an example of a probe having linker sequences on both sides of a specific sequence of a target nucleic acid.
As in the embodiments described above, the probe of the present invention can be variously set with respect to the target nucleic acid, but is preferably set so that the end of the target nucleic acid is captured at the end of the probe. With such a setting, the hybridization efficiency can be increased.
[0080]
The reason will be described below with reference to FIG.
FIG. 7 expresses the localized kinetic energy of each part of the chain molecule indicated by an arrow as “spreading laterally”.
In the probe molecule on the right side of FIG. 7, since 5 ′ is immobilized, the momentum is small, and the momentum increases toward the free 3 ′ end in three dimensions. The 3 'end has the greatest momentum.
For the target nucleic acid molecule on the left, the momentum is the smallest at the center of the chain molecule that is the center of symmetry. When the center of the molecule moves, both ends have to be pulled, so the resistance is large, and the surrounding molecules (such as a solvent) also collide only two-dimensionally. The momentum is greatest at both the 5 'and 3' ends. At the end, there is little resistance in the molecule when moving, and surrounding molecules collide three-dimensionally.
[0081]
A chemical reaction is caused by collision from the right direction with a certain energy or more. The hybridization reaction is no exception, and molecules must first collide with each other. That is, the collision frequency is important.
[0082]
In the case of the above-described reaction system, the portions having the highest local intermolecular collision probability are the 3 'end of the probe and the 5' end and 3 'end of the target nucleic acid.
[0083]
However, if the 3 'end of the probe collides with the 3' end of the target nucleic acid, no hybridization is performed. Therefore, the highest reaction efficiency is obtained by capturing the 5 'end of the target nucleic acid at the 3' end of the probe.
[0084]
<Method for measuring nucleic acid using probe of the present invention: FIG. 6>
FIG. 6 illustrates the method for measuring the amount of probe immobilization according to the present invention.
When the probe of the present invention is used for the microarray, the fixed amount of each probe can be measured, and the probe can be used for measuring the absolute amount of the target nucleic acid captured in each spot.
For example, when the probe of the present invention is fixed as shown in FIG.
As shown in FIG. 6 (2), the target nucleic acid fluorescently labeled with Cy3 is hybridized. An amount of nucleic acid for measuring an immobilized amount, which is fluorescently labeled with Cy5 in such an amount as not to inhibit the hybridization, is also hybridized at the same time.
[0085]
Next, the hybridized microarray is read by a fluorescent scanner. Two images of the fluorescence wavelength of Cy5 and the fluorescence wavelength of Cy3 are obtained, and the fluorescence intensity reflecting the fixed amount ratio of each probe is found from the Cy5 image, and the fluorescence intensity corresponding to the amount ratio of each target nucleic acid is found in the Cy3 image. Obtained (3). Here, if target nucleic acids labeled with Cy3 of a known amount are mixed, the absolute amounts of all target nucleic acids can be measured.
[0086]
The embodiment in which the 5 'end of the probe of the present invention is immobilized on a support has been described. However, it will be readily understood by those skilled in the art that another embodiment in which the 3 ′ terminal side of the probe of the present invention is immobilized on a support can be carried out in the same manner, and has the same effect. Therefore, a probe whose 3 ′ end is immobilized on a support and a method using the probe are also included in the present invention.
[0087]
Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
[0088]
【Example】
[Example 1]
<< Comparison between hybridization at the center of the target nucleic acid and hybridization at the 5 'end of the target nucleic acid >>
<Experimental method>
{Circle around (1)} A 75-mer rE243 sequence labeled with Cy3 at the 5 ′ end was used as a target nucleic acid. The rE243 sequence is a 75-mer sequence consisting of 3 partial sequences of 25 mer, 3 ′ portion, central portion, and 5 ′ portion.
(2) As a probe for the target nucleic acid of the above (1), a CD243 probe complementary to the central 25 mer of rE243 and a CD147 probe complementary to the 5 ′ 25mer of rE243 were used.
[0089]
The probe was evaluated by hybridization using a microarray on which the target nucleic acid and the probe were immobilized. Details of the equipment, instruments, and reagents used in the experiment are described after the experimental results.
[0090]
First, 100 μl of 1 × SSC (containing 0.2% SDS) was prepared so as to contain 2.5 pmol of rE243 labeled with Cy3 at the 5 ′ end, and this was used as a target nucleic acid solution. This target nucleic acid solution was heated at 94 ° C. for 5 minutes before hybridization to perform pretreatment.
[0091]
A hybrid well (described later) was attached to a detection microarray on which CD243 probe and CD147 probe were immobilized. 100 μl of the target nucleic acid solution pretreated immediately before hybridization was injected into the hybrid well, and the microarray was subjected to a hybridization reaction in a 40 ° C. incubator for 60 minutes.
[0092]
Next, the hybridized microarray was taken out of the incubator, and the hybrid well was peeled off, immediately put into a staining vat filled with 1 × SSC, and rocked and washed for 10 minutes using a slide rack and a slide washer. Subsequently, the plate was similarly rock-washed with 0.1 × SSC for 5 minutes, and dried with a blower using Freon gas (HFC134a). This was observed using ScanArray4000, and the scan image was quantified using commercially available quantification software.
[0093]
<Experimental results>
The results shown below are values obtained by averaging the quantitative values of each probe and 4 spots.
[0094]
[Table 1]

[0095]
The above results revealed that the signal intensity was higher when a CD147 probe complementary to the 25mer from the 5 'end of the target nucleic acid was used than a CD243 probe complementary to the central 25mer of the 75mer target nucleic acid. In other words, it can be said that the efficiency is high when hybridization is performed using a sequence complementary to the 5 'end of the target nucleic acid as a probe.
[0096]
<Details of experimental conditions>
・ Detection chip:
An amino group-labeled synthetic oligo DNA was prepared by a method commonly used by those skilled in the art for covalently bonding to a commercially available white glass slide glass substrate. The spotting was carried out using a commercially available spotter having a pin head of 150 μm (pin pitch: 4.5 mm, spot pitch: 0.5 mm), and four spots of each probe were spotted.
[0097]
・ Detection target nucleic acid:
Using a synthetic oligo DNA having the same sequence as the sequence to be detected as a template, a PCR reaction was performed using biotinylated fwd primer and Cy3-labeled rev primer. The PCR product was purified with streptavidin magnetic beads.
[0098]
Streptavidin magnetic beads form a relatively stable bond with biotin even under high temperature conditions to isolate Cy3-modified single-stranded DNA (ss-DNA). That is, the PCR product was captured on magnetic beads and dehybridized in hot water to isolate only the Cy3-modified ss-DNA target nucleic acid (see FIG. 2).
[0099]
・ Hybridization conditions:
Target nucleic acid: Cy3-labeled rE243 2.5 pmol
Reaction solution: 0.2% SDS 1 × SSC 100 μl
Hybrid well: Hybriwell (HBW75) manufactured by Grace BIO-LABS
Reaction temperature: 40 ° C
Pretreatment: 94 ° C for 5 minutes
・ Processing after washing:
Slide washer (Jujo Field Corporation SW-4)
・ Observation conditions:
GSI lumonics scanarray4000 was used, and the setting of LASER / PMT was 100% / 100%, respectively. The resolution is set to 5 μm.
・ Quantification method:
Quantification was performed using commercially available microarray quantification software (QuantArray).
[0100]
<Detailed array information>
The sequences used are described below. For the sequence design, an artificially designed sequence is used in consideration of the cross-hybridization characteristics and the Tm value.
(Notation is 5 '→ 3')
(1) Target nucleic acid sequence
・ 5'-end Cy3-labeled rE243 75mer
TgAAgTTCCgCTTTggAgTggCTAAggAgCAAATATAAATgCgATggCTTTggTTAACTgCCTTCCCCTCCAAA (SEQ ID NO: 7)
(2) Probe sequence
・ 5'-terminal amino group-labeled CD147 probe (complementary to 5'-end 25mer of target nucleic acid)
TTagCCACTCCAAAAgCggAACTTCA (SEQ ID NO: 8)
・ 5'-terminal amino group-labeled CD243 probe (complementary to the central 25mer of the target nucleic acid)
AgCCATCgCATTATATTTTgCTCCC (SEQ ID NO: 9)
[Example 2]
<< Example of reaction specificity of probe and extension direction of common complementary sequence >>
<Experimental method>
{Circle around (1)} A 75-mer rC243 sequence labeled with Cy3 at the 5 ′ end was used as a target nucleic acid. The rC243 sequence is a 75-mer sequence consisting of three partial sequences of the 3 'part, the central part and the 5' part of 25 mer each.
[0101]
(2) As a probe for the target nucleic acid of (1) above, a C243 probe 75 mer which perfectly matches rC243, a C037 probe 75 mer which matches 25 mer from both ends of rC243 but has a mismatch at the center 25 mer, A 243 + CED probe 50 mer complementary from the 5 'end to 50 mer of rC243, a 037 + CED probe 50 mer complementary from the 5' end to 25 mer of rC243 but mismatched with the central 25 mer of rC243, 50 mer from 3 'end of rC243. The 014SD + 243 probe 50mer that is complementary to and the CD243 probe that is complementary to the central 25mer of rC243 were used.
[0102]
The probe was evaluated by hybridization using a microarray on which the target nucleic acid and the probe were immobilized. Details of the equipment, instruments, and reagents used in the experiment are described after the experimental results.
[0103]
First, rC243 labeled with Cy3 at the 5 ′ end was prepared in 100 μl of 1 × SSC (containing 0.2% SDS) so as to contain 5 pmol, and this was used as a target nucleic acid solution. This target nucleic acid solution was heated at 94 ° C. for 5 minutes before hybridization to perform pretreatment.
[0104]
A hybrid well was attached to a detection microarray on which a probe was immobilized. Immediately before, 100 μl of the pretreated target nucleic acid solution was injected into the hybrid well, and the microarray was subjected to a hybridization reaction in a 40 ° C. incubator for 60 minutes.
[0105]
Next, the microarray after hybridization was taken out of the incubator, and the hybrid well was peeled off, immediately put into a staining vat filled with 0.1 × SSC, and rocked and washed for 5 minutes using a rack and a slide washer. Dry with a blower using HFC134a). This was observed using ScanArray4000, and the scan image was quantified using commercially available quantification software.
[0106]
<Experimental results>
The results shown below are values obtained by averaging the quantitative values of each probe and 4 spots.
[0107]
[Table 2]

[0108]
From the above results, the 5′-terminal immobilized probe (probe that hybridizes using complementarity with the 5 ′ end of the target nucleic acid) has high hybridization efficiency regardless of the chain length of the sequence that matches the target nucleic acid. It became clear. However, it was revealed that if only the 5'-terminal 25mer was matched, the reaction would occur even if there was no reaction specificity at the central 25mer (037 + CED probe). That is, it was revealed that the reaction specificity at the central 25-mer portion could not be determined because the PM / MM ratio (perfect match / mismatch ratio) was small.
[0109]
It is also clear that the hybridization efficiency of the 3 'end capture probe (a 50-mer probe that uses the same target nucleic acid end, but a probe that captures the 3' end of the target nucleic acid; 014SD + 243 probe) is extremely low. became. The hybridization efficiency of the CD243 probe complementary to only the central 25mer portion was even lower.
[0110]
<Details of experimental conditions>
・ Detection chip:
It was prepared by a method commonly used by those skilled in the art for covalently bonding an amino-labeled synthetic oligo DNA to a commercially available slide glass substrate. The base material used was an ordinary white plate slide glass. For the spotting, using a commercially available spotter having a pin head of 150 μm (pin pitch: 4.5 mm, spot pitch: 0.5 mm), four spots of each probe were spotted on the substrate.
[0111]
・ Detection target nucleic acid:
Using a synthetic oligo DNA having the same sequence as the sequence to be detected as a template, a PCR reaction was performed using biotinylated fwd primer and Cy3-labeled rev primer. The PCR product was purified with streptavidin magnetic beads.
[0112]
Streptavidin magnetic beads form a relatively stable bond with biotin even under high temperature conditions to isolate Cy3-modified single-stranded DNA (ss-DNA). That is, the PCR product was captured on magnetic beads and dehybridized in hot water to isolate only the Cy3-modified ss-DNA target nucleic acid (see FIG. 2).
[0113]
・ Hybridization conditions:
Target nucleic acid: Cy3-labeled rC243 5 pmol
Reaction solution: 0.2% SDS 1 × SSC 100 μl
Hybrid well: Hybriwell (HBW75) manufactured by Grace BIO-LABS
Reaction temperature: 40 ° C
Pretreatment: 94 ° C for 5 minutes
・ Processing after washing:
Slide washer (Jujo Field Corporation SW-4)
・ Observation conditions:
GSI lumonics scanarray4000 was used, and the setting of LASER / PMT was 100% / 100%, respectively. The resolution is set to 5 μm.
・ Quantification method:
Quantification was performed using commercially available microarray quantification software (QuantArray).
[0114]
<Details of sequence information>
The sequences used are described below. For the sequence design, a sequence artificially designed in consideration of cross-hybridization and Tm value is used.
(Notation is 5 '→ 3')
(1) Target nucleic acid sequence
-5'-end Cy3-labeled rC243 75mer
gATTCgTCgTgTgCCTTTTgACTgTTggggAgCAAATAATAAATgCgATggCTTTggTTAACTgCCTTCCCCTCCAAA (SEQ ID NO: 10)
(2) Probe sequence
・ 5 'terminal amino group labeled C243 probe 75mer (perfect match for target nucleic acid)
TTgTgAggggAAAggcAgTTAAAcAAgccATcgcATTTATATTTgcTcccAAcAgTcAAAAggcAcAcgAcgAATc (SEQ ID NO: 11)
-5'-terminal amino group-labeled CD037 probe 75mer (only the central 25mer of the target nucleic acid is mismatched)
TTgTgAggggAAggcAgTTAAAcAATTAATcAcggAcTgTcTTcgTTgccAAcAgTcAAAAggcAcAcgAcgAATc (SEQ ID NO: 12)
-5 'terminal amino group labeling 243 + CED probe 50 mer (complementary to 50 mer from 5' end of target nucleic acid)
AgccATcgcATTTATTATTGcTcccAAcAgTcAAAggcAcAcgAcgAATc (SEQ ID NO: 13)
-5'-terminal amino group labeling 037 + CED probe 50mer (matches 25mer from the 5 'end of target nucleic acid, mismatches with 25mer at the center of target nucleic acid)
TTAATcAccgAcTgTcTTcgTTgccAAccTcccATTcAccATTgTcAgAg (SEQ ID NO: 14)
-5'-terminal amino group labeling 014SD + 243 probe 50mer (complementary to 50mer from 3 'end of target nucleic acid)
TTgTgAggggAAggcAgTTAAccAAAgccATcgcATTTATATTTgcTccc (SEQ ID NO: 15)
・ 5 'terminal amino group labeling CD243 probe 25mer (complementary to the central 25mer of the target nucleic acid)
AgCCATCgCATTATATTTTgCTCCC (SEQ ID NO: 16)
[Example 3]
<< ED extension probe and target nucleic acid 5 'end capture probe >>
<Experimental method>
{Circle around (1)} A 75-mer rC219 sequence labeled with Cy3 at the 5 ′ end was used as a target nucleic acid. The rC219 sequence is a 75-mer full-length sequence composed of three partial sequences of a 25-mer 3 'portion, a central portion, and a 5' portion.
[0115]
(2) Probes for the target nucleic acid of the above (1), which are grouped into the following groups A to H:
(A): control probe;
A probe having a sequence corresponding to the central 25 mer of the target nucleic acid.
(BE): ED extension probe;
In addition to the sequence corresponding to the 25mer of the target nucleic acid central portion, a sequence extended from the 3, 5 ', 18 and 21mer continuously at the 3' end of the sequence so as to be complementary to the sequence of the corresponding target nucleic acid is further provided. Probes of 35, 40, 43, and 46 mer, respectively.
(FH): target nucleic acid 5 ′ end capture probe;
In addition to the sequence corresponding to the central portion of the target nucleic acid 25mer, a mismatch sequence is inserted at the 3 'end of the sequence, and a 50-mer having a sequence complementary to the sequence of 5, 10, 15 mer from the 5' end of the target nucleic acid Probe,
It was used.
[0116]
Each of the probe groups A to H includes a “central part PM probe” designed so that the “sequence corresponding to the central part 25mer of the target nucleic acid” is complementary to the central part of the target nucleic acid; Part and a “central part MM probe” designed to be mismatched. By comparing and examining the reactivity of such two types of probes, the reaction specificity of the various probes described above to the target nucleic acid central part 25mer can be evaluated.
[0117]
The probes were evaluated by hybridization using a microarray on which the target nucleic acid of (1) and the probe of (2) were immobilized. Details of the equipment, instruments, and reagents used in the experiment are described after the experimental results.
[0118]
First, 5 μmol of Cy3 labeled rC219 at the 5 ′ end was prepared in 100 μl of a commercially available hybridization buffer to prepare a target nucleic acid solution.
[0119]
A hybrid well was attached to the detection microarray on which the probe was immobilized. 100 μl of the target nucleic acid solution pretreated immediately before the hybridization was injected into a hybrid well, and the microarray was subjected to a hybridization reaction in a 50 ° C. incubator for 3 hours.
[0120]
Next, the microarray after hybridization was taken out of the incubator, and the hybrid well was peeled off, immediately put into a staining vat filled with 1 × SSC, and rock-washed for 10 minutes using a rack and a slide washer. Subsequently, the plate was similarly rock-washed with 0.1 × SSC for 5 minutes, and then dried with a blower using HFC134a.
[0121]
This was observed using ScanArray4000, and the scan image was quantified using commercially available quantification software.
[0122]
<Experimental results>
The results shown below are values obtained by averaging the quantitative values of each probe and 4 spots.
[0123]
[Table 3]

[0124]
[Table 4]

[0125]
CD in Tables 3 and 4 indicates the sequence name of the target nucleic acid 75mer central part 25mer, and the CD means a sequence complementary to the 219 probe in the rC219 sequence, which is the target nucleic acid used in this example. .
In the target nucleic acid 5 'end direction extension type probe, the probe length is different for each probe, and the total length of +10 mer, +15 mer, +18 mer, and +21 mer is 35 mer, 40 mer, 43 mer, and 46 mer, respectively. .
[0126]
Regarding the target nucleic acid 5 ′ end capture probe, control 25 mer is CD (only the central 25 mer of the target nucleic acid), +5 mer, +10 mer, and +15 mer are 20 mer, 15 mer, and CD mer, respectively. A 50-mer probe having a 5-mer, 10-mer, and 15-mer complementary sequence from the 5 'end of the target nucleic acid, sandwiching the 10-mer mismatch region.
Tables 3 and 4 show the results obtained on the same microarray, and direct comparison between them is possible.
[0127]
The results are summarized as follows.
(1) Target Nucleic Acid In the 5 'end extension type probe, the signal of the PM probe increased about 2-fold at +10 mer, whereas the signal of the MM probe hardly increased.
[0128]
(2) Target nucleic acid In the 5'-end-extended probe, the signal of PM and the signal of PM were increased at +15 mer or more, and the sequence specificity began to be lost.
[0129]
(3) In the target nucleic acid 5′-end capture probe, signal enhancement was already observed at the target nucleic acid 5′-end capture length of 5 mer. Up to 10 mer, only the PM signal increased and the MM signal hardly changed.
[0130]
(4) In the target nucleic acid 5 'end capture probe, MM signal enhancement is observed at the target nucleic acid 5' end capture length of 15 mer.
[0131]
(5) When comparing the target nucleic acid 5 'end extension probe with the target nucleic acid 5' end capture probe, the target nucleic acid 5 'end extension probe +10 mer (match length 35 mer) and the target nucleic acid 5' end The +5 mer (30 mer match length) of the capture probe shows almost the same hybridization intensity, and the target nucleic acid 5 'end capture probe +10 mer (match length 35 mer) has a match length of the target nucleic acid 5' end extension type. The signal is the same as that of the probe +10 mer, but the signal of the target nucleic acid 5 ′ end capture type is stronger (actually, the measured value of the end capture type under the conditions is saturated, so the signal intensity is even stronger).
[0132]
・ Detection chip:
It was prepared by a method commonly used by those skilled in the art for covalently bonding an amino-labeled synthetic oligo DNA to a commercially available slide glass substrate. The base material used was an ordinary white plate slide glass. For the spotting, using a commercially available spotter having a pin head of 150 μm (pin pitch: 4.5 mm, spot pitch: 0.5 mm), four spots of each probe were spotted on the substrate.
[0133]
・ Detection target nucleic acid:
Using a synthetic oligo DNA having the same sequence as the sequence to be detected as a template, a PCR reaction was performed using biotinylated fwd primer and Cy3-labeled rev primer. The PCR product was purified with streptavidin magnetic beads.
[0134]
Streptavidin magnetic beads form a relatively stable bond with biotin even under high temperature conditions to isolate Cy3-modified single-stranded DNA (ss-DNA). That is, the PCR product was captured on magnetic beads and dehybridized in hot water to isolate only the Cy3-modified ss-DNA target nucleic acid (see FIG. 2).
[0135]
・ Hybridization conditions:
Target nucleic acid: Cy3-labeled rC219 5 pmol
Reaction solution: GlassHyb hybridization solution (CLONTECH)
Hybrid well: Hybriwell (HBW75) manufactured by Grace BIO-LABS
Reaction temperature: 50 ° C
・ Processing after washing:
Slide washer (Jujo Field Corporation SW-4)
・ Observation conditions:
GSI lumonics scanarray4000 was used, and the setting of LASER / PMT was 100% / 100%, respectively. The resolution was set at 5 μm.
・ Quantification method:
Quantification was performed using commercially available microarray quantification software (QuantArray).
[0136]
<Details of sequence information>
The sequences used are described below. For the sequence design, a sequence artificially designed in consideration of cross-hybridization and Tm value is used.
(Notation is 5 '→ 3')
(1) Target nucleic acid sequence
-5'-end Cy3-labeled rC219 75mer
gATTCgTCgTgTgCCTTTTgACTgTTACTgAACTTTTTCCATgCCggTCAATTggTTAACTgCCTTCCCCTCCAAA (SEQ ID NO: 17)
(2) Probe sequence
Control probe;
(A) Probe corresponding to the central 25mer of the target nucleic acid
・ 5 'terminal amino group labeled CD032 probe 25mer (mismatch probe)
ATTACgCACgAAATTACACCggTAC (SEQ ID NO: 18)
・ 5 'terminal amino group labeled CD037 probe 25mer (mismatch probe)
TTAATCACCgACTgTCTTcgTTgCC (SEQ ID NO: 19)
・ 5'-terminal amino group-labeled CD219 probe 25mer (complementary to the central 25mer of the target nucleic acid)
TTgACCggCATggAAAAAAgTTCAgT (SEQ ID NO: 20)
・ 5 'terminal amino group labeled CD243 probe 25mer (mismatch probe)
AgCCATCgCATTATATTTTgCTCCC (SEQ ID NO: 21)
ED extension probe;
(B) Probe corresponding to a sequence extended by 10 mer toward the 5 'end of the target nucleic acid in addition to the central 25-mer of the target nucleic acid
-5'-terminal amino group labeling 032 + 10mer probe 35mer (25mer mismatch at the center of the target nucleic acid, 10mer complement at the 5 'side of the target nucleic acid)
ATTAcgcAggAAATTAcAcggTAcAAcAgTcAAA (SEQ ID NO: 22)
-5'-terminal amino group labeling 037 + 10mer probe 35mer (25mer mismatch at the center of the target nucleic acid, 10mer complement at the 5 'side of the target nucleic acid)
TTAATcAcccAcTgTcTTcgTTgccAAcAgTcAAA (SEQ ID NO: 23)
-5'-terminal amino group labeling 219 + 10-mer probe 35-mer (complementary to central 25-mer of target nucleic acid, complementary to 10-mer on 5 'side of target nucleic acid)
TTgAccggcATggAAAAgTTcAgTAAcAgTcAAA (SEQ ID NO: 24)
-5'-terminal amino group labeling 243 + 10-mer probe 35-mer (25-mer mismatch at the center of the target nucleic acid, 10-mer complementary to the target nucleic acid 5 'side)
AgccATcgcATTTATTATTgcTcccAAcAgTcAAA (SEQ ID NO: 25)
(C) Probe corresponding to a sequence extended by 15 mer toward the 5 'end of the target nucleic acid in addition to the central 25-mer of the target nucleic acid
-5'-terminal amino group labeling 032 + 15mer probe 40mer (25mer mismatch at the center of the target nucleic acid, 15mer complementary to the 5 'side of the target nucleic acid)
ATTAcgcAggAAATTAcAcggTAcAAcAgTcAAAggcAc (SEQ ID NO: 26)
-5 'terminal amino group labeling 037 + 15 mer probe 40 mer (target nucleic acid central part 25mer mismatch, target nucleic acid 5' side 15mer complementary)
TTAATcAcccAcTgTcTTcgTTgccAAcAgTcAAAggcAc (SEQ ID NO: 27)
-5'-terminal amino group labeling 219 + 15mer Probe 40mer (complementary to the target nucleic acid central part 25mer, target nucleic acid 5 'side 15mer complementary)
TTgAccggcATggAAAAAAgTTcAgTAAcAgTcAAAggcAc (SEQ ID NO: 28)
-5'-terminal amino group labeling 243 + 15-mer probe 40-mer (25-mer mismatch at the center of the target nucleic acid, 15-mer complementary to the 5'-side of the target nucleic acid)
AgccATcgcATTTATTATTgcTcccAAcAgTcAAAggcAc (SEQ ID NO: 29)
(D) Probe corresponding to a sequence extended by 18 mer toward the 5 'end of the target nucleic acid in addition to the central 25-mer of the target nucleic acid
-5 'terminal amino group labeling 032 + 18mer probe 43mer (25mer mismatch at the center of the target nucleic acid, complementary to the 18mer at the 5' side of the target nucleic acid)
ATTAcgcAggAAATTAcAcggTAcAAcAgTcAAAggcAcAcg (SEQ ID NO: 30)
-5'-terminal amino group labeling 037 + 18mer probe 43mer (25mer mismatch at the center of the target nucleic acid, complementary to the 18mer at the 5 'side of the target nucleic acid)
TTAATcAcgAcTgTcTTcgTTgccAAcAgTcAAggcAcAcg (SEQ ID NO: 31)
-5'-terminal amino group labeling 219 + 18mer probe 43mer (complementary to target mer nucleic acid central 25mer, complementary to target nucleic acid 5'-side 18mer)
TTgAccggcATggAAAAAAgTTcAgTAAcAgTcAAAggcAcAcg (SEQ ID NO: 32)
-5 'terminal amino group labeling 243 + 18 mer probe 43 mer (25mer mismatch at target nucleic acid center, 18mer complementary to 5' target nucleic acid at target nucleic acid)
AgccATcgcATTTATTATTgcTcccAAcAgTcAAAggcAcAcg (SEQ ID NO: 33)
(E) Probe corresponding to a sequence that is extended by 21 mer toward the 5 'end of the target nucleic acid in addition to the central 25-mer of the target nucleic acid
-5 'terminal amino group label 032 + 21 mer probe 46 mer (25mer mismatch at the center of target nucleic acid, 21mer complementary to 5' side of target nucleic acid)
ATTAcgcAgAAATTAcAcggTAcAAcAgTcAAAggcAcAcgAcg (SEQ ID NO: 34)
-5 'terminal amino group labeling 037 + 21 mer probe 46 mer (25mer mismatch at the center of the target nucleic acid, 21mer complementary to the 5' side of the target nucleic acid)
TTAATcAcccAcTgTcTTcgTTgccAAcAgTcAAAggcAcAcgAcg (SEQ ID NO: 35)
-5'-terminal amino group labeling 219 + 21 mer probe 46 mer (complementary to target mer nucleic acid central part 25 mer, target nucleic acid 5 'side 21 mer complement)
TTgAccggcATggAAAAAgTTcAgTAAcAgTcAAAggcAcAcgAcg (SEQ ID NO: 36)
-5 'terminal amino group labeling 243 + 21 mer probe 46 mer (25mer mismatch at the center of the target nucleic acid, 21mer complementary to the 5' side of the target nucleic acid)
AgccATcgcATTTATTATTgcTcccAAcAgTcAAAggcAcAcgAcg (SEQ ID NO: 37)
Target nucleic acid 5 'end capture probe;
(F) Probe in which a sequence corresponding to the central 25mer of the target nucleic acid and a sequence complementary to the 5mer from the 5 ′ end of the target nucleic acid are linked via a mismatch region 20mer
The portion intended for the mismatch was replaced with T having the least content of the four types of bases contained in this region.
[0137]
* Reference CED probe sequence: In the part of 20 bases from the left of AAcAgTcAAggcAcAcgAcgAATc (SEQ ID NO: 38)
A: 945%
T: 15%
c: 6 30%
g: 4 20%
-5 'terminal amino group labeling 032 + 5'5 mer probe 50mer (25mer mismatch at the center of the target nucleic acid, 5mer complementary from the 5' end of the target nucleic acid via the mismatch region of Tx20)
ATTAcgcAgAAATTAcAcggTAcTTTTTTTTTTTTTTTTTTTTTTgAATc (SEQ ID NO: 39)
-5 'terminal amino group labeling 037 + 5' 5 mer probe 50 mer (25 mer mismatch at the center of the target nucleic acid, 5 mer complementary from the 5 'end of the target nucleic acid via the mismatch region of Tx20)
TTAATcAcccAcTgTcTTcgTTgccTTTTTTTTTTTTTTTTTTTTTTgAATc (SEQ ID NO: 40)
-5'-terminal amino group labeling 219 + 5'5mer probe 50mer (complementary to the target nucleic acid central 25mer, 5mer complementary from the target nucleic acid 5'end via the Tx20 mismatch region)
TTgAccggcATggAAAAAgTTcAgTTTTTTTTTTTTTTTTTTTTTTTTgAATc (SEQ ID NO: 41)
-5 'terminal amino group labeling 243 + 5'5 mer probe 50mer (25mer mismatch at the center of the target nucleic acid, 5mer complementary from the 5' end of the target nucleic acid via the mismatch region of Tx20)
AgccATcgcATTTATTATTgcTcccTTTTTTTTTTTTTTTTTTTTTTgAATc (SEQ ID NO: 42)
(G). A probe in which a sequence corresponding to the central 25mer of the target nucleic acid and a sequence complementary to the 10mer from the 5 'end of the target nucleic acid are linked via a mismatch region 15mer
-5'-terminal amino group labeling 032 + 5 '10-mer probe 50-mer (25-mer mismatch at the center of the target nucleic acid, 10-mer complement from the 5'-end of the target nucleic acid via the mismatch region of Tx15)
ATTAcgcAgAAATTAcAcggTAcTTTTTTTTTTTTTTTTAcgAcgAATc (SEQ ID NO: 43)
-5'-terminal amino group labeling 037 + 5 '10-mer probe 50-mer (25-mer mismatch at the center of the target nucleic acid, 10-mer complementary from the 5'-end of the target nucleic acid via the mismatch region of Tx15)
TTAATcAccgAcTgTcTTcgTTgccTTTTTTTTTTTTTTTTTAcgAcgAATc (SEQ ID NO: 44)
-5'-terminal amino group labeling 219 + 5 '10-mer probe 50-mer (complementary to the target nucleic acid central 25-mer, 10-mer complementary from the target nucleic acid 5' end via the Tx15 mismatch region)
TTgAccggcATggAAAAAAgTTcAgTTTTTTTTTTTTTTTTTAcggAcgAATc (SEQ ID NO: 45)
-5'-terminal amino group labeling 243 + 5 '10-mer probe 50-mer (25-mer mismatch at the center of the target nucleic acid, 10-mer complementary from the 5'-end of the target nucleic acid via the mismatch region of Tx15)
AgccATcgcATTTATTATTgcTcccTTTTTTTTTTTTTTTTAcgAcgAATc (SEQ ID NO: 46)
(H) A probe in which a sequence corresponding to the central 25mer of the target nucleic acid and a sequence complementary to the 15mer from the 5 'end of the target nucleic acid are linked via a mismatch region 10mer
-5'-terminal amino group labeling 032 + 5 '15-mer probe 50-mer (25-mer mismatch at the center of the target nucleic acid, 15-mer complementary from the 5'-end of the target nucleic acid via the mismatch region of Tx10)
ATTAcgcAggAAATTAcAcggTAcTTTTTTTTTTTggcAcAcgAcgAATc (SEQ ID NO: 47)
-5'-terminal amino group labeling 037 + 5 '15-mer probe 50-mer (25-mer mismatch at the center of the target nucleic acid, 15-mer complementary from the 5'-end of the target nucleic acid via the mismatch region of Tx10)
TTAATcAcccAcTgTcTTcgTTgccTTTTTTTTTTTTggcAcAcgAcgAATc (SEQ ID NO: 48)
-5 'terminal amino group labeling 219 + 5' 15 mer probe 50 mer (complementary to target nucleic acid central 25mer, complementary to target nucleic acid 5 'end from target nucleic acid 5' end via mismatching region of Tx10)
TTgAccggcATggAAAAAgTTcAgTTTTTTTTTTTTggcAcAcgAcgAATc (SEQ ID NO: 49)
-5'-terminal amino group labeling 243 + 5 '15-mer probe 50-mer (25-mer mismatch at the center of the target nucleic acid, 15-mer complementary from the 5'-end of the target nucleic acid via the mismatch region of Tx10)
AgccATcgcATTTATTATTgcTcccTTTTTTTTTTTggcAcAcgAcgAATc (SEQ ID NO: 50)
[0138]
【The invention's effect】
Since the probe has a sequence complementary to the common sequence in addition to the sequence specific to the target nucleic acid,
1) The probe length can be set long. This allows the hybridization reaction to be performed at a higher temperature. Therefore, it is difficult for the target nucleic acid to have a stable structure during the hybridization reaction, and the reactivity of the target nucleic acid is increased.
[0139]
2) All detection probes have a common sequence. On the other hand, a labeled nucleic acid for detection that hybridizes to the common complementary sequence portion of the probe is prepared. The labeled nucleic acid is hybridized, and the amount of the immobilized probe can be measured.
[0140]
3) The ratio of the immobilized amount of each probe can be measured, and the amount of the target nucleic acid detected by all the probes can be measured by using a standard sample whose absolute amount is determined.
[0141]
In addition, by using a probe further provided with a linker sequence as the probe, hybridization is more likely to occur and the reaction speed is increased. A probe that can react at a low temperature can be obtained that has a low internal molecular structure for its length. In addition, highly sensitive nucleic acid detection can be performed.
[0142]
[Sequence list]

[Brief description of the drawings]
FIG. 1 is a diagram showing an example of use of a target nucleic acid having a common primer sequence for gene detection.
FIG. 2 is a diagram showing a sequence configuration of a target nucleic acid.
FIG. 3 is a diagram showing an example of a conventional probe for detecting a target nucleic acid.
FIG. 4 is a diagram illustrating a form of hybridization using an extended probe.
FIG. 5 is a diagram illustrating a form of hybridization using an end-capture probe.
FIG. 6 is a diagram illustrating a method for measuring a probe fixed amount.
FIG. 7 is a diagram for explaining the reason why the hybridization efficiency is improved by capturing the end of a target nucleic acid.
FIG. 8 is a diagram illustrating an outline of a comparative experiment of hybridization at the central portion and hybridization at the 5 ′ end of a target nucleic acid.
FIG. 9 is a diagram schematically illustrating a comparative experiment on the reaction specificity of a probe and the extension direction of a common complementary sequence.
FIG. 10 is a diagram illustrating an outline of a comparative experiment on an ED extension probe and an end capture probe.

Claims (6)

A probe for measuring a target nucleic acid having a common sequence at both ends of a specific sequence,
The probe includes, in addition to the specific complementary sequence complementary to the specific sequence, a common complementary sequence that is complementary to all or a part of the common sequence and shorter than the specific complementary sequence. Having a nucleic acid;
The common complementary sequence is linked directly or via a linker to the 3 ′ end and / or 5 ′ end of the specific complementary sequence; and
One end of the probe is immobilized directly on a support for immobilization or via a linker for immobilization,
A probe. The probe according to claim 1, wherein the specific complementary sequence has a chain length of 10 to 50 bases, and the common complementary sequence has a chain length of 5 to 30 bases. 3. The probe according to claim 1, wherein the nucleic acid is selected from the group consisting of deoxyribonucleic acid, ribonucleic acid, and peptide nucleic acid. 4. The probe according to any one of claims 1 to 3, wherein the linker is selected from the group consisting of a long-chain compound such as an alkyl chain, a nucleic acid consisting of an adenine base, a nucleic acid consisting of a thymine base, and a polypeptide chain. Probe. The probe according to any one of claims 1 to 4, which is measured using a known amount of a fixed amount nucleic acid containing a sequence complementary to the common sequence defined in claim 1 and labeled with a labeling substance. A way to
1) a step of bringing the probe into contact with the fixed amount measurement nucleic acid and specifically hybridizing the two;
2) measuring the production of the hybrid in step 1 by a measuring means based on the labeling substance;
3) calculating the amount of the probe on the support based on the measurement result obtained in the step 2,
A method comprising: A probe according to any one of claims 1 to 4, which is immobilized on a microarray, and a known amount of immobilization comprising a sequence complementary to the common sequence defined in claim 1 and labeled with a labeling substance A. A method for measuring the abundance of the target nucleic acid on the microarray using the nucleic acid for measuring the amount,
1) preparing a sample containing the target nucleic acid labeled with a labeling substance B different from the labeling substance A;
2) a step of bringing the probe, the sample of step 1, and the nucleic acid for measuring an immobilized amount into contact with each other, and specifically hybridizing;
3) measuring the production of the hybrid in step 2 by a measuring means based on the labeling substance used;
4) calculating the abundance of the target nucleic acid in the sample based on the measurement result obtained in step 3;
With
The immobilization ratio of the probe between each spot on the microarray is calculated from the measurement result of the labeling substance A (calculated value A),
A hybridization amount of the known amount of the target nucleic acid used as the target nucleic acid to the probe is calculated from the measurement result of the labeling substance B (calculated value B),
A method comprising extrapolating the abundance of each of a plurality of types of target nucleic acids present in the sample from calculated values A and B.

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